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ALLERGY
INVESTIGATIONS ON AEROALLERGENS OF IMPHAL WITH SPECIAL REFERENCE TO RESPIRATORY ALLERGY  

Study was conducted to investigate the aeroallergens of Imphal with special reference to respiratory allergy. The flora of Imphal was never free from flowering throughout the period of investigation. These plants were catagorised into four groups viz. trees, shrubs and undershrubs, climbers and herbs (wild and cultivated). Out of 257 species of wild and cultivated angiospermic plants collected, trees contributed 57 species, shrubs and undershrubs 68 species, herbs 115 species and climbers contributed 17 species. Plants like Hibiscus rosa sinensis, Acacia nilotica, Callistemon linearis, Canna indica, Rosa spp, solanum melongena, Lantana camera, Duranta plumeri, Bougainvillea specatbilis, etc. were found flowering almost round the year. From the allergenic point of view, the following plants are common in Imphal and its suburbs-tress (Melia azadirachta, Mangifera indica, Cassia fistula, Callistemen linearis, Eucalyptus globosus, Carica papaya etc.) shrubs and undershrubs (Rosa indica, Datura metal, Lantana camera, Ricinus communis. Climbers - Cucurbita maxima, Lathyrus sativus, etc.) and herbs (Ageratum conyzoids, Amaranthus spinosa, Artemisia vulgaris, Chenopo- dium album , C.ambrisoides, Coriandrum sativum, Cannabis sativa, Cyperus rotundus, Cynodon dactylon, Erogrostis gangetica, Oryza sativa, Poa annua, Setaria glauca, Targetes erecta, Plantago major, Xanthium strumarium, etc) In Imphal the pollen content of the atmosphere was found to be maximum from July to August, minimum from April to May and moderate from November to January. On the basis of the atmospheric pollen incidence four principal pollen seasons in Imphal could be identified. Trees predominate from February to April, shrubs and undershrubs from December to February, grasses from July to September and weeds from August to October. Among the pollen types identified from the air of Imphal, Poaceae (Cynodon, Digitaria, Eleusine, Oryza, Panicum, Setaria, Imperata and Zea), Cyperaceae (Cyperus), Alnus, Callistemon, Artemisia, Eucalyptus, Carica, Syzygium, Azadirachta, Coriandrum, Morus, Xanthium, Albizzia, Amaranthus, Ageratum, Lantana, Mangifera, Ricinus, Cassia, Rumex, Bauhinia, Brassica, Chenopodium and Acacia, were proved allergenically significant in the published literature. In this region allergenic pollens of weeds which cause symptoms of rhinitis or asthma, were found to be most important followed by those of grasses and trees. Aspergilli-Penicilli ranked second in total yearly contribution. The maximum concentration of Aspergilli-Penicilli spores were recorded during March and October 1989. In the studies where culture plate method was employed to study outdoor air-spores, Aspergillus spp. have been found to contribute to a significant proportion of the total colonies. Six Aspergillus species were identified. Aspergillus niger ranked third in yearly total population of the four Alternaria species identified Alternaria . Alternata ranked fourth in their contribution to yearly total population. Mucor globosus was recorded throughout the investigation period except in June 1988 but was found to be of less significance. Of the four Cladosporium species identified, Cladosporium cladosporioides occupied first place while C. herbarium occupied second place in their yearly total population. Seasonal periodicities of 20 allergenic fungal spores and 15 aller- genic fungal species as compared with meteorological parameters for two years were recorded. In general continuous rain washed off airborne spores. The number of dermatophagoides (Dermatophagoides farinae and D. pteronyssinus) in house dust showed a periodical increase and decrease. Maximum numbers were recorded in August, September , October, February, March and April. The peak growth of two mite species took place in August-September while minimum in the months of January to April. The role of microorganisms (fungi and bacteria) found in the nasal swab of the patients attending ENT OPD revealed no correlation between the nasal swab microflora of patients and respiratory allergy. However, record of allergic case history among the patients attending ENT OPD and Chest and TB Clinics of Regional Medical College Hospital, Lamphel (Imphal, Manipur) and clinical examination of patients gave some clues for diagnosis of respiratory allergy.



ATMOSPHERIC POLLEN & SPORES SURVEY IN RELATION TO THE INCIDENCE OF ALLERGENIC DISEASES AT GWALIOR(M.P.)  

A survey of air borne pollen grains, fungal spores and other bio- components in relation to the incidence of allergenic diseases at Gwalior (M.P.) was undertaken from April 1988 to March 1991. The study revealed that about 310 angiospermic plant species are dominant in Gwalior. The pollination periods of these species vary from season to season; maximum number of trees and shrubs exhibit flowering during February-March, and herbs and grasses in August-September. A total of 67 pollen and 35 spore types were observed in the air in the year 1989, whereas in 1990 a total of 61 pollen and 23 spore types were recorded. A good number pollen and spore types were common during both the years. Occurrence of these biocomponents was directly related with the flowering of plants, climatic factors and other sources. Pollen of certain taxa such as Asteracease, Malvaceae, Amaranthaceae, Chenopodia- ceae, Poaceae etc. were found to be present in the air throughout the year. A total of 155 patients suffering from various allergic disorders were diagnosed and intradermal skin sensitivity test with 92 allergens made. Maximum allergic attacks were recorded during winter and summer seasons. Perennial disorders were commoner than seasonal. Most of the allergic patients did not have allergic family history. Allergic incidences were common among housewives followed by students, labourers, social workers, etc. More attacks were observed in the patients in the 31-40 years age group. Patients exhibited more sensitivity to insect allergens followed by pollen, fungus, dust and others. Smokers exhibited more allergic incidences than non-smokers. The mode of diet, i.e. vegetarian and non-vegetarian did not have effect on allergic manifestations. During the dominance of insects and other biocomponents in the air, the allergic disorders caused by them were also prevalent among patients. Pollen grains of anemophilous plant species are directly concerned with flowering of plants and climatic factors.




ANATOMY
INTEGRATED APPROACH OF YOGA THERAPY IN ATOPIC BRONCHIAL ASTHMA  

Study was carried out to observe the effects of an integrated approach of yoga therapy on atopic bronchial asthma patients, when compared to a group of control subjects, who were followed up at the same intervals, but who did not practice yoga. There were 67 patients in the `yoga' group, however, some of the data were not available for the whole group. On the whole, at various stages of the follow up there was a suggestion of improvement in terms of a significant reduction in their medication, increase in peak flow rate, in breath holding time, and in chest expansion. The FEV1/FVC percentage. ratio. The indices of bronchial lability changed as follows precent rise in PER decreased (indicating improvement), percent fall in PFR showed changes in both directions, and the sum of the two showed an increase. The eosinophils (per 100 cells) and the absolute eosinophil counts showed a general trend of reduction. The skin prick test results showed that at each stage of the follow up the number of patients showing improvement (reduced response to allergens) was more than those showing worsening. Out of a total of 23 patients for whom the serum IgE data were available, 14 showed no change, four showed a decrease and five showed an increase. Among the six control group patients, the small sample size makes it difficult to make any definite statements. There was a significant reduction in medication, an increase in PFR, and in breath holding time. The FEV1/FVC percent ratio, as well as eosinophil counts did not show a definite change. The indices of bronchial lability (% rise, % fall) showed a trend of reduction. The skin prick test results showed that at each stage the number of patients who improved were more than those who worsened. Serum IgE levels tended to increase more often than decrease or showed no change. In conclusion, it can be said that the improvement among the 'yoga' group was more apparent than for the 'control' group, with respect to the number of eosinophils per 100 cells, the absolute eosinophil count, and serum IgE levels. However, it must be emphasized that these conclusions are preliminary. Further studies on large number of control group patients are essential.



DEVELOPMENT OF EXSTROPHY COMPLEX: AN EMBRYOLOGICAL AND ANATOMICAL STUDY WITH A VIEW TO IMPROVE THE SURGICAL TREATMENT  

Aims of the investigation included study of the entire anatomical de- rangement of the patients suffering from exstrophy complex by using clinical as well as other investigative tools including surgical dissection, study of the histological status of the abnormally developed tissues and organs with special reference to detrusor and bladder neck region, study of human embryos by dissections and various investigative tools with special reference to genito urinary system and related systems which are affected in Exstrophy complex, assessment of the accuracy of the existing theories of the genesis, correlatation of the observations made during the study and develop a hypothesis of genesis of exstrophic malformations and development of better surgical treatment of exstrophic malformations so as to correct the total problem with minimum number of operations. The anatomical observations seem to indicate that the theories of genesis are grossly inadequate. It appears from the indirect evidence that the embryogenesis is likely to be very different than so far assumed. A number of variations are noted in the anatomical features of the conventional groups of esxtrophic malformation and may explain inconsistent results of surgery in the same category of patients in same hands. The displaced sphincter apparatus was indentified and confirmed to be so by the histolo- gical studies. It was repaired to give high continence rate in cases of incontinent epispadiatics and exstrophic bladder cases in a single stage repair in an unselected series.



IMMUNOHISTOCHEMICAL STUDY OF THE DORSAL LATERAL GENICULATE NUCLEUS IN ALBINO RAT  

Immunohistochemical studies for analysing the presence and development of the neurotransmitters/neuromodulators-substance P (SP), leu-enkephalin (ENK), serotonin (SER), glutamate (GLU), vasoactive intestinal polypeptide (VIP), and cholecystokinin (CCK)-have been conducted on the lateral geniculate nuclear (LGN) complex of albino rats at gestational day 18 (E 18), various postnatal ages and in the adult. The present study shows the unequivocal presence and the sequential changes in the profile of substance P, serotonin, enkephalin and glutamate while VIP and CCK were not demonstrable at any of the age periods. SP immunoreactivity is found to increase from E18 upto 20 day post- natal (DPN) and decrease thereafter to adulthood whereas the SER and ENK immunoreactive fibres and terminals seen as occasional fibres at 1, 5 and 10 DPN are better visualised from 20 DPN and gradually increase upto the adult period. Immunoreactivity of GLU fibres and cells is seen at 40 DPN and increases in the adult. No VIP and CCK immunoreactive fibres or cells are seen at any age period. The vaxing and waning of the undecapeptide SP during the postnatal period of rat besides indicating an evolutionary process may reflect its trophic role in the LGN complex during development. This prompts speculation of a possible therapeutic future in conditions of neuronal degeneration and atrophy encountered in this region in clinical practice. The presence of SP in the adult though in decreasing intensity and SER, ENK and GLU suggests their role in visual processing in dLGN and perhaps in endocrine and behavioural functions of IGL and vLGN. SER, ENK and GLU appear later in this sequential order and increase with maturity. Further studies are required to analyse the role of the various neuropeptides and neurotransmitters existing in the afferent pathways of the LGN complex.




ANAESTHESIOLOGY
A STUDY OF CELLULAR CHANGES FOLLOWING FOCAL CEREBRAL ISCHAEMIA IN MACACA RADIATA MONKEYS  

An investigation was undertaken to study the cellular changes following focal ischaemia in Macaca radiata monkeys. The electron microscopic investigation of the structural changes in the caudate nucleus and the insula of the brains from monkeys with right middle cerebral artery (RMCA) occlusion for half, 4, 12, 24 and 48 hours and 2 weeks and one monkey with 12 hours of reflow following 12 hours of occlusion confirmed our earlier observations. Upto 24 hours the edema is only cytogenic and only after that period it becomes vasogenic. Blood reflow following 12 hours of RMCA occlusion does not benefit the tissue. The presence of paracrystal- line array in the vascular endothelium only at half, 4 and 12 hours perhaps gives a clue to the agents which could be the signal in stroke pathophysiology. The sudden change in shear stress on occluding RMCA could have triggered an increase in serotonin which polymerized the actin or an immune reaction which resulted in deposits of immunoglobulins.




BIOCHEMISTRY
STUDY OF AIR-OXYGEN AS CARRIERGAS IN ANAESTHESIA  

The study was carried out to determine the usefulness of air oxygen as carrier gas in balanced anaesthesia technique. A total of 60 patients of both sexes in the age group of 16-60 years with an ASA grading of I/II who were to undergo anaesthesia for more than one hour were selected for the study. Anaesthesia was given from the Pedius B Usha Drager anaesthesia apparatus. The patients were divided in two equal groups; one group on spontaneous ventilation and the other on contro- lled ventilation using pancuronium as per requirement of surgery. Clinical parameters remained within normal limits during anaesthesia in both groups. Recovery in both groups was good and the incidence of post-operative complications was low. The awakening time was 5.12 + 0.57 min in patients breathing spontaneously compared to 2.32 + 0.84 min in those on controlled ventilation. No patient had awareness of any intraoperative event. The use of air as carrier gas may be superior to nitrous oxide as the side effects encountered while using nitrous oxide were not seen. Further, the Pedius B machine can be easily oeprated in remote areas. The disadvantage of using air as carrier gas was in the form of loss of mild analgesic and anaesthetic effects of nitrous oxide necessitating higher doses of volatile and intravenous anaesthetics. The Pedius B machine also had some disadvantages such as water condensation, argon accumulation etc. with the oxygen concentrator.



STUDY OF DOPAMINE METABOLISM IN PARKINSON'S DISEASE  

Urinary excertion of vanillye mandelic acid(VMA) & homovanillic acid (HVA) was estimated in 108 patients with parkinsonism and in 39 controls for VMA & 22 controls for HVA. Urine was collected in acidified containers. The VMA and HVA levels in both morning and 24 hr collection showed positive correlation. There was no significant difference in daily HVA and VMA levels of normal controls. Although there was a decline with age in HVA and VMA levels the difference was not significant. In normal healthy individuals no significant difference in HVA and VMA levels was found between males &femals Urine sample stores at -20 deg.C showed no decline in HVA and VMA levels for several weeks. Amongst 108 patients with parkinsonianism, analysis was done in 43 patients before initiation of therapy and in 65 patients after therapy. In 10 patients follow up was done. The drugs used were Sinemet, Pecitane, Benspar, Desrenyl and Eldepsyl. No significant difference was found between normal control & Parkinson's patients either before or after treatment. The mean excretion of HVA in normal controls, patients on drugs & patients not on drugs was 2.9 + or - 2.6, 11.5+ or - 9.9, 4.3 respectively. Urinary HVA levels were significantly higher in treated patients while there was no difference in untreated patients.



EVALUATION OF A FISH-OIL DIET AS A CURATIVE AND PROTECTIVE MEASURE AGAINST OCCLUSIVE VASCULAR DIESEASES  

Study was aimed at finding possibilities of preventing or reducing atherosclerosis by the intake of diet rich in fish oil. The fish oil for this study was obtained from the flesh of marine fish, Hilsa ilisa. Atherosclerosis was induced in rabbits experimentally and status of atherosclerosis thus induced was monitored. Investigations were carried out to find out the potentiality of hilsa fish oil rich diets in preventing such induction in rabbits. Future investigations were undertaken on the possibilities of using diets rich in hilsa fish oil in reducing the atherosclerotic conditions in rabbits in which this disease has already been induced experimentally. Studies on the role of lipid components, particularly the fatty acid composition of hilsa fish oil rich diet in preventing and curing atherosclerosis in rabbits were also taken up. The results of the experiments showed that feeding rabbits with hilsa oil supplemented diets, imparted in these animals a resistance towards the deposition of atherosclerotic plaque in the thoracic aorta and coronary arteries, even in the presence of atherogenic concentration of cholesterol and tryglycerides in plasma and platelets. This effect seems to be related to the rise in the proportion of fatty acids of n-3 series of 20 and 22 carbon chain lengths, particularly that of EPA, in the lipids of plasma and platelet due to such feeding. The results of the study showed that feeding atherosclerotic rabbits with diets containing about 10% of hilsa oil, inspite of the presence of 0.45% cholesterol, can regress the atherosclerotic conditions in terms of reducing the atherogenic factors like the contents of plasma total cholesterol, plasma tryglycerides, plasma LDL-cholesterol and platelet cholesterol load on one hand, and elevating the antiatherogenic factors like plasma LDL-cholesterol content and proportion of phospholipid carried to LDL, on the other. This diet can actually regress the thickly deposited fatty material from the thoracic aorta and coronary arteries of the atherosclerotic rabbits. This reversibility of atherosclerotic plaque by dietary treatment is a very important new finding of the present study. The results of the study thus reveal that a hilsa oil rich diet may be useful for the prevention and abatement of hyperlipidemia and atheros clerosis in man and that raw hilsa oil might be effectively used as therapeutic agent in the management of hyperlipidemia and atherosclerosis in human patients.



EFFECT OF VITAMIN B6 AND MAGNESIUM SUPPLEMENTATION IN EXPERIMENTAL AND CLINICAL OXALATE UROLITHIASIS  

The beneficial effect of pyridoxine and magnesium oxide administration for eight weeks was studied in hyperoxaluria induced (fed 100mg sodium glycolate/100g bw/day) young adult male wistar rats. Significant hyperoxaluria, hypocitraturia decrease in specific activity of liver transaminase with a concomitant increase in the specific activities of liver glycolic acid oxidase (GAO), glycolic dehydrogenase (GAD) and lactate dehydrogenase (LDH) was observed in sodium glycolate fed group; while pyridoxine supplementation (2.0 mg/100g bw/day) to this group lowered urinary oxalate by 43% and increased citrate by 24%, it also significantly decreased liver GAO and normalised transaminase and GAD. Combined treatment of pyridoxine (2 mg) and MgO (150 mg) per 100g body weight per day to sodium glycolate fed rats normalised hyperoxaluria which is beneficial in retarding calcium oxalate urolithiasis. The clinical study which included assaying the blood and urine specimens of 19 stone-formers after 0, 30 and 60 days of a combined administration of MgO (500 mg/day) and pyridoxine-HCl (10 mg/day) confirmed the beneficial effect of this therapy by lowering hyperoxaluria and promoting citrate excretion, both of which are important factors regulating calcium urolithiasis.



STUDIES ON THE BIOCHEMICAL MECHANISM OF ACTION OF TRADITIONAL MEDICINES (VARUN AND CHOTAGOKHURU) ON OXALATE METABOLISM IN RATS  

Induction of hyperoxaluria and calcium oxalate calculi was carried out in rats by intraperitoneal injection of hydroxyproline (OH-proline) and feeding hydroxyproline in diet. The effect of Ayurvedic preparations viz. varun and chota gokhura on dissolution of renal calculi, urinary excretion of calcium, oxalate, inorganic phosphorous, uric acid, citrate and inhibi- tory activity towards calcium oxalate monohydrate (COM) crystal growth, enzymes of oxalate biosynthesis viz. glycolic acid oxidase (GAO), glycolic acid dehydrogenase (GAD), lactate dehydrogenase (LDH) in liver and kidney, calcium and oxalate uptake by intestinal BBMV and calcium and oxalate up- take by renal tubular BBMV was studied to assess any alteration in renal handling of calcium and oxalate. Sixteen rats were taken and divided into four groups each containing 4 rats. Twenty four hours urine of all rats was collected at 0 day (Basal) by placing them individually in metabolic cages. 4-OH-L-Proline (2.5g/kg B.W.) was administered intraperitonealy (10ml/kg B.W) to all rats. Group I: animals were sacrificed after 1 day of OH-proline injection; Group II: animals were sacrificed after 2 days of OH-proline injection; Group III: animals were sacrificed after 7 days of OH-proline injection; Group IV: animals were sacrificed after 15 days of OH-proline injection. Twenty four hours urine specimens were collected from individual animals prior to sacrifice at specific time intervals. Liver, Kidney and Intestines were excised at sacrifice and placed on ice. Twenty four hour urines collected were analysed for creatinine, uric acid, phosphorous, calcium and oxalate. Calcium oxalate crystals formed in kidneys after three consecutive intra- peritoneal injections of hydroxyproline (2.5/kg B.W) were examined under polarised light microscope. One rat was sacrificed after each injection and kidneys were removed to examine microscopically, the presence of calcium oxalate crystals. Urine samples of remaining rats were collected after 5, 10, 15 and 20 days of 3rd injection to ascertain the level of hyperoxaluria, which declined between 10 and 20 days. Therefore, to maintain hyperoxaluric level during the drug trial, sodium glycolate (100 mg/100 gm B.W) was orally administered (by gastric intubation) after 10 days of 3rd OH-proline injec- tion. The dried fruits of chota gokhuru were taken and ground and from the co- arse powder an extract was made. To one part of powdered drug, sixteen parts of water was added. The mixture was boiled till 1/4th was left. The contents were filtered through muslin cloth and evaporated to dryness. An extract was made from the residue with Gum Acacia (1-2% in DDW) and tween-20 (1-2 drops per dose of 5g/kg B.W). The drug was administered by gastric intu- bation in morning hours. Eighteen rats were given pellet diet and water ad lib and divided into 3 groups, each of six animals; Group I: were normal saline treated, Group II: were OH-Proline + sodium glycolate + gokhru treated and Group III: were OH-Proline + sodium glycolate treated. Twenty four hour urine of each rat was collected at i) 0-day ii) after 3 injections and iii) after 1 week of 3rd injection and analysed for creatinine, oxalate phosphorous, calcium and uric acid. On the 11th day of 3rd injection, sodium glycolate (100 mg/ 100 gm B.W) was orally administered to Group II and Group III animals. Chota Gokhuru extract was administered to Group II animals after 3 days of sodium glycolate feeding. Before starting the drug, 24 hour urine was collected to check the oxalate level in urine. One intraperitoneal injection of OH-proline to male rats (B.W 200-250 gm) significantly increased in the urinary oxalate excretion within 24 hours of injection. However, the other parameters (viz. calcium, phosphorous and uric acid) were not altered. OH-proline administered for 3 consecutive days showed a significant decline in the oxalate excretion after 10-20 days. Sodium glycolate feeding (100 mg/100 gm B.W) started subsequently after 10 days of three OH-proline injections, was able to maintain hyperoxaluria for next 30 days, to enable to study the effect of the drug. Drug (Tribulus terrestris) administered orally gastric intubation was able to significantly lower hyperoxaluria within seven days and remained unaltered thereafter. Calcium oxalate crystals viewed under polarised microscope after the OH- proline injections were not observed after drug treatment similar to their sodium glycolate fed controls indicating thereby that the dissolution of P calcium glycolate crystals was not due to the drug administration but due to the loss tapering of OH-proline effect. It may be concluded that chota gokhuru administration was not effective for the dissolution of calcium oxalate crystals but helped in lowering hyperoxaluria in experimental model.



BIOCHEMICAL STUDIES IN YOUNG ADULT RAT BRAIN AFTER SIMULTANEOUS EXPOSURE TO CADMIUM AND ALCOHOL  

Cadmium is known to be potentially toxic to most of the organs of the adult animals. Under normal circumstances very little cadmium reaches the brain in adults because of the selective permeability of blood brain barrier. The susceptibility to cadmium toxicity has been recognised to be influenced by a number of physiological and nutritional factors. One such factor is ethanol whose influence on cadmium neurotoxicity is not widely reported. Study was carried out to find the structural and functional alterations in young adult rat brain after co-exposure to cadmium and alcohol. Cadmium (1mg/kg body weight) and ethanol (2g/kg body weight) were administered alone as well as in combination to different groups of rats, intraperitoneally for a period of one week. Exposure to either cadmium or ethanol alone did not have any significant effect on the body weight but caused a detrimental effect on the growth of the animals when given in combination. Only cadmium exposure caused an appreciable accumulation of cadmium in brain. This accumulation was further enhanced significantly (3- fold) when cadmium was given alongwith ethanol. The increase in cadmium accumulation was maximum in corpus striatum followed by cerebral cortex and other regions of the brain. Marked increase in the uptake and accumulation of cadmium observed after co-exposure to cadmium and ethanol led to a significant depletion in the levels of zinc and copper in these two regions of the brain. Exposure to either cadmium or ethanol alone did not have any significant effect on lipid peroxidation and antioxidant defense system in any of the regions of the adult rat brain but when given in combination, caused a significant increase in lipid peroxidation and markedly decreased the activities of antioxidant defense enzymes like glutathione peroxidase, superoxide dismutase, catalase and glutathione reductase and the levels of glutathione (GSH) in corpus striatum and cerebral cortex regions of the brain. Total thiols and ascorbic acid were reduced only in corpus striatum. Exposure to cadmium alone did not show any significant effect on lipid composition of the brain, but a marked decrease in total lipid (attributed to the reduction in the levels of myelin specific lipids like non- esterified cholesterol, phospholipids and galactolipids) was observed in corpus striatum and cerebral cortex after co-exposure to cadmium and ethanol. In contrast, a significant increase in total lipids, phospholipids, galactolipids and cholesterol was observed in various regions of the brain after exposure to only ethanol. The ratio of cholesterol to phospholipids, a major determinant of membrane fluidity, decreased in these regions of the brain after exposure to only ethanol as well as combined exposure to cadmium and ethanol. Structural alterations observed in terms of increased lipid peroxidation and decreased lipid contents after cadmium and ethanol co-exposure had a profound effect on the activities of membrane bound enzymes such as (Na+-K+)-ATPase and acetylcholinesterase indicating functional impairment. The results of the present study imply that ethanol renders the adult brain more susceptible to cadmium neurotoxicity. Corpus striatum and cerebral cortex are more vulnerable to the toxic effects of cadmium under the influence of ethanol than other regions of the brain.



MECHANISM OF ACTION OF ANTITUBERCULOUS DRUGS ISONIAZID (INH) AND RIFAMPICIN(RIF) ON GLUTATHIONE AND ITS METABOLISM IN MYCOBACTERIA  

Study was carried out to delineate the mechanism of action of antituberculous drugs, isoniazid (INH), rifampicin (RIF) through glutathione (GSH) and related enzymes in mycobacteria. The presence of GSH, its metabolic enzymes viz. gamma glutamyl transferase (GGT), gamma glutamyl cysteine synthetase (GGCS) and glutathione synthetase (GSH) and excretion of GSH out of the cells has been established in M. smegmatis. The presence of GSH was established in M. smegmatis both by chemical and enzymatic assays and was further confirmed by covalent binding of N-ethylmaleimide to cellular GSH. Maximum GSH content was observed on second day of the growth which decreased steadily with the age of culture. It was observed that 80 to 90 percent of nonprotein thiols were present in the form of GSH. The activities of four GSH metabolizing enzymes viz. GGT, GGCS, GSH synthetase and GSH-S-transferase were established in 10,000xg and 100,00xg super- natants of M. smegmatis. GGT of M. smegmatis is a peculiar enzyme in demonstrating only hydrolytic activity and not the transpeptidase activity. M. smegmatis cells excrete non protein thiols (NPSH) into the medium which was observed to increase steadily from second to seventh day of growth. Isoniazid (INH) and rifampicin (RIF) deplete cellular GSH in M. smegmatis but have a diverse mode of action. Depletion caused by INH is significantly higher as compared to RIF.NIH dependent depletion of cellular GSH in M. smegmatis is due to an increase in the hydrolysis of GSH as demonstrated by increased activity of GGT, increased activity of glutathione-S-transferase, a decrease in its biosynthesis, as demonstrated by decreased activities of GGCS and GSH synthetase and finally increased export of GSH out of the cell. However, RIF dependent depletion in GSH was due to a decreased biosynthesis of GSH as demon- strated by decreased activities of GGCS and GSH synthetase in M. smegmatis. RIF was found to have no effect on the hydrolysis of GSH and its export out of the cell.



CORRELATION OF ANTIFUNGAL ACTIVITY OF IMIDAZOLE DERIVATIVES WITH CYCLIC AMP AND LIPIDS OF DERMATOPHYTES  

Studies were undertaken to find out the correlation of antifungal activity of imidazole derivatives with cAMP and lipids of dermatophyte i.e. Microsporum gypseum. Cyclic AMP levels were modulated by different modulators and the effect of modulated cAMP levels were studied on lipid metabolism and susceptibility of cells to imidazole derivative. Theophyl- line, prostaglandin E (PGE), dibutyl cAMP, aminophylline, fatty acids and glycerol increased the intracellular levels of cAMP while atropine decreased it. The increase or decrease in cAMP levels was due to the effect of different modulators on the activity of either adenylate cyclase or phosphodiesterase or on both of these enzymes. The lipid composition of M. gypseum was examined in modulated cells and phospholipid content was observed to increase in the presence of theophylline, PGE, dibutyryl cAMP and aminophylline and it decreased with atropine while no change was seen with fatty acids and glycerol. These observations were further corroborated by incorporation of (14 C)-acetate into total phospholipids in the presence of theophylline, dibutyryl cAMP, PGE1, aminophylline and atropine. There was net increase in the synthesis of total lipids and total phospholipids in the presence of above modulators except in case of atropine where decreased synthesis was observed. The increased and decreased activities of key enzymes of phospholipid biosynthesis i.e. glycerol kinase and acyltransferase in case of aminophylline and atropine respectively further confirmed the correlation of phospholipid synthesis and concerned enzymes. The susceptibility of cells to clotrimazole decreased in the presence of various additives (aminophylline, atropine and unsaturated fatty acids). Less susceptibility is due to more resistance of the cells to this antifungal agent in the presence of cAMP modulators. It has been concluded that there is a correlation between intracellular cAMP levels and lipid biosynthesis and lipids play some role in the anti- fungal action of imidazole derivative.



ROLE OF SECOND MESSENGERS IN PLATELET HYPERSENSITIVITY OBSERVED IN HYPERCHOLESTEROLAEMIC HUMAN SUBJECTS  

Incubation of human platelets with cholesterol-poor, Cholesterol- normal and Cholesterol-rich liposomes revealed that: acquisition or depletion of platelet membrane-cholesterol was highly selective; variation in membrane cholesterol content with respect to values found in unmodified normal platelets, was paralleled by the observed changes in cyclic nucleotides, amiloride sensitive cytoplasmic pH, phosphorylation of 2l kD protein as well as phospholipase 'A2' activity and no change in phosphoinositol metabolism and the membrane cholesterol modulated changes in cyclic nucleotides, Na+/H+ exchange and phospholipase `A2' activity was completely inhibited when platelets were pretreated with quinacrine (specific phospholipase `A2' inhibitor) before exposure to various liposomes. Based on these results, we suggest that membrane-cholesterol modulated phospholipase `A2' activity may be the basic mechanism responsible for hypersensitivity of platelets in hyper- cholesterolemic patients (Type IIa). Further we also propose that this phospholipase `A2' activity may be regulated by 2l kD protein phosphory- lation/dephosphorylation mechanism initiated by variation in platelet membrane-cholesterol content.



DETERMINATION OF OXALATE IN URINE & PLASMA WITH NA+ INSENSITIVE OXALATE OXIDASE PURIFIED FROM SORGHUM LEAVES  

The determination of oxalate in urine and plasma by oxalate oxidase from mosses, barley, banana peel and beet stem requires the pretreatment of urine and plasma to remove cations and anions found in these biological fluids, which complicate the procedure and consequently the sensitivity and reproducibility of the method suffers. An oxalate oxidase has been purified from leaves of 10-day old seedling plants of grain sorghum CHS-5, which is insensitive to these urinary inorganic ions viz. Na+, K+, Ca++, Mg++, Fe++, Zn++, Pb++, Cl-, PO4--, SO4--, HCO3-, CO3-- etc. These improved characteristics of the sorghum enzyme make it ideally suitable for its use in the deermination of oxalate in urine and plasma. A simple enzymic colorimetric method for determination of urinary and plasma oxalate has been developed using the sorghum enzyme. The method simplifies the total analytic procedure by eliminating the need for a separate step to remove cations and anions from urine plasma, prior to oxalate assay. The method is rapid and does not require expensive specialized equipments and therefore suitable for oxalate anasysis in a large number of samples in clinical laboratories.



OXYGEN RADICAL AS A POSSIBLE MECHANISM OF DRUG TOXICITY ANDROLE OF SUPEROXIDE DISMUTASE AND ASCORBIC ACID AS BIOLOGI- CAL DEFENSE  

Investigation were carried out to study the correlation of drug induced superoxide radical production, lipid peroxidation methemoglobinemia as well as interaction of superoxide dismutase and ascorbic acid. Ascorbic acid is a potential scavenger of superoxide radical. Some commonly used drugs like sedatives, narcotics, antiamoebics produce increased superoxide in the liver microsomes of rats and guinea pigs by the NADPH - cytochrome P450 reductase - cytochrome P450 electron transport system, commonly known as the mixed function oxidase. Rats can synthesize ascorbic acid. These drugs stimulate ascorbic acid production in the rat apparently to scavenge the increased amount of superoxide formed. In the guinea pig, a species incapable of producing ascorbic acid, administration of these drugs results in superoxide toxicity, as evidenced by lipid peroxidation in diferent tissues including liver, kidney, lung, heart and adrenal glands. Lipid peroxidation is highly detrimental to structure and function of the membranes. Ascorbic acid completely prevents lipid peroxidation both in vitro and in vivo. Preparation of microsomes, estimation of superoxide radical, assay of cytochrome reductase, cytochrome P-450 content, estimation of ascorbic acid and lipid peroxidation were carried out using standard procedures.



HUMAN ATHEROSCLEROSIS AND FOAMCELLS  

Study was undertaken with the aim to identify the molecules and molecular mechanism(s) involved in the development of human atherosclero- sis and to study the characteristics of foam cells and their transport into the sub-endothelium. For this, procedure for harvesting and culture of human umbilical arterial endothelial cells was optimised. Scanning electron microscopy revealed homogeneous population of endothelial cells in culture. On interaction with endothelial cells substantial structural changes were noticed in human plasma low density lipoprotein (LDL). Changes in biophysical environment of several amino acids of modified LDL were also noticed by circular dichroism, fluorescence and electronic absorption spectrosopic methods. Lipid peroxidation was measured by second derivative electronic absorption spectroscopy and higher levels of conjugated dienes in LDL and extractable lipids of endothelial cells were found. Circular dichroism, derivative fluorescence and electronic absorption spectroscopic methods established that the circulatory monocytes/macrophages utilised endothelial modified as well as chemically modified LDL and degraded it. Scanning electron microscopic and fluorescence microscopic observation suggested that upon interaction with modified LDL, macrophages got transformed into lipid laden cells (foam cells), about 4-times larger than the macrophages. Presence of foam cell like structures was noticed in atherosclerotic plaques. Results of the present study suggest that at the molecular level the partially modified apo B and peroxidised lipids of LDL are involved in the pathogenesis of human atherosclerosis.



TREATMENT OF VITILIGO BY LOCALAPPLICATION OF FORSKOLIN & BASIC FIBROBLAST GROWTH FACTORENTRAPPED IN LIPOSOMES  

Forskolin, a diterpenoid, reported to stimulate melanocyte prolifera- tion in vitro, was tested to find out its effectiveness in vivo. For this purpose an experimental model was designed in which the melanocytes in the skin (of guinea pig) are selectively destroyed by local application of 4-methoxyphenol, thereby resulting in a gradual loss of pigment in the skin, the condition being similar to vitiligo in human beings in several respects. Following cessation of application with 4-methoxyphenol, the remaining peri-lesional and follicular melanocyes proliferate and reoccupy the dermal-epidermal junction in the depigmented patch and this is reflected visually by the appearance of pigment. When forskolin was topically applied on the depigmented patch at a dose of 7.5 X 10 moles, 3 times a day in glycerine base, no enhancement was found in the rate of repigmen- tation compared to only base applied controls. However, following intradermal injection of 4X10 moles of forskolin, 2 times a day, into the depigmented patch, it was found that the per cent of completely pigmented ear lobes after 12 weeks of treatment was 75% of forskolin treated ear lobes against only 43% in controls. Forskolin was thus found to exert its stimulatory influence on melanocyte proliferation under in vivo conditions also.



ISOLATION & CHARACTERISATION OF METAL BINDING PROTEINS IN HUMAN FETAL MEMBRANES & PLACE-TA AT VARIOUS GESTATION AGES &THEIR ROLE IN DETOXIFICATION*  

Trophoblast and the tissues of th uterus, together from an important organ, the placenta, which provides the growing embryo with nutrition and with oxygen; it also removes waste products from the embryo. It is proposed to study the regional distribution of zinc, copper and cadmium in human fetal membranes and placenta. Isolation and purification and structural characterization of metal binding protein(s) from human placenta and fetal membranes will be done. Antibodies will be raised agaisnt the major metal binding protein(s) and homology of this protein with other metal binding proteins in liver and kidney will be determined besides studies on the role of metal binding protein(s) in detoxification mechanism.



LIPOSOMES AS VEHICLES FOR THE DELIVERY OF LOW MOLECULAR WEIGHT COMPOUNDS TO THE BRAIN & AS IMMUNOADJUVANTS  

Liposomes have been used to deliver a wide variety of biologically active substances to different tissues in vivo. Attempts have been made to deliver liposomes to brain with very limited success beacause of the obstacle imposed by the blood-brain barrier. In this study, attempts were made in the delivery of substances entrapped in multilamellar and uni- lamellar liposomes (phosphatidylcholine: cholesterol: sulfatides, 7:2:1) using 2M mannitol to disrupt the blood-brain barrier.Six to 14-fold increase in the uptake of CAMP phosphodiesterase, a high molecular weight protein, and a 3-fold increase in the uptake of CAMP, a low molecular weight compound was observed in the presence of mannitol. Delivery of entrapped materials to the brain was confirmed by immunofluorescence studies using morphine-BSA. 2M mannitol also resulted in a slow clearance of both free or liposomally- entrapped L25I-PDE fraom brain. In vitro studies using C6 glioma cells showed increased incorporation of 32P label from (Y-32P) ATP into TCA insoluble pellet in the case of liposomal CAMP-protein kinase-(Y-32P) ATP complex as compared to free complex at all time points studied. Studies with metabolic inhibitors along with small unilamellar vesicles (SUVs) containing either morphine-BSA or FITC-BSA indicate that one of the mechanisms of uptake of liposomes by these cells could be endocytosis although fusion also appears to take plac



PEPSINOGEN :CLINICAL AND GENETICS STUDIES  

In all 561 patients affected with peptic ulcer (511 were suffering from DU and 50 with GU) were selected for this study in the duration of 3 years. The endoscopic findings were recorded in a proforma along with other details. All the endoscopically proven peptic ulcer cases were selected for the present study. Information regarding age, sex, dietary habits, smoking, alcoholism, pan chewing and incidence of ulcer were obtained. From all these patients blood and urine samples were collected for biochemical studies. The proportion of affected individuals in all the pedigrees was determined based on the segregation of the condition among the sibs of the propositil where (a) both the parents of the propositil were affected (b) one of the parents was affected and (c) both the parents were normal. Out of 365 cases, 70 (19.18%) were found to be vegetarian and the remaining 295 (80.82%) were non-vegetarians. The percentage of non-vegetarians was foun to be higher. Out of the 544 cases 212 (38.97%) were found to be regular in taking meals when as 332(61.03) were irrugular. The familial and isolated groups of patients differed significantly with regards to the frequencies of cases laking diet regularly and irregularly. the percentage of panchewing cases in 4 groups of DU patients i.e. male femilial, female, familial, male isolated and female isolated were 52.54, 23.73, 18.64 and 5.08 respectively and with tobacco 67.24, 22.41, 8.62 and 1.58. the precentage of excess consumption of chillies and spices in food in 4 groups as mentioned above is 21.83, 2.11, 52.82 and 23.24 respectively. Moderate consumptions of chillies and spices were found to be 16.95% 3.39%, 58.47% and 21.11% respectively in the above mentioned groups. The data on cigarette smoking was available in 516 cases. 300 (58.14) were found to be smokers and 216 (41.86) were non-smokers. A higher precentage of smokers was found to be in isolated male patients. With regards to alcohol consumption out of total 437, 111 (25.40) were alcoholics and the remaining 326 (74.60%) were non-alcoholics. A higher precentage of alcoholics were found in male alcoholics and non-alcoholics. All the pedigrees indicating positive family history of ulcers were examined for identifying mode inheritance of the conditions. To understand the segregation of condition among the family members, parents of the propositii were grouped into 3 categories based on the presence or absence of ulcer in them like 1) AXA (affected x affected) both parents were affected, (2) X N (affected X Normal) one of the parents was affected and (3) N X N (Normal and Normal) both the parents were normal. The proportion of affected sibs was higher 50.00% in the sibships where both the parents were affected as compared to those, where one parent was affected (30.85%) and none of the parents were affected (29.31%) The mean unit pepsinogen activity per ml. per 24 hours values of serum and urine for DU patients, and GU patients and then first degree relatives are given in tables 15 and 16. The mean serum pepsinogen concentration in patients (male & female) familial and nonfamilial were significantly elevated where compared with that of controls. (p>0.001). A similar observation was recorded in uropepsinogen values of DU patients. Where as Uro and serum pepsinogen levels in GU was significantly elevated. Out of 310 serum pepsinogen values 188 (60.64%) were found to be normopepsino- genemic. DU patients and the remaining 122 (39.35%) were hyperpepsinogenemic . Out of 310 uropepsinogen values 48 (15.48%) found to be normal and 262 (84.52%) were with hyper uro pepsinogenuria. uropepsinogen was estimated in general population (720 subjects) of Hyd erabad. Out of 335 symptomatic subjects 130 (38.80%) wer found to be with hyper pepsinogen values and the remaining 191 (57.01) were normal values. Out of 385 asymptomatic subjects 72 (18.70%) were found to be hyper pepsinogenemic and remaining 327 (84.93) were within normal range the relative risk of develop ulcer was found to be 3.1 in individuals with elevated uropepsinogen levels.



SULPHATION & PHOSPHORYLATION OF GLYCOPROTEINS & NON- GLYCOPROTEINS IN THE BRAIN  

Phosphorylation and sulfation of proteins are two means of post- translational modification of proteins. While phosphorylation takes place on tyrosine, serine or threonine residues, sulfation takes place on tyrosine residues alone on the proteins. Phosphorylation serves many important functions such as activation or inactivation of enzymes and cellu- lar signalling receptors. Sulfation of proteins is known to alter the biological activity of proteins and promote the fast axonal transport of proteins. In the present study, phosphorylation of a non-glycoprotein enzyme, a cytoplasmic neutral alpha-D-mannosidase from monkey brain has been demons- trated. Phosphorylation on serine residues is catalyzed by a cyclic AMP dependent protein kinase. Phosphorylation also alters the cobalt ion sensitivity of the enzyme. An aminopeptidase from monkey brain purified by investigators earlier has been found to cleave enkephalin sequentially to its constituent amino acids. This aminopeptidase is phosphorylated on serine residues by cyclic AMP dependent protein kinase resulting in its inactivation. It has been demonstrated by means of chemical modification of amino acids that tyrosine residues are involved in the catalytic activity of the enzyme. Sulfation of proteins has been demonstrated in the monkey cerebellum and young (10 days old) rat brain. The sulfotransferase respon- sible for transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate was isolated from monkey cerebellum. Both endogenous proteins and exogenous poly(Glu,Ala,Tyr) were sulfated. Using lectin-sepharose affinity chromatography, it was found that both mannose and galactose containing glycoproteins could undergo sulfation. Evidence for differences in the polysaccharide chain structures of the sulfated glycoproteins from monkey cerebellum and young rat brain was also obtained. Modification of tyrosine residues in proteins resulted in partial abolition of phosphorylation and full abolition of sulfation.



DEVELOPMENT OF HOMOGENEOUS ENZYME IMMUNOASSAY FOR THE DETECTION OF AFLATOXIN B1  

Among the fungal toxins, 'aflatoxins', produced by Aspergillus flavus and A. parasiticus group of fungi have received worldwide attention, due to their potentialities as carcinogenic agents in various laboratory and far animals. Various analytical methods have been developed for the detection of aflatoxins in foods, feeds and biological fluids. These include physico- chemical and immuno-analytical methods. The primary objective of the study was to develop a simple homogeneous enzyme immunoassay for the detection of aflatoxin B1 in biologi- cal samples. In the first phase aflatoxin specific antibodies were produced in rabbits. Initially, the requisite antigen i.e. BSA-AFB1 and Poly-D-lysine-AFB1 conjugates were synthesized for raising antibodies. These antibodies were used for aflatoxin detection, either by homogeneous immunoassay or by heterogeneous immunoassay (ELISA). The enzyme horse radish peroxidase (HRPO) was used as a probe for homogeneous immunoassay. The conjugation of aflatoxin B1 to HRPO resulted in 54-72% loss in enzyme activity. In the presence of aflatoxin specific antibodies, the HRPO-AFB1 conjugate showed reversal of its lost enzyme activity (12%). This was used as an analytical tool to quantitate aflatoxin B1. The displacement studies carried out with free aflatoxin B1 and HRPO conjugate in the presence of antibodies indicated poor linearity as compared to the heterogeneous assay. The number of lysine residues available for conjugation in HRPO were determined to be g, based on a novel method developed. However, it was observed that 6-8 residues were involved in conjugation with AFB1. The low level of modulation of enzyme activity by antibody, with respect to HRPO-AFB1 conjugate, could possibly be explained by the limited number of lysine residues on HRPO molecule and its proximity to the active site of the enzyme. Thus, HRPO was found to be limiting as an enzyme label with respect to homogeneous enzyme immunoassay system for aflatoxin B1 analysis.



INVESTIGATION INTO THE MECHANISM(S) OF THE TUMOURICIDAL ACTION OF ESSENTIAL FATTY ACIDS & THEIR METABOLITES IN VITRO  

Investigations were undertaken to study the effect of essential fatty acids (EFAs) and their metabolites on free radical generation and lipid peroxidation to investigate their possible effect on normal and tumor cell membranes by studying their effect on cell membrane bound enzymes such as Na+K+-ATPase and 5'-nucleotidase and on DNA and DNA synthesis in normal and tumor cells in vitro. Normal monkey kidney (CV-1), normal human fibroblasts (CCD-41-SK), human breast cancer (ZR-75-1) and human cervical carcinoma (HeLa) cells were cultured. To study the growth of various normal and tumor cells in the presence of different concentrations of various types of fatty acids, thymidine incorporation studies were undertaken. Experiments were performed with cyclo-oxygenase inhibitor, indomethacin, lipoxygenase inhibitor, NDGA; anti-oxidants, BHT, vitamin E, SOD, different types of prostagladins and calmodulin antagonists to study the mechanism of the tumoricidal action of GLA and other fatty acids in vitro. Assay of superoxideanion and other free radicals was done by nitroblue tetrazolium (NBT) reduction method. The total amount of lipid peroxidation products present in the cells was estimated using thiobarbituric acid (TBA) method. To know whether cis-unsaturated fatty aicds (c-UFAs) can injure cell membrane and thus, bring about their cytotoxic action, the effect of various fatty acids on the activity of membrane bound enzymes such as Na+-K+-ATPase and 5' nucleotidase of tumor (HeLa) cells was studied. The result obtained from the studies indicate that EFAs and their metabolites especially DHA, EPA and GLA can kill tumor cells selectively. They augment free radical generation and lipid peroxidation in ZR-75-1 and HeLa Cells. The cytotoxic action of EPA, DHA and GLA on HeLa cells can be blocked by anti-oxidants such as vitamin E, SOD, BHA and BHT and also by cyclo-oxygenase and lipoxygenase inhibitors namely indomethacin and NDGA as well as by calmodulin antagonists. Inhibitors of the cytotoxic action of GLA and other c-UFAs can also inhibit the production of free radicals and lipid peroxidation process in HeLa cells induced by these fatty acids. Hydroxy and hydroperoxy products of lipoxygenase system derived from GLA but not prostaglandins showed cytotoxic action against HeLa cells. On the other hand, prostaglandins enhanced HeLa cell growth. These results suggest that lipoxygenase products are the mediators of the cytotoxic action of GLA on HeLa cells. Studies performed have thus demonstrated that GLA and other c-UFAs have selective cytotoxicity against tumor cells but do not harm the normal cells. These fatty acids are able to bring about their selective tumoricidal action by enhancing the generation of free radicals and lipid peroxidation process in the tumor cells.



STUDIES ON THE NEUROBIOCHEMIC-AL MECHANISM(S) IN THE DEVELO-MENT OF BEHAVIORAL TOLERANCE TO METHYAL-XANTHINES & THEIR WITHDRAWAL EFFECTS IN MAMMALS  

Studies on the neurobiochemical mechonisms in development of behavioural tolerance to methylxanthine and its withdrawl effect Study was carried out male charles Foster strain albino rats to understand the neurobiochemical mechanism explaining the development of behavioural toleronce to methylxanthine (caffine) and its withdrawl effects at the level of (i) the dynamics of different neurotransmitter viz, GABA and adenosine receptor binding and (ii) introduction of GABA, adenosine with receptor of other neurotransmitters. Rats were divided into 3 groups and animals of group 1 and 2 were treated with caffeine at a dose of 10 and 20 mg/kg/day respectively for 1-16 consecutive days. Group 3 animals (control) were treated with water (vehicle) only Single does (10-20mg/kg) of methylxanthine (cafeeine) increase the locomotor acitivity (LA) of rats in a dose dependent manner. The caffeine induced LA incease further with the duration of treament showing a peak after four consecutive treatment. This increased LA was restored back to control values after 16 and 12 consecutive days of caffeine treatment at a dose of 10 and 20 mg/kg/day, respectively indicating the development to tolerance to caffeine. Pharamacological and biochemical studies suggest that caffeine under nontolerant conditions reduces adenosinergic activity by its antagoistic effect on adenosine receptor which in turn enhances dopaminergic activity thereby reduceing central GABA ergic activity through the inhibition of cholinergic system and results in enhanced locomoter acitivity. The development of tolerence to caffine following 12 or 16 consecutive days of treatment depending on the dose was found to be due to up gulatioin of central adenosine receptor activity and GABAergic activity through the modulation of dopaninargic and cholinergic systems. The withdrawl of caffine following its tolerance decreases LA maximally at 48 h from the time of withdrawl. This decrease in LA under cafeine withdrawl was found to be the effect of increase in central GABAergic activity via the modulation of central dopaninergic and cholinergic activities. It is thus concluded that caffeine induced changes in LA under nontolerant, tolerant and withdrawl conditions are mediated by modulation of not only the central adenosinergic acitvity but also the central GABAergic acitvity through the interaction of dopaminergic and cholinergic systems.



ROLE OF OXIDATIVE DEFENCE MECHANISMS IN NEUROTOXICITY OFSELECTED ENVIRONMENTAL CHEMICALS  

The present study was aimed to; investigate the nature as well as degree of oxidative stress imposed by managnese, endosulfan and acrylamide on the nerve cells/tissue; Understand the mechanism of the neuronal tissue injury and understand the role of antioxidative defence capacity of the tissue particularly GSH) in modulating the neurotoxicity of these chemicals. Impairment of oxidant antioxidant balance has been implicated in the etiology of cerebro vascular disorders and upon exposure to a variety of enviromental chemicals including metals, solvents, pesticides and monomers. Considerable progress has been made in elucidating the mechanisms of free radicals induced membrane damage in various tissues and organs, however free radicals induced injury to the brain, upon exposure to environmental chemicals and in ischemic stroke has not been studied extensively. Since reavtive oxygen species and fatty acid hydroperoxides are physiologically present in brain and platelets and believed to play a regulatory role in membrane functions, it was of interest to investigate the possible involvement of reactive oxygen species in altering the membrane related events upon exposure to a metal managanese, monomer acrylamide and pesticide endosulfan and in patients of ischemic stroke. The observations revealed that Iron Ascorbate treatment in vitro, resulted in a significant increase in LPO and depletion in GSH level both in brain and platelets. This was in parallel to decreased membrane fluidity and elevation of (Ca++) i levels. Free radical scavengers mannitol, thiourea and physio Ehysiological antioxidant vit- E provided partial protection in Iron-Ascorbate induced lipid preoxidation and gluthione oxidation and consequent alterations in membtrane fluidity, (Ca++)i levels and platelet aggregation. Acrylamide at concentrations > 5 mM (in vitro) caused a decrease in GSH levels and increase in LPO and membrane function alterations. However, at lower concentrations (<5 mM) at which acrylamide has previously heen ahown to cause neuronal tissue injury neither caused oxidative damage nor altered neuronal membrane function. Acrylamide and endosulfan exposure in vivo although caused a slight increase in LPO, the intgrity and functions of neuronal membranes remained unaltered both in young and adult rats. The observed increase in 3H spiroperidol binding following acrylamide exposures appears to be independent of oxidative events. The significant decrease in LPO and increased membrane fluidity in human platelets and rat brain preparations in vitro and in vivo suggest the antioxidant like action of manganese. The increased levels of PC and PE, increased 3H spiroperidol binding and deoreased 3H- 5HT binding in young rats as compared to the adults suggest that young rats are more susceptible to manganese mediated neuronal membrane damage. Incrased platelet aggregatino in response to ADP, epinephrine arachidonic acid was observed in ischemic stroke subjects as compared to normals. This was in parallel with incrase in platelet (Ca++)i levels, enhanced rate of MDA formation, oxidation of reduced glutathione and decrease in membrane fluidity. From the present observa observations it may be concluded that oxygen free radicals play a significant role in modulating the membrane related events following exposure to environmental chemicals and in pathological conditions such as ischemic stroke.



STUDIES ON A MEMBRANE GLYCOPROTEIN WHICH CAUSES ANOREXIA  

The study was carried out to evaluate the role of a membrane glycoprotein on the control mechanism of anorexia in normal and diseased condition in swiss albine nice. Bioassay studies revealed the presence of anerexigenic glycoprotein (proteoglycan) in both monocatylednors wheat (Triticum asestivum), rice (Oryza sativa) and barley (Hordeum valgare) as well as dicotyledorions mung sean (Phaseolus radiants and chick pea (Gicer orietinum) plants. However, the proteogly can isolated from the plants of the graminease family (monocot) suggested faster degradation in animal body. A similar loss of appetite response was observed following the administration of protoglycan to the animal model system either by intramuscular/ intravenous/ intradermal/ or intraperitoneal rout. But no anorexigenic response was observed when isolated proteoglycan was administered orally. Majority of the non ionic detergents were not found suitable for the suitable and effective isolation from ming bean sprout preparation. Subcellular fractionation studies clearly revealed its presence in cell surface (plasma) membranes which strengthed the hypothesis that the anorexigenic proteoglycan is anchored to the surface membrane. Overall studies on this anorexigenic proteoglycan suggest that, being a common constituent of the surface membrane it is well accepted by the animal body without any rebound or apparent unwanted effect. Hence it can be hypothesised that with a regulatory control on hunger drive, it could provide a useful tool in the hands of physicians dealing with the problem of obesity (due to overeating) and anorexia in various diseased conditions. In addition, it can also be used in various nutritional research studies as a conventional parallel control group with controlled food intake depicting a true satiety condition.



DEVELOPMENTAL AND FUNCTIONAL ASPECTS OF BRAIN LECTINS FROM PRENATAL HUMAN BRAIN AND POSTNATAL RAT BRAIN  

The study was carried out to elucidate structure and function and biochemical characterization of lectins from human and rat foetal brains. Human factness were obtained from nutritionaly normal ladies undergoing medical termination of pregnancy. Lactose binding (18KDA and 66KDA) as well as beparin binding (29KDA and 45KDA) lectins were pruified from human foetal brain by a simple chromatographic procedure using Q-Sepharose as the matrix. Through both 29KDA and 45KDA lectin were heparin inhihitory, the 29KDA heparin binding lectin showed inhihition towards stachyose as well as mesoglycan while 45KDA hepar in binding lectin showed strong inhihition by sulodexide as well as mesogtlycan. This revealed the complexity of endogenous ligands for 29 and 45KDA lectin. Both 29KDA and 45KDA lectins showed high content of acidic aminn acid. Absence of pysine from 29KDA lectins distinguishes it from other known lectins. The major glycopeptide recognized by 29 and 45KDA lectins was 0.5M N aretyl D glucosamine eluted glycopeptide. Mannose terminated glycopeptide had no effect on these lecitns. Higher contents of 29KDA lectin in cerehral cortex and powest in spinal cord cnrrelates lectin levels with neuronal population. Presence of 29KDA lectin in mitochondrial fraction can be correlated with the lysosomal functioins i.e. as a signal for secretion and release. Myelin basic protein isolated from rat brain showed heparin inlibitery lectin activity. This property can be used to identify the abnormalities of myelin where myelin basic protein leaks in cerebrospinal fluid.



CHEMICAL ANALYSIS OF URINARY CALCULI IN HARYANA  

Chemical analysis of urinary calculi in Haryana. The study was carried out to elucidate the morphological characteristics and chemical composition of urinary calculi formed by people of Haryana and to correlate the chemical composition with the type of calculi and age of stone formers. Quantitative chemical analysis of 400 urinary calculi (240 renal, 66 ureter, 94 bladder) collected from different districts of Haryana revealed the presence of calcium oxalate monohydrate (CaOxM) in all the stones. However the presence of magnesium ammonium phosphate hexahydtate (MAP), hydroxy apatite ( HA) and uric acid (UA) was observed in 83.6%, 93.46% and 77.2% patients respectively. The content of CaOxM was found to be higher in renal stones compared to bladder to renal and bladder stones. The concentration of uric acid was found to be higher in bladder stones as compared to renal and ureter stones. The CaOxM was found as a major component in all types of urinary stones analysed. The presence of CaOxM and HA decreased and MAP and UA increased with the age of the stone formers.



ALUMINIUM PHOSPHIDE POISONING:BIOCHEMICAL MECHANISMS OF NEUROTOXICITY IN RAT  

The study was carried out on Wistar strain albino rats to investigate the biochemical mechanism of aluminium phosphide (ALD) poisonig with special emphasis on mitochandrial dysfunctions in brain and to find out if Vitamine E has any protective effect on aluminium phosphide induced neurotoxicity. Acute aluminium phosphide (10 mg/kg body wt.) exposure resulted in disruption of energy metabolism by altering the various components of electron transport chain in mitochondria and disturbing the carbohydrate homeostasis. A markeddecrease in the activity of cytochrome oxidase was observed which resulted in the decrease in the oxygen uptake. The ATP hydrolysis which lead to depletioin of ATP levels in mitochondria. The components of the electron transport chain, ie the NADH dehydrogenase and dehydrogenase showed a significant decrease in their activities. This energy crisis resulted in a considerable decrease was obsered in the acitivity of hexokinase in various regions of rat brain. A decrease was also observed in the gluconeogenic enzymes viz Glucose-6-phosphatase and fructose 1,6-bisphosphatase. Glucose-6-phosphate dehydrogenase also depicted a marked decrease. ALP exposure resulted in oxidative stress to the different regions of centralnervous System through lipid peroxidation and antioxidant defense system thereby affecting the structure and functiins of synaptic plasma membranes. There was significant increase in the levels of lipid peroxidation in cerebral cortex, cerebellum and brain stem on exposure to ALP. Superoxide dismutase and catalase demonstrated a marked decrease in all the three regions of rat brain. The acitvity of glutathione reductase was significantly decreased following ALP exposure however, glutathione peroxidase remained unaffected. The levels of reduced and oxidized glutathione decreased significantly and total sulfhydryl groups showed a marked decline. The membrane fluidity was adversely effected due to oxidative stress generated following toxic exposure ALP wshich further affected the acitvities of membrane bound enzymes i.e. acetycholine esterase and sodium and potasium ATPase. In a short term study of 15 days, vitamin E (150 IU/kg body wt./day) was found to decrease ALP(1mg/kg body wt/day) induced oxidative stress in rat brain. Vitamine E and ALP administered together resulted in reduced levels of lipid peroxidation in all the region of rat brain compared to only ALP treated group. The activity of superoxide dismutase was enhanced significantly when vitamine E was given with E. This also resulted in decreased glutathione reductase levels as compared to only ALP treated group. ALP exposure exerts a devastating effect on the energy metabolism and generates severe oxidative stress leading to various structural and functional alterations in the cell. Vitamin E showed some protective role in elevating toxic effects of ALP.



INVESTIGATION INTO THE TUMOURICIDAL ACTION OF ESSENTIAL FATTY ACIDS AND THEIR METABOLITES  

The objective of study uptake and distribution of essential fatty acids and their metabolites in normal and tumour cells in vitro using radiolabelled compounds and the effect of essential fatty acids and their metabloities on the concerntations of various anti-oxidants such as superoxide dismutase, catalase and glutathione in tumor cells in vitro. To characterize various lipid peroxides formed in the tumor cells treated with essential fatty acids and their metabolities by HPLC/GC analysis using dervatization methods. The effect of various fatty acids was seen on the growth and survival of tumor cells in vitro (HcLa cell). The percentage of live cells was less as the dose of fatty acids was increased EPA and DHA were found to be the most potent fatty acids, n-6 and n-3 fatty acids were found to have cytotoxic/ cytostatic action on HcLa cells. ALA & EPA fatty acids were mostpotent in inhibiting the growth of SP2/0 cells at all concentrations tested. DHA and OA were also found to affect the proliferation of SP2/0 cells but only to a limited extent. The n-6 fatty acids: LA, GLA, DGLA and AA did not show any inhibitory effect on the proliferation of SP2/) cell at all doses tested. Infact, both DGKLA and AA Stimulated the proliferation of SP 2/0 cells. Both ALA and EPA also decreased the viability of SP 2/0 cells. Both ALA and EPA also decreased the viability of SP 2/0 cells bout only to a limited extent and that too obly at 40 mue g/ ml concentratioin. KB-Ch R-8-5 cells are resistant to the cytotoxic action of vincristine compared to the response of KB-3-1 cells. Of all the fatty acids, EPA and DHA are the most potent in reducing the survival of KB-3-1 and KB-Ch R-8-5 cells in vitro. The cytostaitc/ cytotoxic action of various fatty acids on KB-3-1 and KB-ChR-8-5 cells in further evident from the thymodine incorporation studies. Studies have demonstrated that the sensitivity or resistance of tumor cells to the cytotoxic action of anti cancer drugs and cytokines is determined, at least in part, by the intracellular anti-oxidant concentrations.Thus one of the factors that can contribute to tumor cell drug resistance is their anti oxidant content was studied. This study was performed in SP2/) cells. Of all the fatty acids tested, only ALA and EPA exhibited potent cytotoxic action on SP 2/0 cells. Hence, the effect of ALA and EPA only on the concentrations of a5t ous anti oxidants in Sp 2/0 cells was studied. Results of this study revealed that ALA and EPA ( 5 and 10 mue/ ml) treatment can decrease the concentrations of SOD(superoxide dismutase) to a significant degree in SP 2/0 cells. ALA treatment decreased where as EPA enhanced the activity of catalase at the end of 24 hours of incubation. A Decrease in the levels of glutathione peroxidase (GPX) and gluthathione reductase (GR) with ALA (5 and 10 mue/ml) treeatment where as an increase with EPA treatment (10 mue g/ mg) was observed. A significant change in the levels of glutathione-S transferase (GST) was induced by ALA (at mue / ml dose. Total glutathione levels were elevated in the cells treated with 10 mue g/ ml of ALA and EPA at the end of 24 hours of incubation. These results indicate th that a marked degree of alteratin in the levels of various anti oxidants in SP 2/0 cells can be induced by ALA and EPA treatment.



BIOCHEMICAL MECHANISMS OF ALUMINIUM INDUCED NEUROTOXICITY AND ITS ROLE IN NEURODEGENERATIVE DISEASES  

Aluminium for a long time has been considered an indifferent element form a toxicological point of view. In the recent years the knowledge about the ubiquatous nature of this element and the growing evidence about its neurotoxicity makes it the subject of increasing interest. Neurological dysfunctions in patients of dialysis encephalopathy have been related to high aluminium concentration in the dialysate and to the use of phosphat binding gels containing aluminium. An attempt has been made to drawn a correlation between the biochemical abnormalities, behavioural aberration and histopathological alterations following aluminium exposure. Male abino rats of Wistar strain in the weight range of 140-160 gms were used throught the study of 12 weeks. The various biochemical parameters to be stud 12 weeks. The various biochemical parameters to be studied were standardized. After the respective treatments, animals were fasted overnight and euthanised by decapitation. The brains were removed, rinsed in ice cold isotonic saline and dissected into the following regions according to the guidelines of glowinski and Iverson (1996), cerebral cortex, corpus striatum and hippocampus. The following experiments were conducted Momory Function Test, Intrasynaptosomal Calcium assay, Calcium uptake in synaptosomes, Calcium ATPase assay and preparation of synaptic plasma membranes from rat brain was also done. Memory function test i.e. Passive avoidance and Active avoidance. The animals following chronic aluminium exposure showed a significant decrease in retention time during the passive avoidance memory test.



STUDIES ON REGULATION OF SYNTHESIS AND SECRETION OF GnRH IN THE PLACENTA  

The role of placental steorids, hCG, inhibit and opioid peptides in the regulation and secretion of GnRH by placenta was investigated using both human and rat placenta as model. In the first Phase of the study experiments were designed to evaluate the effects of placental steroids in the GnRH levels in the in vitro incubation studies. These studies involved standardization of RIA for GnRH and application of GnRH RIA for monitor the levels of GnRH under in vitro conclusions following addition of modulators. In the second phase of the study experiment were planned to monitor the level of GnRHR mRNA since it is known that at the pituitary level the regulation is mainly mediated by modulating the mRNA level of GnRH recptor by the gonadal steriods and accordingly human placenta. Both in vitro and in vivo studies, which were carried out to evaluate the efficacy of the GnRH receptor antiserum to inhibit binding of GnRH to pituitary, suggested that the antibody is not able to inhibit binding. Hence, active and passive immunization studies were undertaken to evaluate the effects of long term exposure to antibodies under in vivo conditions. The goal of the studies described herein was to use the primers based on the sequence of the human pitutiary GnRH receptor to amplify, clone and sequence transcript of interest and to determine their homology to the hpit GnRH receptor sequence. The data generated would contribute to the understanding of the similatities and differences between the hypothalamic/ pituitary and placental GnRH axes and this inturn would perhaps contribute to our overall understanding of hCG GnRH regulation.



BIOCHEMICAL INVESTIGATIONS ON THE MECHANISM OF ACTION OF HYPOGLYCAEMIC COMPOUND FROM THE WATER EXTRACT OF THE BARK OF FICUS BENGALENSIS  

A study on the role of different neurotransmitters in regulating the GI motility, indicates cholinergic involvement and partial role of prostaglandin and NO in the mechanism of action of black tea extract. Results indicate that thearubigin fractin of responsible for the prokinetic effect of black tea extract. Exepanol-HCl (drug under development as a prokinetic agent) produced protentiation of GIT both in normal and in hyperglycemic rat.Exepanol HCl (10 mue gram/kg) reduced significantly indomethacin treated ulcerogenic rat. (upto 82%). Exepanol was equipotent as MCP by restoring the gastric emptying time in normal as well as in hyperglycemic rat. Results indicate a role of NO in the mechanism of prokinetic effect of Expanol HCl. Now to extrapolate the data already generated in our laboratory as mentioned above, we are in the process of estimating NO in different parts of GI tract in control group as well as in experimental group treated with graded dose of prokinetic drug, Exepanol HCl. Studies ith ciprofloxacin (a, broad spectrum fluoroquinnolone antibacterial agent) also indicates an involvment of cholinergic mechanism in the prokinetic effect of ciprofloxacin both in normal and in diabetic rat. It is suggested that systemic clinical studies may be initiated to evaluate the potential efficacy of ciprofloxacin in the treatment of GI motility disorders, specially in diabetic gastroporesis. During the course of investigation, some medicinal plants are evaluated for possible prokinetic and or antidiarrhoeal activity. The results so far indicates that edible mushroom s shares prokinetic effect. Seed extract of albizzia ledbeck and root extract of psidium guajava exhibited marked anti diarrhoeal activity in animal model. Its potency is comparable with loperamide, the clinically used antidiarrhoeal drug. Results indicate that the antidiarrhoeal action of the extract may be due to the inhibition of prostaglandin synthesis and release.



A STUDY OF SERUM NITRATE LEVELS IN PREGNANT WOMEN  

The study was planned and carried out in the Department of Physiology and subjects recruited in OPD of Obst. & Gynae, in St. John's Medical College Hospital. In all 155 patients were included in the study, after obtaining the informed consent as required by the Ethical Committee of the Institution. The subject groups were as follows Control (55) first trimester (n=13) second trimester (n=42, 3rd trimester (n=45) of which normotensive (n=32), PET (n=13). The nitrate levels were measured by one step enzymatic assay using nitrate reducfase method. Fasting blood sugar, Hemoglobin level, creatinine were measured using standard laboratory producers. The value of serum nitrate in all the gropus had a skewed distribution and therefore these werre analysed by non parametric tests i.e. Kruskal Wallis and Mannwhitney by using statistics package SPSS 7.5. The serum nitrate levels were highest during 111o pregnancy (median 22.8, IQR 26.2) as compared to first trimester (median 16.1, IQR 22.4). This is an expected finding. The level of nitrate in Preclamptic group were significantly lower (p=02). The renal function of all the subjects were within normal range, and therefore differences could be attributed to the reduced production of the nitrates from Nitric oxide. Although the number of subjects in the hypertensive group is small but the results of present study indicate lower levels of nitrate production in Preclamptic group. Larger study is needed to confirm and extend the findings.



ALPHA ADRENERGIC RECEPTOR LINKED SECOND MESSENGER RESPONSE TO ANTIDEPRESSANTS IN RAT BRAIN  

The present study characterised alpha 1 and 2 adrenoreceptors by using specific radioligands in regions of rat brain. The monophasic displacement by unique pharmacological agents of each subtypes provides strong evidence that atleast two distinct populations exist in rat brain. The denisity of alpha 1 and 2 adrenorecptors was studied in different regions of rat brain after chronic exposure (40 days) to antidepressants (ADs). Tricyclin antidepressants act by downregulating cortical adrenoreceptors but not hippocampal adrenoreceptors. However there was no significant change in the affinity of these receptors. The differential alteration of adrenoreceptors linked second messengers like cAMP and IP3 was also observed in reponse to chronic administration of ADs. The phenomenon of increased synaptic NE content due to blockade of uptake sites by TCAs with concomitant stimlation of post synaptic receptors may be resulted in the subsensitivity of cortical adrenergic signalling. This decreases in alpha adrenergic responsiveness could be the mechanism of action of TCAs and the adaptive phenomenon of desensitisation of receptors may be considered an important factor in the achievement of theraupeutic efficacy of AD drugs after chronic tyreatment. This study has provided a complete profile of biochemically and pharamcologically characterised alpha1 and 2 adrenoreceptors in rat and the data will help in the understanding the mechanism of action of certain drugs and also desigining new drugs which will become effective where adrenergic neurotransmitter is defective.



ANTIOXIDANT VITAMIN SUPPLEMENTS AS PALLIATIVE TREATMENT OF BONE DISORDERS  

Bone disorders encompass a wide spectrum of conditions associated with imbalance of osteociastic and osteobalstic activities. Osteoclasts, the scuplptors of bone operate by producing free radicals acting as chisels. These are responsible for remodeling. Indiscriminate production of free radicals leads to oxidative stress mainly due to lipid peroxidation as seen in our pilot study. The present study probes into the role of antioxidants as a palliative treatment for bone disorders. 50 healthy controls and test group comprising of 198 patients suffering from skeletal disorders like osteoporosis, renal osteodystropy, bone malignancy and fractures underwent baseline assessment of biochemical markers viz. osteoblastic markers serum Alkaline phosphates (ALP), Free Ca++ and Inorganic phosphorus (Pi, osteoclastic marker serum Tartarate rsisitant acid phosphates (TrACP) and Malondialdehyde (MDA) and the antioxidant status serum superoxide dismutase (SOD) and Erythrocyte reduced glutathione (GSH). Each test group was then divided in to gropus A (Evinal 400 mg), B (Celin 500 mg) , C (Evinal + celin ) for antioxidant supplementation for a period of 90 days. The results reveal that there is significant improvement in the biochemical markers serum MDA, serum TrACP, serum ALP and antioxidant status SOD and erythrocyte GSH after antioxidant supplemtntation. Antioxidant vitamins E and C individually or conjointly imoprove the bone status in various skeletal pathologies and hence may serve as cost effective, palliative supplements in additioin to curative treatment.



STUDIES ON MECHANISM OF SIGNALTRANSDUCTION DURING RELEASE OFPROINFLAMMATORY CYTOKINES IL-1BETA AND TNF-ALPHA BY ALVEOLAR MACROPHAGES IN ASTHMA 

In asthma, proinflammatory cytokines released by alveolar marcrophages (AM), such as IL-1 beta and TNF- alpha, are known to play a significant role in airway inflammation. The release of these cytokines may be triggered by various extrinsic stimuli (e.g. asthmogens). Exposure of cells to stimulus is known to cause stimulus receptor coupling, activation of transmembrane signalling and phosphorylation of some target proteins of proteins of protein kinase c (PKC), the key regulatory enzyme of the pathway. Phosphorylation changes the conformation of the proteins, which is necessary for several cellular functions. The expression of these cytokines by AM may be associated with the phosphorylation of some of these proteins. based on these presumptions, the studies were conducted on AM of asthmatic patients and healthy subjects to evaluate the expression and release of IL-1 beta and TNF- alpha at mRNA and protein levels by various stimuli viz. asthmogens (histamine, methacholine chloride; MCC), mitogen (LPS), agonists of PKC PMA), antagonists of PKC (sphingosine) and bronchodilator (salbutamol), followed by identification of target proteins of PKC and changes in their phosphorylatioin. The results suggest a significant increase in IL-1 beta and TNF- alpha levels in cell free BAL fluid of asthmatic patients as compared to the healthy subjects without significant change in total member of cells in the BALF. Apparently, the AM of the AM of the asthmatic patients are primed to the triggers of asthma, the presence of which in the milieu may induce thhe early expression and release of these cytokines in comparison to the healthy subjects, leading to the perpeptuatioin and orchestration of inflammation in the airways. The expression of IL- 1 Beta and TNF- alpha by AM seems to be regulated by PKC mediated mechanism. This gets support by the changes caused by these stimuli on phopshorylation of the target proteins of PKC of 120, 66, 35 and 25 kDa, as identified in this study. It may be concluded that in asthma, alveolar macrophages are primed to the triggers and the expression and release of these ctokines by them could be associated with the phophorlation of 120, 66, 35 and 25 kDa proteins through PKC mediated pathway.



STUDIES ON THE EFFECT OF CHRONIC ETHANOL FEEDING ON TRANSPORT OF FOLATE AND EXPRESSION OF ITS TRANSPORTER IN RAT INTESTINE  

Folate deficiency is the common sign of chronic alcoholism. ntestinal malabsorption of the folate is a contributing factro to alchol induced folate deficiency. The aim of the present proposal was to elucidate the mechanism of intestinal malabsorption of folate during chronic alcoholism. Male Wistar rats were fed 1g/Kg body weight ethanol (20% solution) orally for 12 weeks. Intestinal and renal brush border membrane (BBM) vesicles were prepared to study the binding and transport of 3) folic acid. The kinetic studies of the folate transport process revealed the folate transport exhibited saturable kinetics and was pH, Na+, temperature, divalent cation dependent.Chronic ethanol feeding was found to decrease the binding as well as transport the folic acid by altering various kinetic characteristics of the processes and different factors impart diverse effects to these processes at both the intestinal and renal brush border membrane. Thus the process seems to be complex interplay of the diverse biochemical and physiological factors at the absorptive and conservative sites in the mammalian system. Decreased uptake of folate across the intestinal epithelium was associated with a reduced expression of mRNA of reduced folate carrier (RFC). Moreover, the transport was reduced all across the crypt villus axis arger chronic ethanol feeding. Using vrious activators and inhibitors of different singaling pathways it was observed that folate transport is regulated by cAMP dependent protein kinase A. Ethanol also seemed to exert its effect on transport of folate by interfering with his regulatory pathway. In conclusion, ethanol ingestion results in reduced renal & intestinal transport of folate by decreasing the expression of its transporter and interfering with regulation of the transport process.




CARDIOVASCULAR DISEASES
LEUCOCYTE AND PLATELET DEPOSITION IN ATHEROSCLEROTIC PLAQUES:ROLE OF SUGAR SPECIFICADHESION TO ENDOTHELIAL LECTINGALECTIN 1 AND PROSPECT OF IT'S INHIBITION BY HUMAN ERYTHROCYTE MEMBRANE OLIGOSACCHARIDES  

Objectives of the project were to delineate the role of cell surface carbohydrate epitopes on leukocyte and platelet on one hand and of galactose bingding lectin (galectin-1) of endothelial and underlying cells on the other, in anchoring these cells on blood vessel walls. Also to explore the potential of human erthrocyte membrane glycoproteins as a soruce of glycopeptides and oligoscacchrides that may be useful for inhibiting galectin-1 mediated deposition of blood components on vessel wall. Galectin-1 was purified from human heart tissue by affinity chromatography using lactose-Sepharose. The co purifiction of endogenous glycoproteins along with galectin-1 suggested formation of carbohydrate dependent complexes between the two. It was suggested that when the divalent galectin-1 binds to multivalent endogenous glycoproteins the resulting complexes contain enough suger binding sites to facilitate erythrocyte receptor aggregation during hemagglutination by this complex as well as its attachment to lactose-Sepharose during isoltion of galectin-1 Present results suggest lectin-carbohydrate recognition as primary reason for association of co purified glycoproteins with HHL within intact itssue or after homogenization. Copurified glycoproteins therefore are chosen endogenous complementary glycoproteins for HHL and contain sugar ligands apt for the lectin. Further proof for this is the recognition of coated HPLC separated co purified glycoproteins by purified HHL. In this context the abundance of sialylated as well as free T antigen in copurified glycoproteins points to a preference for T antigen by HHL. Erythrocyte membrane glycopeptides were prepared for use as inhibitors of galectin-1 for preventing tumor spread since some saccharides are easily soluble compared to their membrane glycoproteins precursors. From the point of view of a possible therapeutic potential, erythrocyte derived glycopeptides offer other major advantages such as ease of availability of erythrocytes from discarded blood as well as plasma separation and being non immunogenic in humans.



CENTRE FOR ADVANCED RESEARCH IN CARDIOMYOPATHY  

Sensitivity and specificity of fluorescent labelled antibody absorption (FLA-ABS) and lepromin tests were investigated for early detection of subclinical infection among household contacts of leprosy patients (both paucibacillary and multibacillary). FLA-ABS was found to be very sensitive and specific for the detection of subclinical leprosy infection as FLA-ABS positivity in contacts of both multibacillary and paucibacillary leprosy patients was higher than that of lepromin FLA-ABS positivity was much higher in the contacts of multibacillary leprosy patients than paucibacillary patients. On the basis of FLA-ABS positivity and lepromin reactivity contacts of leprosy patients could be classified into four groups viz. (i) FLA-ABS positive, lepromin negative; (ii) FLA-ABS positive, lepromin positive; (iii) FLA-ABS negative, lepromin positive; and (iv) FLA-ABS negative, lepromin negative. Contacts who were FLA-ABS positive and lepromin negative appeared to be at much higher risk of developing leprosy than the other group of patients as most of the contacts who developed leprosy were from this group. Hence, it can be tentatively concluded that FLA-ABS and lepromin testing help in identifying contacts who are greater risk of developing the disease. Follow up of leprosy patients under treatment indicated that FLA-ABS test remained positive even after clinical subsidence of the disease. FLA-ABS positivity was higher in patients of multibacillary leprosy after subsidence of disease as compared to those with pauci- bacillary leprosy. These findings, however, need to be confirmed by larger studies before any definite conclusion can be drawn regarding the usefulness of FLA-ABS testing in the follow up of treated leprosy patients or the contacts of leprosy patients.



TO STUDY THE EFFECT OF SUSTAINED EOSINOPHILIA ON THE HEART IN RHESUS MONKEYS  

This study was undertaken to develop a method for producing sustained eosinophilia in rhesus monkeys, and to find out its effects on the heart in the presence of specific and non-specific immune enhancement. The effect of sustained eosinophilia after specific deficiency of tryptophan and the possible association between eosinophilia and endomyocardial fibrosis were also studied. Sustainable blood eosinophilia was produced in rhesus monkeys by subcutaneous weekly injections of globular sodium alginate beads coated with coat IgG. Eosinophilia of more than 2000 eosinophils/cubic millimeter was obtained after 12 weeks, the count peaked to about 6000 cells/cubic millimeter at 9 months and this persisted upto 12 months of study. Lymphocyte blast transformation studies carried out at two monthly interval revealed that lymphocytes play an important role in the maintenance of the eosinophilic state as evidenced by increased propensivity to proliferate (as measured by tritiated thymidine incorporation) when challenged with a mitogen (PHA). The highest proliferation of lymphocytes was observed at the end of 12 months suggesting that they were secreting certain factors which helped to sustain the eosinophilia. Persistent blood eosinophilia did not produce any significant histo- pathological changes in the heart. No such changes could be found even when the eosinophilia was associated with non-specific or specific immune enhancement, or tryptophan deficiency.



PRODUCTION OF CARDIAC LESIONS IN D 17 POSITIVE AND NEGATIVE MONKEYS INJECTED WITH GROUP A STREPTOCOCCAL MEMBRANE AND CARBOHYDRATE  

The study was carried out to type a group of monkeys for D 8/17 B-cell alloantigen: creat a laboratory model of carditis using cross-reactive antigen in genetically defined monkeys: and study the antibodies generated against purified streptococcal carbohydrate and membrane antigens alone and in combination in D 8/17 positive and negative monkeys. Adherence of streptococci on oropharyngeal epithelial cells of monkeys before and after sensitization was also studied and cross-reactive antigen generated in circulating immune complexes during various phases of the disease was identified. Of the 117 monkeys typed for D 8/17 B-cell alloantigen 23(19.66%) were positive. The expression of D 8/17 alloantigen on the cells of the monkeys did not change with time after repeated sensitization of the animals with streptococcal membrane or carbohydrate or a combination of these antigens. Repeated injections of streptococcal membrane and carbohydrate antigens did not change the expression of the oropharyngeal epithelial cell receptors binding to streptococci. High levels of anti-membrane and anticarbohydrate antibodies were seen in the sera of the monkeys injected with the respective antigen confirming a heightened humoral immune response to the streptococcal antigens. Anti- membrane and anti-carbohydrate antibodies persisted in the sera of animals sensitized with these antigens for a long time. Streptococcal antigens were present in the immune complexes of animals repeatedly sensitized with streptococcal antigens. Myocardial granulomas were observed in two and focal myocarditis in one of the four D 8/17 positive monkeys injected with streptococcal membrane antigen. Focal myocarditis was observed in two and edema and thickening of valves in one D 8/17 negative monkey injected with streptococcal membrane. Edema of valve cusps was also observed in two and inflammation and thicke- ning in one monkey injected with streptococcal carbohydrate antigen. Edema of mitral valve was seen in only one 8/17 negative monkey. Focal myocarditis was seen in two and edema in mitral valve inone D8/17 positive monkeys injected with a combination of streptococcal membrane and carbohydrate antigen. these data suggest that experimental myocarditis can be produced by repeated injection of streptococcal antigens in rhesus monkeys. Animal injected with membrane antigen apperead to develop lesions predominantly in the myocardium while those injected with streptococcal carbohydrate mainely in the valves. Animal injected with a combination of streptococcal membrane and carbohydrate antigen failed to show any significant lesion. Only minimal change were seen in the myocardium. It thus appeared that genetically identified positive monkeys develop greater lesion compared to the genetically negative monkeys.



DILATED CARDIOMYOPATHY IN INDIA-CLINICAL,IMMUNOLOGICAL, VIROLOGICAL AND MORPHOLOGICAL MARKERS AND ROLE OF LONG TERM METOPROLOL MORBIDITY AND MORTALITY *  

The study was planned to carry out detailed virological, immunological and histological study in 40 patients with dilated cardiomyopathy and to evaluate usefulness of betablocker therapy. The endomyocardial biopsy was done in large number of patients and with its help it was possible to identify patients with inflammatory myocarditis for improvement in the therpeutic approach. The technique in situ hybridization for study of viral antigen in the EMB specimens has been established. Detection of antigen in EMB specimens has been established. Detection of antigen in EMB specimens will help to understand the role of viral infection in causation of dilated cardiomyopathy. This is in the first large study in world literature for evaluation of usefulness of propranolol, a nonselective betablocker, in dilated cardio- myopathy. Though because of certain limitations, a randomized trial could not could be carried out but the observations obtained highly suggest the useful role of propranolol in this disease. Further this also shows that non-selective agents may also be useful. These observations do merit a large multicentre study to evaluate the beneficial effects of propranolol in these seriously sick patients. As such the prognosis in these patient is dismal and curative treatment like cardiac transplant is beyond the reach of our people. Similarly a multicentre study to evaluate the role of immunosuppressive therapy in inflammatory myocarditis is needed. Till today no randomized study is available in literature assessing the role of these modalities in patients with dilated cardiomyopathy.



ROLE OF ALPHA AND BETA ADRENERGIC AGONISTS IN STIMULATION OF HYPERTROPHY IN CULTURED NEONATAL RAT HEART MYOCYTES  

The study was carried out to assess the role of norepinephrine and isoproterenol in the development of hypertrophy in cultured cardiac myocytes of the rat and to find out whether alpha and beta adrenergic antagonists can modulate or block this hypertrophic response. The action of norepinephrine and isoproterenol on the synthesis of contractile and noncontractile proteins in the cultured cardiac myocytes was also studied. Primary culture preparations of neonatal rat cardiac myocytes obtained from one to three day old rats were utilized for the study. Norepinephrine (NE), an alpha and beta adrenoceptor agonists had a very potent effect in producing hypertrophy of cardiac myocytes at a concentration of 2uM, whereas isoproterenol (a beta adrenoceptor agonist) did not produce any such effect as measured by the total protein content. The protein content of the cells increased markedly with NE (199 pg/cell/ day) whereas the increase was least with isoproterenol (35 pg/cell/day) as compared to control cultures (33 pg/cell/day). The increase in protein con- tent with NE was both time and dose dependent with the maximum response occurring on day 11 with 2um drug concentration. Norepinephrine had a stron stimulatory effect on the synthesis of contrac- tile proteins actin and myosin heavy chain in cultured cardiac myocytes whi- le isoproterenol stimulated mainly the synthesis of noncontractile protein The differential response with adrenoceptor agonists and studies with adrenoceptor antagonists (phentolamine and propranolol) indicated that the hypertrophic effect of NE was mediated by its stimulatory effect on alpha adrenergic receptors; phentolamine, an alpha adrenoceptor antagonist completely blocked the hypertrophic response of NE. It is thus concluded that NE has a potent hypertrophic effect on the cultured cardiac myocytes and this is predominantly mediated by its stimulatory effect on alpha adrenoceptors. The present study will provide an excellent model to study the biological effects of various drugs and pharmacological preparations on living heart muscle cells.



EFFECTS OF APOLIPOPROTEIN POLYMORPHISM OF A-IV,E & H ON THE LIPID & LIPOPROTEIN LEVELSRELATED TO RISK FOR CARDIOVASCULAR DISEASE  

Effect of apolipoprotein E polymorphism on the lipid and lipoprotein levels related to risk for cardiovascular disease. The study was carried out on unrelated individuals of Kshatriya (182) and Mala (38) communities from Andhra Pradesh to identify the apolipoprotein (apo) E genotypes and allele frequencies in these population. Among the Kshatriya, five apo E genotypes (3/3, 3/4, 3/2, 4/2 and 4/4) were observed in males and (3/3, 3/4 and 3/2) in females. But in Mala population only three genotypes (3/3, 3/4 and 3/2) were observed in both the sexes. There was no significant gender differences in the frequencies of apo E genotypes in both the populations as well as between tow population groups. The 4/2 and 4/4 genotypes were found to be less frequent and observed only among 2 individuals each (missing in female Kshatriya and both sexes in Mala). The gene frequencies of the three alleles designated apo Summation 2, Summation 3 and Summation 4 for the Kshatriya (male and female pooled together) were 0.0522, 0.8516 and 0.0962 and for Malas 0.0395, 0.9210 and 0.0395 respectively. Gene frequencies for three alleles for the pooled population of Kshatriya and Malas were 0.0500,0.8636 and 0.0864 respectively. In the apo E genenotypes, the apo E4 homo or heterozygotes showed higher levels of cholesterol compared to other genotypes. The influence of each of the three common apo E alleles (Summation 2, Summation 3, Summation 4) on cholesterol and HDL cholesterol levels was evaluated by the test o f average allelic effects. The Summation 2 and Summation 4 alleles showed lowering and and elevating effects respectively on cholesterol levels. But there was no evidence for a consistent relationship between apo E genotypes and HDL cholesterol. The study also supports the assumption that lower Summation 4 frequency may be associated with lowered risk of cardivascular disease in rural population.



JAI VIGYAN MISSION MODE PROJECT ON COMMUNITY CONTROL OF RF/RHD IN INDIA (REGISTRY PROTOCOL)  

Heart Diseases is a preventable chronic disorder of the heart. It is the serious complication of Rheumatic Fever when it is left untreated. The consequences of the disease include increasing disability, repeated hospitalizations and prmature death. This project was organised by the Jai Vigyan Mission Project on control of RF/RHD. A community based survey with both active (School survey) and passive (RF/RHD registry) reporting modes was adopted for the study. For RF/RHD Registry, all reported cases of RF/RHD in this population of 1368715 were listed and population prevalence estimated. In the school survey 25033 school children were actively screened in a meticulous way (first by the project medical doctors, then by cardiologist and confirmation done by an echocadiogram) to estimate prevalence of RHD among childre3n. Extensive health education campaigns on the disease and preventive measures were carried out in the project area. The prevalence of RHD and incidence of RF in the selected regions of Ernakulam district is among the lowest reported thus far from the developing world. It appears to support the view that improved access to health and antibitics results in a decline in the RF and prevalence of RHD.



ESTABLISHMENT OF JAI VIGYAN MISSION MODE PROJECT ON RF/ RHD IN THE DISTRICT OF WAYANAD(SATELLITE CENTRE)  

In connection with the school sruvey for screening RF/ RHD, a sub study was initiated on the anthropometric and blood pressure measurements of school children. A school based cross sectional survey was done where the height, weight and blood pressure measurements of 21000 students of 5 to 16 years of age were taken. on analysis blood pressure in children shows an increasing trend in relationship with age that continues into adolescence. This relationship is seen in systolic as well as diastolic blood pressure. Blood prressure also shows an increasing trend in relationship with height for all age groups. Diastolic hyopertension predominates in children.



CREATION AND EVALUATION OF A CLINICAL STRATEGY TO SCREEN FOR CONGENITAL HEART DISEASE IN NEWBORN  

India faces an impending epidemic of cardiovascular diseases. Major concern in this respect to our country is CVD's are more likely to attack adults in their productive years of life. This has a profound and adverse effect on households, families and society. The prevalence of various cardiovascular ailments in adivasi population has been poorly documented. The health care infrastructure for these people is also very limited. A cost effective preventive strategy will need to focus on bringing down the risk factors both in an individual and in the population at large. With this aim this project was initiated in Wayanad District in Kerala which have high proportion of adivasis. Objective is to study the distribution and determinants of the cardiovascular disease freequency in the adivasi population and to create a model for performing disease surveillance in diffucult to reach areas using telemedicine. The project team would visit the adivasi hamlets and screen for cardiovascular diseases. The screening include measurement of height, weight, resting blood pressure, heart rate etc. and cardiac auscultation. Basic demographic data, income source, predominant diet, tobacco consumption and nutrition is also collected. Follow up will be done for patients with risk factors for CVD




CELLULAR AND MOLECULAR BIOLOGY
CARDIOVASCULAR DISEASE SURVEILLANCE AMONG ADIVASI POPULATION OF WAYANAD DISTRICT 

This project is conducted as a part of the second phase of the registry component of the Jai Vigyan Mission Mode Project on RF/RHD control. It is a satellite project of the Amrita Institute Medical Sciences. Kochi RF/RHD Registry Project and is being implemented at Wayanad district in Kerala. In this phase the project is extended to areas with a relatively poorer health care infrastructure hence the District of Wayanad has been selected. A community based survey with both active (RF/RHD registry) reporting modes was adopted for the study as in the nodal centre, Kochi. The strategies used in the nodal centre, Kochi has been adopted here, but the passive surveillance is done through the entire health care infrastucture abailable in the district.



ROLE OF ELECTROCHEMICAL GRADIENT OF PROTONS IN GROWTH AND DIFFERENTIATION OF CANDIDAALBICANS, A PATHOGENIC YEAST  

Study was undertaken to assess the role of electrochemical gradient of protons in growth and differentiation of Candida albicans, a pathogenic yeast. The results demonstrate that a transient rise in intracellular pH preceded phenotype divergence of C. albicans and the magnitude of change in pHi was greater in the evaginating population destined to diverge as buds when compared to that of the germ tube-forming population. These findings suggest that pHi may be a contributory agent in the differentia- tion of C. albicans. There was a rapid rise in pHi around 135 min. which also coincided with the time of evagination. The transient increase in pHi was followed by a rapid drop. The transition of pHi could be correlated to plasma membrane H+- ATPase activity. The uptake rates of various amino acids e.g. L-proline, L-methionine, L-glutamic acid, L-alanine, L-phenylalanine and glycine varied with the evagination. As compared to other amino acids, the uptake rates of L-methionine were much higher in mycelial form. Results demonstrated that pHi and H+-ATPase play an important role in driving the nutrient transport of C. albicans.



INVESTIGATION INTO THE EFFECT OF PROSTAGLANDINS AND CIS-UNSATURATED FATTY ACIDS ONCELL MEMBRANE BOUND ENZYMES INNORMALS AND HYPERTENSIVES  

Cis-unsaturated fatty acids are known to lower hypertension both in animals and humans, and it has been attributed to modulation of prostaglandin biosynthesis. In essential hypertension, the activity of cell membrane bound enzymes Na+K+-ATPase has also been shown to be altered. Hence, the influence of prostaglandins and their precursors, cis-unsaturated fatty acids (c-UFAs) on the activity of Na+-K+-ATPase was studied in normal individuals and patients with essential hypertension. It was observed that both n-3 and n-6 fatty acids can either augment or inhibit the activity of normal human leukocyte Na+K+-ATPase and 5'-nucleotidase activities and in leukocytes of patients with essential hypertension in vitro. Both cyclo- oxygenase and lipoxygenase inhibitors did not influence the modifying action of n-3 and n-6 fatty acids on the activity of leukocyte cell membrane bound enzymes. These results suggest that the anti-hypertensive action of cis- unsaturated fatty acids can at least, in part, be due to their action on cell membrane bound enzymes.



TUMOUR PROMOTERS-MECHANISM OF PROMOTION  

An understanding of the biochemical mechanism of action of tumour promoters is fundamental to developing methods and strategies for controll- ing the incidence of cancer in humans. Study was carried out for under- standing and comparing the initial events in the mechanism of action of tumour promoters. A series of tumour promoters like phorbol-12-myristate- 13-acetate (PMA), benzoyl peroxide (BP), mezerein and 3-keto bile acid (KA), with different abilities to promote tumours were examined and their rela- tionship studied in different cells in culture i.e. both normal and trans- formed cells, to see intercellular variations, if any, and to overrule cell specific effects. The cell types studied were mouse fibroblast cells NIH 3T3, human epidermoid carcinoma -HeP2 and A431 cells. The cells were grown to 80% confluency in their respective media and were treated separately with standardized doses of tumour promoters i.e. 25 ng/ml of PMA, 125 ng/ml of mezerein,125 ng/ml of KA and 1 mM BP for varying lengths of time. A stimula- tion of poly ADP ribosyl transferase (ADPRT) activity was found with all the tumour promoters in all the cell types studied as compared to the untreated control cells,the effect being more for the more potent tumour promoters and there were individual variations in time when the maximum effect of these tumour promoters was discernible. The tumour promoter induced accumulation of poly ADP-ribose was accompanied by a concomitant drop in NAD levels. The decrease, however, did not always coincide with the time kinetics or the extent of enzyme stimulation. Addition of 3-amino-benzamide (3AB) an inhibitor of poly ADPRT inhibited the stimulatory effect of these tumour promoters on the enzyme activity. As many tumour promoters induce an oxidative burst and cause membrane mediated damage, the intermediacy of active oxygen species was investigated by studying the effect of antioxidants and antipromoters on the enzyme activity. Superoxide anion levels after promoter treatment were also measured. Pretreatment of the cells in culture with enzyme scavengers of active oxygen like superoxide dismutase (SOD), catalase (CAT) and the antioxidant butylated hydroxy toluene (BHT) showed a decrease in poly ADPRT activity to varying extents with PMA and BP., thereby showing that active oxygen species represent intermediates in this reaction. This was supported by a 2.5 fold increase in superoxide anion level with PMA and BP. Antipromoters also showed inhibition in poly-ADPRT activity in the case of PMA and BP treated cells. In order to verify the tumour promoter induced alteration of intra- cellular calcium in the cell types studied, calcium was measured using the fluorescent indicator method. With this technique a substantial increase in calcium was detected to the extent of 2 to 3 fold with PMA and BP respectively in HeP2 and A431 cells. The replication of DNA in A 431 cells as measured by incorporation of labelled thymidine in these cells was stimulated by 36% and 48% by PMA and BP respectively. However, mezerein showed only 21% increase. The effect of these tumour promoters on the subcellular distribution of protein kinase C (PKC) was also observed at 15 min. and 90 min. post promoter treatment. In NIH 3T3 cells, addition of PMA lead to a transloca- tion of the enzyme to the particulate fraction at 15 min. but the effect decreased at 90 min. probably due to down regulation of the enzyme. Simi- lar effect was seen with mezerein whereas BP showed no change. In A431 cells the basal level of PKC was much lower as compared to NIH 3T3 cells but these cells also responded to the tumour promoters in a similar fashion as NIH 3T3. Any protein changes by these tumour promoters in various subcellular fractions as well as the endogenous phosphorylation of proteins was also studied. In NIH 3T3 cells bands corresponding to Mr 66, 60 and 55 KD in PMA, BP and mezerein treated cells were seen. An additional band corresponding to Mr 19 KD was observed with PMA and mezerein treated cells. The most predominant band observed with all these tumour promoters was the Mr 66 KD band. The other predominant bands were in the Mr range of 29 - 14.5 KD. Phosphorylation pattern of the proteins essentially showed substrates at 116 KD and in the region of histones i.e. Mr 29 - 14.5 KD. Untreated A431 cells, as well as on tumour promoter treatment, essen- tially showed similar changes in the protein and phosphorylation pattern as NIH 3T3 cells. The results show that there is a unifying set of control mechanisms to translate the signal by these promoters studied, into intracellular message to the nucleus and thereby result in altered gene expression.



ONCOGENES AND NEOPLASTIC DEVELOPMENT  

The aims and major objectives of the study included identification of oncogenes in human cancers by cell transformation, studies on oncogene amplification and expression and study of molecular events of neoplastic conversion and determination of role of oncogenes in cell proliferation. Tumor specimens were transported in cold saline and used for DNA or RNA extraction within 3 hours of removal from the patient. Primary cultures of Swiss embryo fibroblasts were maintained and cell transformation studies were carried out in rat embryo fibroblasts . Attempts were made to assess the extent of possible amplification and expression of oncgenes. For these studies, in vitro random primer labeled 32p-oncogene probes of myc, ras and fos genes were used. The tumor DNAs and RNAs were isolated from tumors of scalp, cheek and pinis. Analysis of tumor DNAs by DNA-DNA and RNA-DNA hybridizations indicated amplification and increased expression of the myc gene in these tumors. However, the ras onco- gene was neither amplified nor expressed increasingly in these tumors. In another set of experiments, the role of oncogenes in cell-cycle progression was determined during the course of exposure of quiescent cells to mitogens. To determine the effect of DMSO on the expression of early growth-response genes in serum-stimulated cells, the levels of mRNAs of IL-6, c-fos genes were quantitated by RNA- DNA hybridization. The results showed that the expression of the proto-onocogene c-myc and that of the cytokine IL-6 were completely blocked by DMSO treatment of cells from the beginning of mitoge- nic stimuli. No inhibition of myc an diL-6 expression was observed when the cells were exposed to DMSO at later stages (after 3 h) of serum-stimulated growth. The serum-induced expression of fos gene was completely abolished by DMSO treatment of MEF during the first 30 min of mitogenic stimuli. DMSO did not have significant effect on PMA-mediated expression of the fos gene. DMSO drastically inhibited serum-induced DNA synthesis, it failed to inhibit DNA synthesis in the presence of PMA and FCS. In order to get insight into the biochemical basis of neoplastic develop- ment, normal rat embryo cultures were co-transfected with oncogenes myc and ras. A 48 KDa secreted protein identifed as plasminogen activator inhibitor showed a drastic decrease upon transformation. A 45 KDa secreted protein identified as DNA synthesis inhibitor also showed a decrease upon transformation.



MOLECULAR BIOCHEMISTRY OF PLATELETS IN HEALTH & DISEASE  

Study was carried out to probe the molecular pathology of the platelets in hyperaggregatory states, especially in coronary artery disease, in a 2-pronged way. The physical alteration in platelet plasma membrane including measurements of membrane microviscosity and lipid order parameter investigated. Effect of relevant drugs (e.g.propranolol) on these parameters was studied and effect of agonists like thrombin, phorbolsters or calcium ionophores (A 23187) on platelet cell biology and stimulus-response coupling system was investigated including assay of activity of protein kinase C, measurements of intracellular Ca and phosphoinositide turnover. 14 C serotonin uptake and release studies were done to assess level of platelet activity. Platelet aggregation and secretion (of 14C-serotonin) was found to be hightened in acute myocardial infarction. Propranolol suppressed both these responses. Propranolol enhanced anilinonaphthalene sulfonate(ANS) binding to platelet membrane. Scatchard analysis showed an increase in number of bind- ing sites for ANS in propranolol treated platelets.From steady-state fluore- scence polarisation studies using, diphenyl hexatriene(DPH) propranolol was found to decrease the membrane microviscosity in a dose-dependent manner, with a simultaneous fall in lipid order parameter and rise in fusion activation energy for viscosity. On the contrary, platelets from acute myocardial infarction were found to have decreased membrane fluidity, as well as fusion activation energy. Preliminary results showed that propranolol depressed protein kinase C activity in thrombin stimulated platelets but not in platelets stimulated with phorbol esters. Propranolol was, however, found to inhibit activities of phospholipase C and calpain. The results of the study indicated that patients from acute myocardial infarction have altered platelet membrane microviscosity. Propranolol, a drug widely used in cardiac disorders for beta-blocking property, has direct perturbing effect on platelet membrane bilayer, and possibly, on platelet transmembrane sigma transduction.



EVALUATION OF POSSIBLE GENOTOXIC POTENTIALS OF PAN MASALA  

In recent years, pan masalas have emerged in Indian market as safer alternatives to tobacco chewing. The list of ingredients makes a very strong case against the product and to confirm possible risk associated with pan masala consumption, a detailed project encompassing in vitro animal and human studies was initiated. In vitro experiments included three different parameters and experiments with aqueous and DMSO extracts in presence and absence of the S-9 activation system. The results on frequency of chromosome aberration (CA), sister chromatid exchanges (SCEs) and micronucleated cells (MNC) very clearly showed that treatment of CHO cells with aqueous as well as DMSO extracts of pan masala `plain' variety (PM) and one with tobacco (PM-T), resulted in statistically significant elevation in the levels of all three parameters. Frequency of CA in bone-marrow of mice fed with PM/PM-T at 3 dose levels for 1 to 12 weeks showed insignificant elevation at lower doses and significant elevation at higher doses. Most convincing results were obtained with human studies. With a two issue: three parameter protocol, all three parameters i.e. CA and SCE in blood lymphocytes and MNC in exfoliated buccal mucosa, provided a very strong evidence for genotoxic effects of pan masala consumption.



MOLECULAR APPROACHES TO CARDIAC HYPERTROPHY IN HUMAN HEART  

Study was aimed to understand the molecular mechanisms underlying cardiac hypertrophy and myocardial failure in human hearts to identify the molecular signals which are responsible for the onset and maintenance of cardiac hypertrophy and to correlate the presence, if any, of such molecular factors (hypertrophic factors) with the increase in various cellular events associated with hypertrophic growth. The expression of genes which are specially triggered during early stages of hypertrophy both in lower mammals as well as in human beings has been studied. Enhanced expression of proto oncogenes such as c-fos, c-myc, c-ras and heat shock protein gene HSP70 occur in all human heart tissue samples obtained from patients with ventral septal defect (VSD) or Tetralogy of fallot and artrial septal defect (ASD). This may probably explain the activation of cellular events in myocardial cells during hypertrophy. Also, changes in the expression of myofibrillar protein gene such as MLC2, in the tissue of various cardiac disorders have been presented. The appearance of a high molecular weight protein in the sera of patients with various cardiac anomalies has raised the possibility of identifying the early occurrence of cardiac disorder in human beings. The preliminary studies involving the antibody raised against this cardiac hypertrophic specific protein for the inhibition of hypertrophy development in rats has opened new avenues in the diagnosis of hypertrophy development.



HEAVY METALS-MEDIATED MODULATION IN INSULIN BIOSYNTHESIS AND RELEASE  

The study was carried out in wistar strain male albino rats to identify the metals which are known to produce either reversible or irreversible impairment in the release of insulin form islets of langerhan and establish a correlation between serum/panereas- metal concentration and the extent of impourment in the insulin secretion from islets. Effeorts were also made to study metals mediated alteration in the transdueion of extr4acellular signals responsible insulin release and investigate the mechanism by which metalions impair the bioregulation ann seeretion of insulin. The administration (intraperitoneal; ip) of nickel chloride (NiCl2: 75 Mue mole/ kg), to rats caused dose dependent rapid and transient rise in blood glucose post administration. The increase in the blood glucose levels levels was accompanied with hyperglucogonemia and marginal increase in insulin leading to a sharp decrease in the insulin-glucagon ratio. The NiCl2 administration also caused diminished glucose tolerance in experimental animials. The response of glucose load in Ni-treated rats was maximum at 0.5 hr. and remained elevated at all subsequent time intervals compared to cotrols receiving either Ni or glucose. The nickel administration also cused significant increase in pancreatic lipid peroxidation at all time intervals with concomitantdecrease in GSH and Zn levels. Significant positive correlation was observed between lipid peroxidation and blood glucose levels. Significant but decreasing correlation was also observed between lipid peroxidation and GSH, lipid peroxidation and Zn and blood glucose and GSH levels. Enhancement in lipid peroxidation, along with the impairment in not-enzymatic defence status including levels of GSH and Zn could be related to Ni-induced oxidative stress. Similar to the elevated patterns of blood glucose and lipid peroxidation, a significant increase in the activity of constitutive nitric oxide synthase (cNOS) in adrenals and brain tissue were observed after Ni administraiton. Contrary to the brain and adrenals, the activity of cNOS was reduced almost to three fold in pancreas. The decrease in cNOS activity in pancrease was accompanied with the significant increase inthe inducible form of the NOS (iNOS) activity. The interplay of NOS in Ni-induced hyperglycemia was further evident by the observation that pretreatment with NG- monomethyl arginine, an inhibitor of NOS, significantly reversed the Ni-enhanced blood glucose levels. To further confirm the role of NO in Ni-induced hyperglycemia, administration of L-arginine, a precursor of No formation, prolonged the Ni-enhanced blood glucose level compared to Ni-treated animals. The role of cNOS in insulin re3lease is well documented, however, the cytotoxic action of iNOS pancreas could thus be playing an importment role in modulating hormone release. These results collectively provide for the probable role of ROS and NO, a key reactive molecule in Ni-induced hyperglycemia.



TO RAISE MONOCLONAL ANTIBODIESAGAINST POLY ADP-RIBOSE AND TOUSE THEM FOR IMMUNOHISTOCHEMICAL STUDIES OF POLY ADP-RIBOSYLATION IN TUMOUR TISSUES  

To raise monoclonal anntibodies against poly ADP-ribose and to use them for immunohisto chemical studies of poly ADP ribosylation in tumour tissues: The study was carried out for in vitro synthesis of the polymer, poly ADP- ribose lench by immunofluorescence analysis in patients of squamous cell tumours and explore the possible role of poly ADP- ribosylation in cancer diognostics with the main objective of understanding the importance of poly ADP-ribosylation in cancer diagnostics and early detection and control of cancer. A polymer of poly ADP-ribose with a chain length of 7 resideres was synthezed with the help of poly ADP-ribose transferase extracted from calf liver. With a method using MBSA two monoclonal antibodies could be produced against this polymer by immunizing Balb C mice with antigen and fusing with SP2/O Ag-14 mouse myeloma cells. The antibodies did not cross react with related compounds, such as DNA, RNA and synthetic nucleic acid hompolymers and ADP-ribose mononer. However they recognized shorted chain as well as long chins polymers of ADP-ribose for the antigen- antibody reaction (3 to 25 residues of ADP-ribose). This antibody when used to measure the poly-ADP-ribose with 3-30 residues of ADP-ribose in squamous cell tumours, clearly demonstrated the presence of poly ADP-ribose in tumors by immunofluorescence. The intnesity of the fluorescence was related to the degree of malignancy.



ACTION OF CIS.PLATINUM ON UNICELLULAR ORGANISMS  

Action of cisplatinum on some unicellular organisms. A study was conducted to investigate the mode of action of cisplatinum on unicellular ciliate species on order to understand the action of this drug on eukaryotic cells and some aspects of its metabolism. Several different species of genus Stylonchia collected from wild habitat were treated with anticancer drug, cisplatinum in the dose of 150 mue g/ml for 2 h. Studies on macronucleus and micronucleus were carried out to study the mechanism of action of the drug. Different species exhibited variable sensitivity towards the antitumour drug cisplatinum in post treatment resistant cells showed that higher resistant was not due to poor mental uptake; these cells showed comparatively higher uptake; Likewise, the germ line nuclei bound at least 3 times more of platinum when cells were treated in situ. The DNA lesions in the micronucleus caused by extremely low platinum dosages were deterimental to the completion of meiosis and formation of a new functional somatic macronucleus. DNA characterization of platinum damaged micronuclei showed unusual fragmentation to low mol. wt. components. Such damage was irreversible leading to the loss of micronuclei and production of amicronucleate strains. it appeared that micronuclear components like non histone chromosomal proteins facilitate higher platinum binding leading to fragmentation of high mol.wt. micronuclear DNA.



ONCOGENES, TRANSCRIPTION FACTORS AND EXTRACELLULAR FACTORS : A RELATIONSHIP WITH CARDIAC HYPERTROPHY  

Oncogene transcription factors and extracellular factors. The study was carriedout to determine the role of proto-oncogenes, transcription factors and extracellur factors in the development of cardiac hypertrophy in rats. Efforts were also made to study the mechanism of action of these factors and a protein kinate c (PKC) mediated signal transduction pathway in the induction of cardiac hypertrophy. The 182 KDa serum protein appearing at an earlier stage of hypertrophy was found to be responsible for the induction of protooneogenes and muscle specific genes. The immunopreeipitation studies of nuclear extract indicated that 182 KDa serum protein is synthesized and transloeated into the nucleus within 15 min afeter aortic constrictin. The signal transduction to be muscle specific. The very high level of 182 KDa protein on Ist and 3rd days of neonatal rats also suggested to its hypertrophy specific nature. Activation of PKC within 10 min in 182 KDa protein injected animals along with the activation of proto-oncogenes and transcription factors indicated that the signal transcription pathways provoked by the 182 KDa serum protein could be via PKC mediated pathway. These fidings suggest that the 182 KDa protein synthesized immedicately after the imposition of pressure overload could be the molecular signal for the activation of various events like induction of protooncogenes, transcription factors and muscle specific genes associated with the development of cardiac hypertrophy. The transcription stimulating factor of 45 KDa appeared 3days after the onset of hypertrophy at the time when 182 KDa protein occurs at fairly sufficient levels. This 45 KDa protein probably take over to maintain specific genes resulting in increased muscle protein synthesis to maintain the hypertrophic state.



STUDIES ON GENE REGULATION ANDEXPRESSION OF TRANSPORT AND BRUSH BORDER ENZYME PROTEIN INANIMALS INDUCED FOR MALABSORPTION  

Gene regulation and expressin of brush border enzymes and transport proteins in animals induced for malabsorption. This study aimed at investigaing the underlying mechanism of malabsorption in male Wistar strain albino rats (80-100g) exposed to chronic alcohol ingestion and giardiasis. The results indicated that Giardia lamblia infection in rats induced malabsorption of glucose and decreased the activities of the brush border disaccharidases and alkaline phosphatases which was primarily due to phosphatase, sucrase and lactase in rat intestine. there across the villus axis in response to giardia infection. chronic ethanol feeding also induced solute malabsorption, which was presumably due to morphological alterations in the intestine. G. lamblia induced idental, changes in mRNA levels of the brush border enzymes and also sodium dependent D-glucose levels in both control and ethanol fed rat intestine.



DNA POLYMERASES IN DEVELOPING AND AGING RAT BRAIN : LEVELS AND MECHANISM OF MODULATION OFENZYME ACTIVITIES  

The activity levels of various DNA plymerases in isolated neuronal and astroglial cells from brain of young (4 days), adult (6 months) and old (>2 years) rats, were assessed using the stragegy based on their differential sensitivities towards various inhibitors such as BuAdATP, BuPhdGTP and dideoxy TTP using activated calf thymus DNA and poly (dA).oligo (Dt)12-18 as template primers. From these results it may be broadly concluded that at all stages studied and in both types of brain cells beta polymerase appears to be the predominant one although other DNA polymerases also seem to be present in detectable amounts. It is known that PCNA activates pol delta but not pol . Western Blot analysis of whole brain bomogenates for assessing the PCNA activity at different ages did not show any difference in the levels with age. Further work could not be continued due non availability of purified PCNA. Since beta polymerase appears to be the predominant polymerase present in adult and aging brain cells, further experiments were designed to examine how this polymerase activity is modulated at different ages, especially its ability to remove a specific 3 end mismatch from a synthetic oligodeoxynucleotide duplex and extend the primer strand to the predicted length. It was observed that on the addition of purified recombinant rat liver B polymerase to the neuronal extracts, the extension activity was restored and the age dependent decrease in activity was not seen. This age dependent primer elogation activity was also studied with unlabelled oligo duplexes incubated under standard conditions with one of the dNTPs radioactive. This study suggests that probably beta polymerase activity is the limiting factor in DNA repair during aging.



MECHANISM OF TUMOUR SUPPRESSION BY NICOTINAMIDE  

Nicotinamide (NA) a naturally occurring vitamin and a protease inhibitor, has been reported to be affective against skin ailments. NA also suppresses the tumor development and cell growth in different model systems in vivo/ in vitro. But NA has limited use as its mode of action is not well documented. A knowledge of the stages of neoplastic development can be exploited to predict the effective approaches to cancer therapy. In order to make the better use of NA as tumor suppressing agent we tried to elucidate the mechanism of action and involvement of stages of tumor development. Topical application of NA suppressed TPA induced mouse skin tumor promotion in two stages tumurigenesis protocol. NA modified TPA altered ODC activity, DNA synthesis, TG activity, endonoclease activity in mouse epidemis. These are the marker events suggests that probably these stages of tumor development are affected by NA. Since NA modified all the critical stages of tumor development altered by tumorigenesis, it becomes a candidate of choice for detailed study of cancer control.



IMMUNOPROPHYLACTIC PROPERTIES OF 30kDa SECRETORY PROTEIN OF M.TUBERCULOSIS H37Ra IN DIFFERENT ADJUVANT SYSTEMS ANDIMMUNOPHENOTYPING DURING IMMUNIZATION/INFECTION  

Immunoprophylacic properties of 30kDa secretory protein of M.tuberculosois H37Ra in different adjuvant systems and Immunophenotyping during immunization/ infection (No. 53/2/98-BMS). Six formulations viz. poly lactide-co-glycolide (PGL) microspheres, demethyldioctadecyl ammonium bromide (DDA), liposomes, liposomes containing monphosphoryl lipid A and coated with alum (L-LIPA-AL) or without alum (L-LIPA) were evaluated to promote cell mediated immune responses towards 30 kDa secretory protein of mycobacterium tuberculosis H37Ra in comparison to standard Freund's incomplete adjuvant (FIA). Two adjuvant fromulations of 30kDa L-LIpa-AL and 30kDa-PGL showed maximum reactivity in terms of lymphoproliferation w.rt. other adjuvant fromulations and also exhibited a Th1 shift. Flowcytometry analysis of spleen of immunized animals revealed the capacity of these two adjuvant formulation to activate T cells subjects like CD4 and CD8 T cells. The upregulation of B7 costimulaytory molecules (B7 & B7-2) after immunization further proved the ability of the two vaccin3e formulations to activate antigen presenting cells. The imunostimulatory nature of these two vaccine formulations was also reflected in their capacity to reduce the bacilli load from the lungs of the experimentally infected mice. This study demonstrates PLG and L-LIPA-AL as potent adjuvants for future subunit vaccine development against tuberculosis.




CHOLERA AND ENTERIC DISEASES
CENTRE FOR ADVANCED RESEARCH ON AGING AND BRAIN (CRAB)  

Various DNA repair pathways relevant to brain tissue were examined during development and aging. It was found that Base Excision Repair (BER), a mode or repair to take care of minor base changes, mismatches and small gaps in DNA, is drastically reduced in aging brain cells. During the period of this centre , the locus of defect in these pathways in aging nerve cell has been identified. Thus, supplementation of the neuronal extracts from aging brain with DA- Polymerase beta, DNA ligase along with thymine glycosylase and Apurinic/ apyrimidinic endonuclease in some cases, restored in BER acitvity in vitro. Further, aging neurons were found to show enhanced pol beta activity and BER acitvity following electroporation of pure pol beta. These observations are considered to be of far reaching consequences. However, in the case of Non-Homologous End Joining (NHEJ) mode of repair in brain cells was found to be highly error prone and very feeble in joining blunt and mismatched end while ends while cohesive ends are joined with reasonable efficiency which has decreased with age. Attempts to find the locus of defect in this case have been unsuccessful so far. In a separate study, the presence and the role of two newly discovered DNA Polmerases, the pol lemda, pol mue and Topisomerase II and III in developing and aging brain was studied. Both pol lemda and mue are present in different regions of brain and in isolated neuronal cells. No marked changes in levels of these enzymes were found with age while pol lemda in isolated neurons could be found only in young. DNA topoisomerase were analyzed during development and ageing. Topo I, II and III show a decrease during ageing. Topoisomerase II beta shows significant expression throughtout the developmental stage, whereas Topo II alpha levels decreased in post natal rat pups. The analsis of expression of Topo II alpha while neurons expressing Topo II beta. This data suggest that Topo II alpha playing most significant role in proliferating cells, but Topo I beta may be involved in non-proliferative functions of the cells. One of such function, DNA damage and repair activity is measured in siRNA mediated Topo II alpha and beta knockdown cells, the relsuts showed that Topo II beta along with polymerase beta, Ku70. WRN protein plays important role in peroxide induced Double strand breaks repair. Similarly Topo II beta along with polymerase beta were involved in repair of ENU mediated DNA damage. These studies implicate important role of Topoisomerase II beta and polymerase beta in corrobaration in neurons. hence these enzymes may participate in various DNA repair processess, the decrease of both Topo II beta and polymerase beta ageing may contribute to observed deficiency of DNA repair activity of neurons in ageing.



CRYPTOSPORIDIOSIS IN DIARRHOEAOF INFANTS AND CHILDREN  

In the initial part of the study not a single cryptosporium was isolated from 375 stool samples of children. Thus, the original aims were modified to include study of other organism cuasing diarrhoea. A total of 1260 stool samples of children were examined for parasitic, bacterial and viral pathogens with special reference to `Cryptosporidium'. It was observed that in the 200 children admitted to the hospital for diarrohea, none showed evidence of cryptosporidiosis. In the 1060 samples from field areas, 12 (1.13%) were positive for `Cryptosporidium'. Cryptosporidium lead to both acute as well as chronic (persistent) diarrhoea and the clinical features consisted of diarrhoea (91%), abdominal pain (50%), anorexia/vomiting (50%) and fever (25%). The prevalence of `Rota virus' was 15.5% in the hospital group while it was lower (9.2%) in the field area group. Serogrouping revealed that group A subgroup I was the only subgroup present in this area. The incidence of 'Shegella sp.' and 'S. typhimurium' was similar in both the groups of children. 'Each.coli' was the most common bacterial isolate in both the groups. Serotyping of strains from the hospitalized children revealed that EPEC was the commonest group (25.5%). Among the other pathogenic enteric parasites, 'Giardia lamblia' was much more common in the children from rural areas (15.1%), compared to hospital group (3%). The prevalence of 'Entamoeba histolytica' was similar in both the groups (3.4% and 2% respectively).



INVESTIGATION ON THE ISOENZYMEPATTERN OF ENTAMOEBA HISTOLYTICA (ZYMODEMES) IN SYMPTOMATIC AND ASYMPTOMATIC CASES OF AMOEBIASIS  

Strains of E. histolytica were isolated from the patients of amoebiasis under two categories viz Acute ameobic dysentery (AAD) and non-dysenteric amoebic colitis (NDAC). The stool samples positive for E. histolytica were inoculated into Boek and Drbohlav medium for xenic cultures. They were regularly subcultured till profuse growth of each isolate was obtained. From each category the amoebae were picked up, 8 from each isolates. The remaining xenic culture was used for pathogenicity in animals (rats). Ther weanling NIN-Wister strains were taken for the assessment of pathogencity for both xenic cultures (Parent) and clone-cultures. The cloning was done according to method of Srivastvaet. al. (1990). A success rate of 80% 60% and 83% were obtained for clonal-cultures obtained from acute amoebic dysettery (AAD), Non-abscess respectively. The clone cultures along with parent-cultures were tested for pathogenicity in rat caeca. The degree of ulceration was scored according to Neal (1956) scoring method. The smear of feacal speciment was prepared for the positive presence of amoebae. It was observed that there is difference in the degree of ulceration caused by parent-cultures (xenic) and clones derived from them. The zymodema pattern of clone cultures was done by PAGE electrophore- sis based on the isoenzyme pattern of four enzymes viz. phosphoglucomutase (PGM), Hexokinase (HK) Maleic enzyme (ME) and glucose phosphate-isomerase (GPI). The absence of alfa-band and presence of beta-band in PGM along with advance band in HK was taken as marker for pathogenicity. All the 16 clones derived from xenic- cultures were characterized and it was observed that zymodema II is pathohgenic zymodema found in this part of the country. This report is in contrast on other report from North-East India which showed that symodema XIV was the pathognic zymodema for Indian population. The studies were based on zymodema analysis of xenic-cultures obtained from North-East part of the country.



CHARACTERISATION AND IMMUNOREACTIVITY OF MAJOR CYSTICERCOSIS AND ADULT T.SOLIUM WORM ANTIGENS  

Crude soluble extract (CSE) antigen from Cysticercous celulosae was eva- luated by the enzyme linked immunosorbent assay (ELISA) for the detection of antibodies in serum and cerobrospinal fluid (CSE) samples from patients suspected clinically of neurocysticercosis (NC), neurological disorders other than NC and controls. The results were compared with indirect heamagglutination (IHA) test and matched with retrospective analysis of proven diagnosis of these patients. ELISA and IHA were found to be positive repspectively in 88 and 84 percent of CSF and 92 and 87.2 percent of serum samples from proven NC patients. However, CSE antigen was also found to be reactive in a very high precentage of serum samples from patients other than NC by both ELISA and IHA tests. With CSF samples, IHA test was found to be absolutely specific while by ELISA test CSF antigen was reactive in CSF samples from 5 non-cysticercosis patients, one each suffering from disappearing CT scan lesion, tubercular meningitis (culture negative), chronic meningitis, bening intracranial hypertension and non compressive myelopathy. HOwever, possibility of neurocysticercosis cannot be absolutely ruled out in such patients. Crude soluble extract antigan gave 23 peaks when reacted with homologo- us hyperimmune serum raised in rabbits, by two dimensional immunoelectropho- resis and multiple (25-30) bands with molecular weight ranging from 8 to 2 kDa were observed by sodium dodecyl polyacrylamide gel electrophoresis. The CSE antigen was purified by sephadex G-200 column chromatography and major purified fraction was subjected to western blot analysis using positi- ve serum pool from five surgically confirmed NC patients and-negative serum pool from five normal healthy control subjects. Antigenic fractions with molecular weight of 18, 20, 24 and 65 kDa were found to be reactive with positive serum pool and non-reactive with control serum pool.



CLINICO-EPIDEMIOLOGICAL STUDIES IN CEREBRAL CYSTICERCOSIS WITH SPECIAL REFERENCE TO IMMUNODIAGNOSIS AND EVALUATION OF TREATMENT  

The objectives were to study the epidemiological aspects of neuro- cysticercosis and clinical manifestations of the disease in Indian subjects, to evaluate the various modes of treatment viz. praziquantel, palliative (symptomatic) and surgical therapy and to study the dynamics of the immune response in the treatment and prognosis of neurocysticercosis. Within a period of five months from July to November 1989, 15 patients of neurocysticercosis were enrolled in the study. Patients were hospitalised and clinical and CT investigations performed. All cases had repeated stool examination for the presence of ova of Taenia solium. Out of 15 patients, 5 were vegetarians. Of the 10 non-vegetarians, three had never eaten pork, did not eat beef and two had neither eaten pork or beef Four out of 15 patients had close contact with domestic animals, 3 with dogs and one with chickens. None of the patients had direct contact with pigs or cattle. All patients were negative for the presence of ova of T. solium in the faeces inspite of repeated examination. Seizures were the most common presenting feature of which generalised seizures were more frequent than focal or mixed type. Signs and symptoms of raised intracranial tension associated with visual disturbances were also frequently observed. The lesions seen on C.T. scan of the brain were of the following types: (1) Multiple ring like lesions with enhancement on contrast C.T., (2) Multi- ple calcified rice like lesions situated in the brain parenchyma, (3) Iso- dense lesions with extensive white matter oedema and chinked lateral ventricles, (4) Intraventricular enhanced cystic lesions, (5) Granuloma. Specific anticysticercus antibody in serum and CSF was estimated by the ELISA test. Of a total of 12 patients who were tested for the presence of antibodies, a significant antibody response was seen in the serum and CSF in 7 cases each. Symptomatic patients with type (1) and (3) lesion on C.T. scan were given praziquantel 50 mg/kg in three divided doses for 15 days. Decadron 2-3 mg twice a day was also given along with praziquantel. Symptomatic patients with type (2) C.T. lesions and negative immunological response were treated with anticonvulsants alone. Surgical therapy, i.e. removal of cyst was reserved for patients with type (4) lesion on C.T. Evaluation of treatment was based on clinical criteria, CT picture and immunological response. Follow up was inadequate to arrive at definite conclusions. Some preliminary observations which need further validation are as under. The disease was seen in vegetarians and Muslims, thus substantiating the observations by other Indian workers and contrary to well documented studies from other parts of the world who found the condition to be related to eat- ing of undercooked infested pork. Granulomas were seen on C.T. in one case, this has not been reported earlier in Indian patients. Only 60% of cases showed a significant antibody response, one of two cases that worsened after praziquantel and one case that remained the same did not show adequate antibodies either before or after treatment. The possibility of anergy in these cases warrants a study of their immune status. Three cases out of 10 treated with praziquantel were refractory to treat- ment and even worsened. It is not clear whether the patients had an exacer- bation of the disease per se or developed hypersensitivity to praziquantel. Six cases out of 10 treated with praziquantel continued to require anti- convulsants. No relationship was observed between the clinical response and changes in C.T. morphology after praziquantel therapy.



COMPARATIVE STUDY OF VIRULENCEPROPERTIES OF ENVIRONMENTAL AND DIARRHOEAL ISOLATES OF AEROMONAS SPP.WITH SPECIAL REFERENCE TO THEIR ENTEROPATHOGENICITY  

A total of 147 strains of 'aeromonas' of which 54 were isolated from cases of acute diarrhoea and 93 from environmental sources (44 were from water and 49 from fishes) were speciated to 'A.hydrophila' A.sobria' and 'A.caviae' on the basis of only 7 simple biochemical markers, the remaining being common for all of them. 'A.caviae' and 'A.sobria' were predominant in environmental and clinical sources respectively, while the frequency of 'A.hydrophila' was sparce in both the sources, while 56% of the strains produced enterotoxin prior to passage, while the remaining 44% (approx) did so only after 1-3 passages through rabbit gut. The environmental isolates of 'A.hydrophila' and 'A.sobria' produced significantly more enterotoxin (P<0.005) than their clinical counterparts, however, the 'A.caviae' strains did not show such sourcewise difference. Further, 'A.hydrophila' and 'A.sobria' produced significantly more enterotoxin than 'A.caviae'. Nearly 73% of strains produced beta-haemolysin irrespective of their species in the initial experiments, while the remaining did so only after passage. 'A.hydrophila' and 'A.sobria' elaborated relatively more (16-128 HU/ml) haemolysin than 'A.caviae'. Only a small percentage of 'A.sobria' and 'A.caviae' produced alpha-haemolysin. The 31 non- haemolytic strains were distributed into the three species, majority being 'A.caviae'. No correlation could be observed between production of haemolysin type and sources of isolation of the strain. nearly 67% of the beta and 31% of alpha/non- haemolytic strains produced enterotoxin prior to passage, but on passage all of them switched over to production of beta- haemolysin and enterotoxin. Forty four of 97 strains of 3- species of 'aeromonas' caused haemagglutination independent of source of origin. Proportionately more 'A.hydrophila' showed HA than 'A.sobria' and 'A.caviae', whereas equal proportion of clinical and environmental isolates of 3 species showed variety of HA pattern indicate that this property may not be used to assign a isolate to a particular source. Majority strains, mostly showing MS, MFS and MFGS-HA patterns caused accumulation of fluid independent of origin in the initial test. The nontoxic strains, mostly NHA or showed MGS-HA or other patterns because enterotoxin producers after 1-3 consecutive passages through RIL. Twelve strains of 'Aeromonas', 4 each of 'A.hydrophila', 'A.sobria' and 'A.caviae', when tested for adherence to rabbit intestinal mucosa, adhered to independent of species designation or source of isolation. Inhibition of HA or adherence by different sugars indicate that haemagglutinin and adhesin are probably the same molecule. The data thus indicate that all strains of aeromonas whether environmental or clinical in origin are potentially enteropathogenic irrespective of their species. There exists some correlation between HA and enterotoxicity and HA and adhesin.




CONTRACEPTION
ELECTROLYTE REFLUXES IN THE INTESTINAL MUCOSA OF VITAMIN ADEFICIENT RATS AND MICE IN RESPONSE TO BACTERIAL TOXINS  

Electrolyte refluxes in the intestinal mucosa of Vitamin A deficient rats and mice in response of bacterial toxins. The study was carried to determine the Na+, Cl, Ca++ and 3-O- methyl D- glucose fluxes and the key, second merrengers controlling these fluxes in vitamin deficient in male albino mice and Wistar strain male albino rats exposed to different bacterial toxins and to see the reversal of changes, if any after vitamin A supplemenlation. Vitamin A deficiency was produced in animals by feeding them vitamin A deficient diet. This was reflected by cessation in weight gain and confirmed biochemically by reduction in lever and plasma Vitamin A levels compared to controls.There was a significant increase in malondialdehyde levels also the levels of antioxidant enzyme superoxide dismutare in Vitamin A deficient animals thore by making the animals more prone to infection and disease. Both heat labile and heat stable enterotoxins of Escherichia coli decreased the Na+, Cl- and D- glouse absorption and increased Na+ and Cl- secretion in small intestine whereas there was a significant increase in Ca2+ absorption in ST toxin treated animals compared to control. The study related to toxin treatment in vitamin A deficient animals could not be carried out. However, the findings of the study clearly demonstrated that the mucosal barrier of the gastrointestinal tract can be severly impaired by vitamin A deficiency making it more likely to be continously exposed to Gijher level of environmented challange




ENDOCRINOLOGY
DNA BASE MODIFICATION IN HORMONE ACTION  

Control of gene expression in higher organisms is related to the methylation of cytosine in DNA. The pattern of methylation is inherited and is under the control of hormones in hormone dependent tissues. Study has been initiated with the investigate the pattern of methylation and demethylation in steriod hormone dependent and gonadotropic hormone dependent tissues. Such a study would give us information on mechanism of action of hormones at gene level. It is proposed to use two model systems for these studies. One of the systems would be to use sterioid hormone dependent tissue, ventral prostate and uterus. The pattern of methyl transferase and methyl cytosine groups in the DNA following hormonal deprivation and in response to steroid hormones would be studied. In the second system the pattern of methylation of DNA in gonadotropin hormone dependent tissue liketestis will be studied addition to the studied on the regulation of the enzyme DNA-methyl transferase in the gonadotropic hormone dependent tissues. Studies will also be undertaken on the pattern of DNA-methylation in specific ornithine decarboxylase gene of ventral prostate in relation to the levels of hormones.




ENVIRONMENTAL HYGIENE AND OCCUPATIONAL HEALTH
PLASMA ANGIOTENSION II LEVELS IN NORMAL SUBJECTS AND PATIENTS WITH RENOVASCULAR ANDCHRONIC RENAL PARENCHYMAL HYPERTENSION  

Measurement of peripheral plasma renin activity and renal vein renin activity has been used to diagnose the potentially curable cases of renovascular hypertension. Plasma renin activity (PRA) is an indirect measure of renin-angiotensin system (RAS) since Ang-II is the main vasoactive harmone responsible for hypertension. Recent evidences have indicated the existence of two types of renins, active and inactive in the plasma the former corresponding to Ang-II. Study was therefore conducted for establishing and standardising the radioimmunoassay and bioassay of Ang-II and evaluating its diagnostic and prognostic value in comparison to plasma renin activity in patients of renovascular and renal parenchymal hypertension. Peripheral Ang-ii levels were estimated in normal subjects and the effect of age , sex, posture and dietary salt was evaluated as also in renal venous blood in patients of renovascular and chronic renal parenchymal hypertension was estimated in some of the samples to correlate the values with Ang-II levels. Significant decrease was observed in PRA and Ang-II levels at higher age in normal males and females. Low salt diet led to significant increase in plasma angiotensin-II accompanied by directionally similar changes in plasma renin activity in normal valunteers. The mean peripheral values for Ang-II were found to be higher in patients of renal parenchymal hypertension. The predective value of renal peripheral vein Ang-II levels for favourable surgical outcome could be evaluated only in patients of renovascular hypertension. Lastly the value obtained by the procedure developed in the laboratory and those obtained by using Ang.II test kit (Buhlmann lab. Switzerland) could not be compared as the kit did not work because of the delay in transit and expiry of the radiolabelled AngII the results of the study reveal that Ang-II and /or PRA measurements are required as supportive tests for the diagnosis of renovascular and renal hypertension. Ang-II can be measured by bioassay as well as by RIA with almost equal sensitivity and specificity however bioassay appeared to be more cost-effective.



EFFECT OF LEAD ON THE DEVELOPMENT PROCESSES OF IRON DEFICIENT RATS  

The study was carried out on Wister strain albino rats (40 + or - 5g body wt) to investigate the effect of lead exposure (through different routes) on foetal development at various gestational stages in iron defi- cient rats so as to provide information on the fetotoxic and embryotoxic effects of lead exposure. Pregestational lead exposure (1g/1 as lead acetate/nitrate) for 30-60 days through drinking water was found to delay the onset of estrus cycle, more so, in iron-deficient rats. Their mating with normal males resulted in reduced gestational weight gain and enhanced incidence of resorptions probably due to the higher lead uptake in maternal blood and feto-placental unit. Iron deficient diet to rats also affected the blastular hatching in vitro from zona pellucida when lead (10 mu mol/1) was added to the incuba- ting medium. However, iron deficient diet did not seem to affect the impla- ntation sites. Lead intoxication in iron deficient rats also affected organogenesis (6-14 days of gestation) and fetal development (15-20 days gestation). After exposure to lower doses of lead (25 and 50 mg/kg) a dose- dependent feto-placental transfer of lead was seen, it did not produce significant embryo and fetotoxic effects. However higher doses of 100 and 200 mg/kg produced significant dose-dependent impaired gestational development viz increased number of resorptions and reduced litter-size, apart from soft tissue and skeletal deformities. such deformities were more pronounced in iron-dificient lead nitrate treated groups. Intravenous lead administration (single dose; 25mg/kg) on days 9 and 15 of gestation resulted in reduced litter size crown -rump length and fetal weight higher resorptions and abnormalities like hydronephrosis, haemorr- hagic coelomic cavity megacephaly etc in iron deficient groups as compared to those fed normal diet. The 50 mg/kg lead treatment (intravenously) on days 9 and 15 of gestation resulted in higher embryonic deaths (compara- tively more in iron deficent rats). The study thus revealed a dose-dependent feto-placental lead uptake more so in iron-deficient rats reflecting possibility of higher embryo and fetotoxic effects in iron deficiency.



CHEMICAL & BIOLOGICAL EFFECTS OF OXYGEN RADICALS DUE TO EXP-OSURE TO BENZENE & ANTIOXIDANTPOTENTIALS IN LIVER & SERUM DURING OXIDATIVE STRESS*  

The efficiency of hydroquinone (HQ) and 1,2,4-benzene-triol (BT) iron chelates to act as prooxidants was studied in Wister strain albino rats. Evaluation was done by studying (i) the potential of HQ and BT to release iron from ferritin and whether the iron released could catalyze oxidative reaction; (ii) efficiency of HQ and BT in catalysing bleomycin-dependent DNA degradation, glutathione degradation and lipid peroxidation in vitro; and (iii) the antioxi- dant potentials in serum and liver of rats exposed to benzene. Administration of benzene consecutively for 10 days to rats of either sex resulted in decrease in antioxidant potentials in serum. Serum uric acid and albumin showed significant decrease in all groups exposed to benzene. Alpha-tocopherol in serum and liver did not exhibit significant changecompared to control. Exposure to benzene also led to incrase in lipid peroxi- dation and decrease in free sulph-hydryl groups. Serum ferroxidase activity, total iron content (TIC) and total iron binding capacity (TIBC) measured in female rats exposed to benzene showed significant decrease in ferroxidase activity without any change in TIC or TIBC. The decrease in antioxidation potentials observed may be due to the oxidation reaction exhibited by the benzene metabolites, particularly HQ and BT resulting in oxidative stress in benzene treated rats. Lower concentrations of BT resulted in the release of iron from ferritin which was able to induce lipid peroxidation and catalyze bleomycin-dependent DNA degradation. The presence of oxyradical scavengers ie albumin, catalase or superoxide dismutase significantly inhibited iron release from ferritin by BT. It was also found that iron complexes with HQ and BT catalyzed bleomycin-dependent degradation of DNA. Iron catalyzed bleomycin-dependent degradation of DNA was enhanced 12-fold in the presence of glutathionyl hydroquinone (GHQ) and three-fold in the presence of glutathione (GSH). The degradation of DNA was linear with the increase in concentrations of GHQ or GSH, other factors being constant. The presence of oxyradical scavengers viz thiourea, mannitol, albumin, superoxide dismutase, catalase and dimethyl sulphoxide caused significant inhibition of degradation by GHQ and iron. It is concluded that the poolyphenolic metaabolites of benzene enhance the availability of FE(II) to bleomycin. Such FE (II) was possibly made available due to reduction of FE (III) by polyphenols thus protecting the FE (II) from hydrolysis to hydroxides and further oxidation. It is also concluded that GHQ is a potent prooxidant, capable of enhancing iron dependent formation of super- oxide radical and also due to its faster autooxidation to glutathionyl benzosemiquinone may facilitate formation of adducts with DNA which is one of the prerequisites of benzene toxicity and carcinogenesis.




EXPERIMENTAL MEDICINE AND SURGERY
ERGONOMIC EVALUATION OF DIF- FERENT NODES OF LOAD CARRY ANDMAXIMUM PERMISSIBLE LOAD FOR INDIAN WOMEN ENGAGED IN MANUALMATERIAL HANDLING  

Ergonomic evaluation of different modes of load carrying maximum permissible load for Indian Women engaged in manual material handling. The study was carried out to document the existing modes of manual load carrying by Indian women and determine the phsiological cost involved during the process. The basic approaches (field and laboratory studies) were employed for the study. Several paramenters like personal and medical history. sites of pain the body, etc. were acertained through a questionnaire. Anthropometry, physiological cost of the work, work rest cycle, walking speed, environmental heat load and energy consumption were determined by direct measurements on the subjects in the field. Women involved in construction activities carried the load on the head shoulder. waist and hand mode were observed. Besides these, women had adopted several other modes for carrying water like head and hand. head and waist. waist and hand etc. Ninety three women engaged in construction activities and 7 women engaged in water carrying activities were selected at random for detailed study. Anthropometric details were recorded on 47 women workers from construction and 25 women workers from water carrying activities. In the case of women engaged in construction activities the average body height, weight and age were 149.7(+-1.7)cm, 42.8(+-5.5)kg and 28.5(+-7.1)yr. respectively. The percentage of fat, lean body weight and body surface area was 21.2(+-3.7)%, 34.0(+-.09)kg and 1.5(+-0.1)m*m, respectively. The average energy intake was 1355 kcal. The average walking speed in the field was 3.4 Km/h and the maximum heart rate of 163 beats/min was observed for women engaged in block carrying. Heart rates during other activities like carrying concrete for column and beam, carrying concrete for block making and mud transferring were between 110-135 beats/min. The O2 demand varied from 0.771/min (45.31% of VO2 max) to 1.411/min(82.7% of VO2 max) being lowest at ground level and maximum after climbing to the 4th floor. The maximum aerobic power obtained was 1.711/min. A regression equation between heart rate and O2 consumption was derived. In case of women carrying water the average body height, weight and age was 150.0(+-5.3) cm. 50.4(+-10.9) kg and 30.5(+-8.1)yr respectively. The fat content lean body weight and body surface area was 25.6(+-5.9)% of the whole body weight as compared to those involved in construction activity. The women involved in water carrying had work experience ranging from a few months to 25 years and more. The water was being carried by HANDA and load varied from 10 to 50kg, while in case of construction activity the weight varied from 9-34kg. Women carrying water reported the back, elbow, upper arm, calf and knee joints asthe major sites of pain. Similar sites of pain were also reported by the construction workers. About 6.5% women water carriers reported miscarriage while miscarriage was reported by 8.3 women involved in constructioin activity. The average walking speed of fro water carrying was 3.8 km/h which was slightly higher than that of construction group. The average working heart rate was 129 beats/min The average maximum heart rage and O2 consumption for women carrying water during maximum aerobic power was 182.3 beats/min and 1.471/min respectively being lower than that of construction workers. This indicates that the physical work capacity of the construction workers was superior to that of women engaged in water carrying activity.



PROTECTION AGAINST ASCENDING PYELONEPHRITIS BY IMMUNISATIONWITH PILI AND K-ANTIGEN OF ESCHERICHIA COLI  

The study was carried out on female Wistar strain albino rats (Wt. 180+20g) to produce ascending pyelonephritis using uropathogenic strain of Escherichia coli, to isolate and purify pili and K-antigens and to find out whether immunization with pili and K-antigens provides protection against ascending pyelonephritis. Ascending pyelonephritis was produced successfully in the rat model by inoculating uropathogenic E.coli 06 into the bladder with and without ligation of the left ureter. Histopathology of the kidneys showed signifi- cant increase in severity score on days 7 and 14 post-infection in both obstructed and unobstructed kidneys. Purified capsular polysaccharide K-antigen from uropathogenic E.coli contained less than one per cent contamination of nucleic acid and protein. Coupling of K-antigen with bovine serum albumin (BSA) produced a high molecular weight complex eluted into void volume of sepharose 6B column. Immunization with two doses of K-antigen-BSA conjugate at four weeks inter- val protected 60 per cent rats from ascending pyelonephritis. However, either K-antigen or BSA alone failed to protect the animals against the disease. SDS-PAGE analysis of purified pili antigen showed a single band at molecular weight of 17 KDa. When tested pili antigen was found to be immunogenic. Animals receiving either active or passive immunization with pili antigen had comparatively low severity score compared to non- immunized infected animals sacrificed on days 7 and 14 post-infection. Pyelonephritis produced damage in the kidney and the transport of glucose and amino acids through renal brush border membrane was also altered (reduced). The changes in the uptake of glucose, L-alanine, L-lysine and L-proline amino acids were partially restored after immunization with pili antigen.




FILARIASIS
STREAMING POTENTIALS AND THE MECHANISM OF ACCLERATED BONE HEALING  

Studies were carried out with the objectives to standardize an animal model for obtaining accelerated fracture healing and a method for confirming the formation of extra callus formation over the control cases (un- stimulated). To understand the mechanism of acclerated fracture healing, experiments were further conducted to measure the strain on bone surface in response to electrical stimulation, and for measurement of strain generated potentials. A new pulsed radio frequency electric field pattern evolved from bone studies was adopted to accelerate fracture healing in rats. The output of a specially designed signal generator was capacitatively coupled to the fracture site using a pair of stainless steel electrodes. In a series of experiments performed on rats, fractures were induced in the femoral shafts and electrical stimulations were applied to one leg. Bone mass formed in the gap was estimated by measurement of the cortical thickness and by ultrasonic attenuation. It was found that the stimulated side showed greater bone mass than the contralateral control. This device offers a simple and reliable method of accelerated healing, immediately after fracture.




GASTROENTEROLOGY
A CLINICO-EPIDEMIOLOGICAL STUDY OF FILARIA RELATED ORTHOPAEDIC MANIFESTATIONS  

A population of about 16010 in filariasis endemic area for around Anji PHC, was surveyed with the help of staff of the National Filariasis Control Programme, Wardha and Community Medicine Department Mahatama Gandhi Institute of Medical Sciences, Wardha. Night blood smear was positive for 731 cases. Thorough personal, family and clinical history and examination was done and out of these 160 cases were having Orthopaedic or surgical manifestation. The patients were classified in 3 groups. Group A - Orthopaedic manifestations of non-filarial etiology. (No signs and symptoms suggestive of filariasis) - 102 could be followed. Group B - Orthopaedic manifestation of probably filarial etiology - 21 could be followed. Group C - Orthopaedic + Surgical manifestation (filarial cases). Serological investigations showed 148 were positive for filarial anti- body. 20 had eosinophilia, 8 were positive for ASO, 4 for CRP, 11 for RA factor and all were negative for sickling, 135 were found to be sterile after culture, 63 patients showed radiological changes. In Group A & B: 57 & 13 patients were treated with either Aspirin, DEC or Aspirin + DEC for 6 months, 79% showed good response to Aspirin + DEC, 58% for Aspirin, 48% to DEC. In group C in 22 patients - synovial biopsy showed non-specific synovitis, 6 were positive for filarial antibody, 2 for RA factor and with chemotherapy none showed response none that patients presenting with orthopaedic manifestations in the area endemic for filariasis test for filariasis should be done for better management of patients. Re-ELISA was negative in 12% in group A, 20% in group B, and none in group C. Hence it is suggested that different orthopaedic manifestations in endemic area based in immunodiagnosis may be helpful for better management by appropriate treatment.



EFFECT OF SODIUM NITROPRUSSIDEINDUCED HYPOTENSION IN THE PERFORMANCE OF LIENORENAL SHUNTS FOR PORTAL HYPERTENSION *  

A prospective controlled randomised study was carried out in 48 (35 male and 13 female) patients to determine the role of sodium nitro- prusside (SNP) induced hypotensive anaesthesia, in reducing the operative blood loss and portal pressure during elective proximal lienorenal shunt operations for portal hypertension due to extra hepatic obstruction (EHO) or non-cirrhotic portal fibrosis (NCPF). Twenty five patients received SNP intraoperatively to reduce the mean systolic blood pressure by 26 per cent whereas 23 patients matched for age, sex, body weight and preoperative clinical, biochemical and hematological parameters served as controls (did not receive SNP). Blood loss, the number of units of blood transfused and portal pressure at various stages of the procedure were measured together with the postoperative drainage from the abdominal and chest tubes till they were removed. The portal pressure showed a consistent and significant decrease of 15 per cent with the 26 per cent reduction in the mean systolic pressure. SNP induced hypotension produced a significant decrease in the operative blood loss (mean 701 + OR - 633 ml compared to 1319 + OR - ml; P<0.05) and in the number of units of blood transfused (1.3 + OR - 1.3 compared to 2.17 + OR - 1.10; P<0.05). There was a significant decrease in the operating time from 292 to 265 minutes with the use of SNP. In subjective assessment, the operating field was clearer and procedure was easier in operations where SNP was used. There was no significant alterations in the blood gas measurements, nor were any complications due to SNP infusion. There was no deterioration in the liver function in patients and no alteration in cerebral or renal function during or after surgery in patients who received SNP. In addition, there were no differences in the postoperative drainage from both sites, haemoglobin and haematocrit in both groups of patients. It is concluded that SNP induced hypotension reduces the operative blood loss and transfusion requirements during splenectomy and lineorenal shunt surgery in patients with portal hypertension due to EHO and NCPF. The technique is safe, simple and can be adopted easily by any qualified anaesthesiologist.



CHRONIC CALCIFIC PANCREATITIS:A PROSPECTIVE STUDY OF RESPONSE TO SURGICAL TREATMENT 

Patients with a clinico-radiological diagnosis of chronic calcific pancreatitis (CCP) were studied to evaluate the effect of surgical intervention on pain and the exocrine and endocrine functions. Of the 126 patients (104 males and 22 females; mean age 36.98 years), 40 had alcoholic pancreatitis, 81 tropical calcific pancreatitis, 3 had carcinoma of the pancreas initially presented as chronic pancreatitis and 2 had pancreas divisum. One hundred and twenty patients presented with abdominal pain (of pancreatitis) with an average duration of 4.63 years, 33 had diabetes mellitus, 19 had pancreatic steatorrhoea, 23 obstructive jaundice and 10 patients had leftsided portal hypertension (L-PHT). Pancreatic calcification was observed in 37 of the 126 (29.4%) patients on plain x-ray of the abdomen, while ultrasonography showed dilated pancreatic duct and calcification in 49 (33.6%) patients. Endoscopic retrograde cholangio pancreatography (ERXP) done on 50 patients revealed dilated duct and/or calcifi-cation in all 50 patients. All patients with pain were initially managed conservatively with absti- nence from alcohol, small frequent carbohydrate rich diet, oral pancreatic enzymes and H2 receptor blockers. Sixty nine of the 126 patients not respondingto conservative tretment underwent surgery - modified Puestow's operation in 56,Duval's operation in seven and distal pancreatectomy in six. Internal drainage for symptomatic pancreatic pseudocysts was carried out in 17, biliary enteric anastomosis for obstructive jaundice in 22 and splenectomy for L-PHT in 10 patients. A total of 50 patients could be followed up for periods ranging upto 5 years. Postopertive relief of pain was recorded in 45 (90%) patients. Pain recurred in 4 (8%) patients at the end of the 5 years. Five of the 11 (45.4%) patients with overt diabetes mellitus improved in terms of insu- lin requirement and normalisation of blood glucose values. It is thus concludedthat the drainage operations for CCP do result in a consistent long-term improvement in abdominal pain and diabetes mellitus. Quantitative estimation of inorganic and organic constituents of 8 8 pancratic stones showed that Ca2+ (31.37 + 4.07 mg/dl) and oxalate (5.06+2.18 mg/dl) were the major inorganic constituents and proteins (83.0 + 13.5 ug%) and mucopolysaccharides (6.96 + 1.77 mg%) the major organic constituents.



PLASMA FIBRONECTIN AND COMPLEMENT LEVELS IN PATIENTS OF FULMINANT HEPATIC FAILURE (FHF) AND SUB-ACUTE HEPATIC FAILURE (SAHF)  

Using highly sensitive and specific ELISA methods which were developed and standardized, plasma levels of soluble fibronectin(Fn), breakdown product of C3 complement(C3d) and breakdown product of properdin factor B (Ba) were estimated in 64 patients with fulminant hepatic failure(FHF), 29 with subacute hepatic failure(SAHF) and 32 healthy controls without any history of liver diseases. Etiological analysis of patients revealed hepatitis B, hepatitis C and hepatitis NANB-NC infection in 18.8, 42.2 and 39.0 aper cent patients respectively with FHF and 31 34.5 and 34.5 per cent patients respectively with SAHF. None of the patients had hepatitis A infection. Plasma Fn levels were significantly reduced and C3d significantly increased in patients of both groups as compared to healthy individuals, irrespective of viral etiology. However, there was no change in plasma Ba levels as compared to normals levels. Serial estimations of plasma Fn in 19 survivors and in all other patients daily, till death, showed that the rate of rise was significantly more amongst survivors compared to non-survivors. The initial Fn levels were inversely correlated with the aspartate amino transferase, serum alanine amino transferase and prothrombin time in both survivors and non-survivors.



CALCIUM HOMEOSTASIS IN ISOLATED PARIETAL CELLS OF MAN:IN NORMAL AND ALTERED PHYSIOLOGICAL STATES  

The study was carried out to ascertain the basal status of intracellular free calcium and calcium transport across the plasma membrane in an isolated parietal cell population derived from patients with and without duodenal ulcer. Multiple endoscopic gastric mucosal biopsies from the corpus of the stomachin patients undergoing endoscopic evaluation for epigastric pain/reevaluation ofduodenal ulcer were processed and isolated parietal cells were studied. Gastricbiopsy tissue taken 5 cm away from the lesion in subjects with suspected gastriccarcinoma but having normal acid levels were used as controls. Patients taking calcium antagonists and those concurrently taking H2 antagonists were excluded. Relevant clinical details including smoking habits, alcohol intake, and drug history were recorded. Intracellular calcium was estimated in 21 patients using fura-2-acetoxymethyl ester. Calcium influx was determined in 26 patients and efflux in 12 patients (in 10 patients both influx and efflux could be performed together) by using radioactive calcium. Acridine orange retention was used to assess acid production by parietal cells in 20 patients. A homogeneous population of viable (>75%) parietal cells was obtained with a yield of 1.75x10 cells/ml. The cells had characteristic eosinophilic cytopla-sm and concentric nucleus. The purity of the preparation was demonstrated by a modest three-fold rise in K+-dependent paranitrophenyl phosphate activity from the "crude" to the pure after precoll density gradient. Patients with an active duodenal ulcer had a higher intracellular free calcium (239.75 nmol/10 cells) as compared to patients who had nonspecific endoscopic findings (mean 143.36 nmol/10 cells) and patients with normal endoscopy (mean 145.6 nmol/10 cells). The acridine orange retention as a measure of acid secretion was higher but comparable in patients with active duodenal ulcer (32.55%) and nonspecific endoscopic findings (35.32%) as compared to individualswith normal endoscopy. Only calcium influx at 20 min was significantly more in patients with duodenal ulcers as compared to the control group and those with nonspecific changes at endoscopy. There was no difference between the groupss (with active duodenal ulcer, non-specific changes at endoscopy and normal endoscopy) in calcium influx at 0 and 60 min; calcium efflux at 0,20 and 60 min. None of the parameters tested showed consistent variations that could be attributed to smoking, alcohol intake or presence of nonspecific changes at endoscopy. It is thus concluded that in the unstimulated state calcium homeostasis in isolated parietal cells of pateitns with duodenal ulcer shows only a minimal difference as compared to controls.



STUDY OF ASSOCIATION OF CAMPYLOBACTER PYLORIDIS IN PATIENTS UNDERGOING GASTROSCOPY FOR BENIGN PEPTIC AND DUODENAL ULCER  

Antral mucosal biopsy tissues of 100 patients each of non-ulcers dyspepsia,benign gastric ulcer and with other diseases of gastrointestinal tract were studied for analysing the association of Helicobacter pylori with disease. The biopsy samples obtained on endoscopy from apparently healthy region of the antrum away from the ulcer site served as control. The biopsy samples were subjected to urease tests (indigenously standardised), culture and histopatho- logy. H. pylori was found to be significantly associated in patients with gastritis and gastric, duodenal ulcers and gastric carcinoma. Brain heart infusion agar with 7 per cent horse serum, growth supplements and Butzler antibiotic supplements supported the growth of H.pylori.SDS-PAGE analysis revealed the presence of all the major proteins except the 120kDa protein which was not present in all the isolates. A liquid and a semi- solid urease test standardized indigenously was found to be comparable and cost effective than the commercially available CLO (Campylobactor Like Organism) test. Cytopathic effect was produced by broth-culture filterates of H.pylori confirming the production ofa cytotoxin in HeLa cell line. Intraperitoneal injections of cell sonicates or live suspensions of H. pylori produced lethalityin Balb/c mice. However, injections of heat treated suspension did not produce such effects confirming the production of heat labileby H. pylori. Analysis of data on clinical history of patients failed to reveal the role of alcohol and smoking as associated risk factors in the ulceration process in the patients with H.pylori infection. Irregular dietary habits seemed to play arole in the ulceration process. PCR analysis showed the presence of similar DNA sequences in both Campylobacter jejuni and H.pylori.



INCIDENCE OF DELTA INFECTION IN ACUTE AND CHRONIC LIVER DISEASES  

Antidelta antibodies were evaluated in 254 HBsAG positive patients to study the correlation of delta infection with severity of liver diseases. The incidence of delta coor super-infection was also estimated using the IgM anti HBc test. Age of patients varied from 1 to 78 years. Of the 254 patients, 148 had acute viral hepatitis and 92 had chronic liver disease (chronic hepatitis, cirrhosis or hepatocellular carcinoma). Eight patients who had received multiple blood transfusion and had no evidence of liver diseases and 6 asymptomatic patients were also HBsAG positive. Antidelta positive was found in 17.3 per cent(44/254) patients. These 44 patients included 23(16.08%) with acute viral hepatitis, 17(18.47%) with chronic liver disease, 3 recipients of multiple blood transfusion and one asymptomatic HBsAG carrier. Patients with delta infection were aged 3 to 63 years. Of the 44 patients with delta infection, 6 gave history of possible parenteral transmission through dental visits, injection etc., 6 had received blood transfusion, 2 were medical professionals and 1 patient had chronic renal failure with hepatitis. Mortality in delta positive patients was found to be 13.63 per cent(6 of 44), while in delta negative patients it was only 8.09 per cent (17 of 210). Of the 44 antidelta antibody positive patients 26(59.09%) had co-infection and 18(40.9%) super-infection. Delta co-infection was seen more commonly in patients wit acute liver disease while super-infection was more common in those with chronic liver disease.Though incidence of encephalopathy as also mortality was higher amongest delta positive patients compared to delta negative patients with HBsAg related acute or chronic liver disease, it was not significant.



SEQUENTIAL ASSAY OF OXYFREE RADICALS AND NITRIC OXIDE IN EXPERIMENTAL PERITONITIS  

Sequential assay of oxygen free radicals, flitrates and citrulline in experimental peritonitis. Sequential production of oxygen free radicals, nitrates and citrulline was studied in New Zealand Wistar strain rats of average weight (200-250 g) after creating peritonitis with caecal perforation. These parameters were also studied after closure of the perforations and peritoneal lavage (ie reversal of peritonitis). Average survival time of rats after induction of peritonitis was 5.33 h. Baseline values of oxygen free radicals, nitrates and citrulline (for comparison) were obtained in sham operated rats (controls) subjected to lpaarotomy and closure. After creating peritontis sequential assays were performed from 1 to 6 h at 1 h intervals. Reversal of peritonitis was carried out at different intervals after induction of peritonitis (from 1-6 h post peritonitis ) and sequential assays were carried out at every 1 h after reversal of peritonitis. There was a steady increase in the value of oxygen free radicals from 0 to 3 h and in the nitrates and citrulline levels from 0 to 4 h, thereafter they showed a gradual fall. Peak levels of oxygen free radicals were seen at 3 h and for nitrates and citrulline at 4 h post peritonitis. Maximum mortality was observed between 3-4 h post peritonitis. After induction of peritonitis all values were higher than the base line values indicating that it was peritonitis which caused the increase. Reversal of peritonitis produced significant fall in all values. This study thus clearly indicated that the values of oxygen free radicals, nitrates and citrulline were significantly increased in peritonitis and were brought down signifiantly with intervention in the from the reversal of peritonitis.




GERIATRICS
DIETARY FACTORS IN THE AETIOLOGY OF GALLSTONES: A CASE CONTROL STUDY  

The study was carried out to assess the validity and reproducibility of food frequency questionnaire (FFQ) as a tool for examining the association between various dietery factors and diseases. The study group comprised 100 individuals each ( with no history of illness or modified dietary pattern) from three communities middle income group (MIG), low income group (LIG) and urban slum (US). The FFQ was validated against seven day dietary recall method. The past dietary habit was assessed by using FFQ and data on daily intake for a period of seven consecutive days were collectd utilizing the dietary recall method. The level of agreement between the FFQ and recall was measured by calculating the median difference between the findings from the two methods and the percentage of individuals classified by FFQ to within +/- 1 day per week of the recall. In the reproducibility study the FFQ was administered to a sub sample of indivisuals twice, once at the beginning and again after six months. The median difference between the tow methods for any food item in the data obtained from individuals belonging to LIG was zero. In MIG no median difference was observed in 67% of the food items. The percentage of individuals belonging to LIG, classified by the FFQ to within +/- 1 day per week of frequencies reported in 7 days of recall, ranged from 33.3% for other vegetables to 99.9% for cereals, while those belonging to MIG ranged from 10.3% for other vegetables to 100% for cereals. Similarly individuals belonging to urban slums ranged from 8.6% for fruits to 100% for cerels. In the reproducibility study no significant difference in intake between the tow interviews was observed for most of the food items. The FFQ was found to be fairly valid and reproducible.



FAMILY ENVIRONMENT IN THE ELDERLY WITH CHRONIC RESPIRATORY DISEASE  

The respective study was carried out in 100 elderly patients (aged 60 years and above) suffering from chronic obstructive airway disease to determine the relationship between the family environment and characterstics of the disease (viz. its duration and severity), erception of the famil environment by the patients and caregivers, and medical support services available. The caregivers included sons, daughters, spouses and close relatives. Using semistructured interview schedules significant correlation was observed between the duration and severity of the disease, abailablity of medical support services and patients perception of his/ her family environment. A fair degree of agreement was observed in all the subscales of the family environment scale (FES). In the relationship dimension, high scores was recorded in cohesion and expressiveness indicating to a large extent the commitment, help and support that family members provide to each other and that the faily members are encouraged to act openly and express their feelings directly. Low scores of both patients and caregivers on the issue of family conflict also indicated a supportive family environment. High scores on the independence subscale predicted a tendency to be assertive and self sufficient. The study thus revealed that both the elderly patients and caregivers felt quite positively about their families and preceived their families as supportive characterzed by high cohesion and expressiveness and low conflict associated with family members better adjustment and greater ability to deal with stress, espicially when coping with personal physical illness.




HAEMATOLOGY
NUTRITIONAL AND HEALTH STATUS OF ELDERLY LIVING AT HOME IN DELHI  

The dietary profiles and nutritional, socio-econimic and health status as also the lhalth behaviour and morbidity profile were assessed in non- institutionalalized elderly (228 men and 205 women) aged 60 years and above. The instruments used in the studyn included a semistructural predesigned and pretested interview schedule to elicit information on the demographic porfilem socio-econimic status self assessment of health status, general health behaviour,morbidity and diaetary profile, anthropometric measurements, blood pressure measurment and estimation of haemoglobin and blood sugar levels. The elderly lived in all categories of women v.z:- high income (HIG), middle income (MIG) and low income (LIG- including slum dwellings). Of the 433 elderly studied, 47(10.8%) alone and 386 (89.2%) lived with the family. Nutritional supplements in the form of vitamins, Ayurvedic and homeopathic preparations were taken by 48,40 and 6% elderly living in HIG, MIG and LIG houses, respectively. A higher prevalenc of smoking and alcohol and tobacco consumption was observed among elderly from the low income group.Alcohol intake, tobacco chewing and smoking has more in men, however, 92% of elderly women from the low income group also smoked. A higher prevalence of diarrhoea, dysentery and respiratory infections was reported by elderly (both men and women) from the low income group. Prevalence of cardiovascular diseases was higher among men compared to women, whereas musculoskeletal problemds, arthritis and pain in weight bearing joint were mone comman in women. Other problems of significance reported by elderly (both men and women) from all strata were cataract and impaired hearing. A greater proportion of women (64%) were anaemic compared to men (46%). The prevalence of anaemia was higher in LIG women (85%) as compared to those from HIG (53%) and MIG(58%). Food beliefs and preferences were strong determinants in selection of food items by the elderly. The men daily intake of all the nutirents showed a decline with increase in age. The iron intake poor across the socio- economic strata, with a deficit of 46% for women and 37% for men. In addition, the intake of protein, calcium and vitamin was alos inadequate in LIG elderly. A gradation was observed in the mean anthropometric measurements with increase in economic status. Among the LIG elderlyn 56% suffered from chronic energy deficiency. Conversely 34% elderly from HIG and MIG suffered from different grades of obesity.



FIBRINOLYTIC ACTIVITY OF BLOOD AND EFFUSION FLUIDS IN PATIENTS WITH PLEURAL AND PERITONEAL EFFUSIONS OF MALIGNANT AETIOLOGY  

Fibrinolgic activity of blood and effusion fluid in patients with pleural and paritoneal effusion of malignant etiology. The study was carried out in 91 patients with maligrant effusion (4s with pleural and 46 with peritoneal effusion, 33 patients with tubercular effusion (16 with pleural and 17 with periteneal effusion and 26 patients of hepatic currhosis with ascites to investigate the fibrinolytic activity of plasma as well as effusion fluids. Patients with milignant effusion showed a striking lack of plasminogen activator activity in plasma in resulting state. Post-tornique plasma samples, howere, showed release of plasminogen-activator activity in most of the patients. Study on effusion fluid of malignant etiology showed the resemblance of of their fibrinolytic activity with normal plasma behaviour, i.e., lack of fibrin plate lysis by whole fluid, but presence of lysis by the euglobulin fraction. Patients with normalignant etiology showed contrasting results in fibrinolytic activity of both plasma and effusion fluids. In resting state their plasma samples showed similar response as the normal healthy individuals i.e., presence of plasminogen activator in the euglobulin fraction. The effusion fluids of non-milignant etiology also behaved differently from the fluids of malignant etiology. In most of the instance the whole fluid produced lysis on fibrin plate without the need for euglobulin fractionation. Alpha-2 macroglobulin, Alpha-1-antitrypsin and AT-III were assayed in both plasma as well as effusion fluids. The t-PA inhibitor was also tested in effusion fluids. However,no significant increase of these inhibitor was not observed in plasma or effusion fluid samples from cancerous patients as compared to those with having effusion of non-malignant etiology.



STUDY ON ELECTRON MICROSCOPIC CYTOCHEMISTRY IN ACUTE LEUKAEMIAS  

Blasts from acute lymphoblastic leukaemia (30 cases), acute myeloblastic leukaemia (20 cases) and chronic granulocytic leukaemia in blast crisis (8 cases) were studied for ultrastructural cytochemistry with the aim to detect lineage infidelity if any, and to detect early differentiation particularly to myeloid lineage. It was also aimed to subtype the leukemia depending on the ultrastructural methodology and cyto- chemical reactions. The samples of blood/bone marrow were collected and after separating the blasts on a Ficoll gradient, they were washed with PBS and processed. Cells were divided into five portions, one for ordinary transmission electron microscopy and the other four for the cytochemical reactions namely myeloperoxidase (MPO), platelet peroxidase (PPO), acid phosphatase and non-specific esterase. Out of the 30 cases of ALL studied, two were found to be AML based on MPO positivity and one case was discovered to be M7 based on PPO positivity. Another 4 cases showed characteristic acid phosphatase reaction indicating the T-cell nature of the blasts. All the cases diagnosed as AML except one case of M5 and two cases of M7 showed MPO positivity in the blasts. One case of M2 and one case of M3 showed more than 10% blasts positive for PPO indicating the megakaryoblastic nature and thus intralineage infidelity. Two cases of M7 showed more than 60% blasts positive for ultrastructural PPO. Of the 8 cases of chronic granulocytic leukemia in blast crisis five were myeloid and three of lymphoid blast crisis. The MPO positivity in myeloid blast crisis ranged from 10 to 90%. One of the cases of lymphoid blast crisis showed 10% PPO positivity. The other two cases of lymphoid blast crisis were negative for all four cytochemical reactions. Thus using MPO, 2 cases of ALL could be reclassified as AML on the basis of ultrastructural MPO positive. In cases of AML and CGL-BC it con- firmed the myeloid nature of the blasts. Therefore, ultrastructural MPO is a specific and early marker of myeloid lineage and of diagnostic importance in unclassified leukaemias. Demonstration of platelet peroxidase (PPO) by ultrastructural cytoche- mistry is one of the specific criterion for diagnosing blasts of megakaryo- cytic lineage. It was found that in two cases of AML a significant number of megakaryoblasts (more than 10%) were there. Two cases of M7 showed more than 60% blasts positive for PPO. Using ultrastructural acid phosphatase reaction, monocytic nature of blasts in AML-M4 and AML-M5 could be confirmed. In cases of ALL, acid phosphatase reaction in golgi region indicated the T-cell nature of the lymphoblasts.



EVALUATION OF IMMUNOSUPPRESSION WITH ANTI T-CELL MONOCLONAL ANTIBODY (WM48) IN THE TREATMENT OF APLASTIC ANAEMIA  

Study was conducted to evaluate the effect of T cell depletion with a specific anti T cell monoclonal antibody and determine whether this treatment can induce haemopoietic recovery in severe aplastic anaemia and to compare this treatment with conventional treatment with oxymethalone with regard to cost, transfusion support required, side effects and response to treatment in severe aplastic anaemia. Patients with severe aplastic anaemia treated with oxymethalone have an 18% chance of response. The response in patients with moderate disease is higher (40%). Haemopoietic recovery was seen in 38% of 34 patients treated with antilymphocyte globulin obtained from MERIEUX. This response is similar to reported results in the Western literature. There was a significant difference in response in patients treated with Hoechst anti lymphocyte globulin (ALG) when compared to Merieux (ALG) and this correlated with the ability of the ALG preparation to induce lymphocyte blast transformation in vitro. This is the first clinical study to document a correlation between response to ALG and its ability to induce lymphocyte blast transformation in vitro. Anti T cell monoclonal antibody WM48 was able to produce profound T cell depletion in patients with acceptable side effects. Treatment of aplastic anaemia with 5 mg of anti T cell monoclonal antibody (WM48) daily for 7 days does not induce haemopoietic recovery, despite profound T cell depletion. Lower dose (1 mg daily for 5 days) of anti T cell monoclonal antibody (WM48) produced a response in 3 of 6 patients treated. In a small number of patients there was a correlation between in vitro lymphocyte blast transformation (LBT) to ALG and haemopoietic recovery with immunosupressive therapy. LBT may be a useful test to identify those patients who are likely to respond to immunosuppression. Data from this study suggest that a combination of lymphocyte depletion and stimulation may be necessary for haemopoietic recovery following immunosupression. Further studies on combination of anti T cell monoclonal antibody with haemopoietic growth factors may provide a more effective therapy and help in the understanding of the biology of aplastic anaemia.



ESTABLISHMENT OF FIBRONECTIN LEVELS & CORRELATION WITH SURVIVAL OF INFANTS WITH BIRTHASPHYXIA & SYSTEMIC INFECTIONS*  

Study was conducted to standardise an assay system for measuring fibronectin levels in serum and the extraction of purified fibronectin from donor blood. To study fibronectin levels in low birth weight neonates with special reference to small for date babies and to study the fibronectin levels in neonates and infants having birth asphyxia and/or systemic infection (pneumonia/sepsis). Extraction of fibronectin from plasma using heparin cold precipitation and its assay by IE and ELISA were standardised. Serum fibronectin was assayed in 6 different categories of healthy and sick infants using immuno-electrophoresis (IE) and ELISA [Term appropriate for date (TAFD); preterm appropriate for date (PTAFD); term small for date (TSFD); preterm small for date (PTSFD); birth asphyxia and sepsis (SEP)]. Term appropriate for date infants were assayed for plasma fibronectin also. Comparison of serum fibronectin levels in different groups by the Wilcoxan Rank Sum test indicated no difference in fibronectin levels between term and preterm infants, between preterm AFD and PTSFD, term SFD vs TAFD, Birth asphyxia vs no birth asphyxia. Septic babies on the other hand had a significantly lower fibronectin level than the non-septic babies. Plasma fibronectin levels in TAFD infants were less than half the levels reported in the western literature for new born term infants. However, adult plasma and serum fibronectin levels in this study were similar to those reported in the literature. No satisfactory explanation is available for this difference. Maternal fibronectin may provide us a clue.



MOLECULAR CHARACTERIZATION OF B-THALASSAEMIA ENCOUNTERED IN THE STATE OF UTTAR PRADESH  

The study was carried out on transfusion dependent patients of thalasaemia for molecular characterization of beta- thalassaemia encentered in Uttar Pradesh. Of the 160 patients of thalassaemia and hemoglobinepathics studied 96 were suffering from thalassaemia major, 36 with thalassaemia haemoglobinepathy, 12 with haemoglobinepathy and 16 with thalassaemia trait. Sixty four of the 96 thalassaemia major patients were hemozygons for one of the 5 common mutations reported from India. Amongst 36 patients with thalassaemia haemoglobinepathy, 24 were of e/Trial 11 of s/trial and one of D/Trial. The DNA samples from a total of 244 unrelated chromosomes from patients with beta- thalassemia and thalhaemoglobinopathy were investigated for 10 (5 common and 5 uncommon) mutations by PCR ARMS technique The prevalence of different mutations was IVS 1,5 (G-->C):60.24%, -619bp deletin:11.06%, Codon 41/42:9.01%, Codon 8/9 : 6.55% IVS 1/1 (G-->T): 3.68%, codon 16:2.46% codon 15: 0.82%and Cap +|:0.41%. 4 cases (1.7%) 2 previously known mutation co30(G-C) and IVS 1-1(G-A) were identified. Though these have been described in Indian samples earlier, there are mainly seen in mediteranean and Middle East countries. Also one new mutation was also characterized. The study thus revealed that beeta- thalassaemia and haemoglobinepathies, particularly HbE and to some extent HbS are important genetic problem in Uttar Pradesh. These disorders are present in the natives of U.P. and in not only those who have migrated from Pakistan. The prevalence of 619 bp deletion is much more common in people of Pakistan origin, while in natives of UP IVS(1,%) G-C mutation was the commonst.



STUDY OF HAEMORHEOLOGICAL PARAMETERS IN CEREBROVASCULAR ISCHAEMIA AS COMPARED TO NORMAL CONTROLS  

Cerbrovascular acccident is the leading cause of death and is the most common life threatening neurological disorder. The term 'Cerbrovascular accident' referes to all disorders in which area of brain is transiently or permanentaly affected by ischemia or bleeding and or in which one or more blood vassels or brain are primarily impaired pathological processes. In the majourity of cases of peripherial and cerebral vascular diseases, there is a reduction of blood flow to the affected parts of the body. Blood flow properties of blood (Chien, 1993). The treatment of vascular disease is aimed therefore at improving the flow properties in order to produce a flow behaviour compatiable with tissue survival. Haemorheological factors such as red cell concentration, red cell aggregation and fibrinogen concentration were determined in patients of cerebrovascular diseases with assiciated diabetes, hypertension and ischeaemic heart disease. The altered values were observed to be significantly higher in comparision to their controls. The observed values of haemorheological parameters in diabetic and non diabetic parents with reference to normal controls exhibited increasing levels of whole blood viscosity, plasma fibrinogen and red cell deformation with decreased levels of red cell aggeration and packed cell volume.




HEALTH SERVICES RESEARCH
PREVALENCE AND MOLECULAR CHARACTERIZATION OF ALPHA- THALASSAEMIA IN UTTAR PRADESH  

One hundread ninty normal healthy individuals were screened first for alpha gene deletion, the commonest one accounts the major prevalence among Asian Indian reported by Garewal et al 1994. On screening 125 samples seven were found with one alpha gene defect (4.2%). Two hundred two beta thalassaemia traits were screened for - alpha 3.7 kb, deletion. Out of 202 samples tweenty three were found positive for this deletion (11.5%). A total of 392 samples were screened out of which 31 were found positive for - alpha 3.7 kb deletion (7.9%). Thirty four samples of thalassaemia patients were screened for alpha gene deletion. These thirty four samples include eleven cases of Thalassaemia major and twenty three case of Thalassaemia Intermedia. Out of eleven thalassaemia major cases in two subjects - alpha 3.7 kb deletion was present on both the chromosomes (- alpha 3.7 kb/- alpha 3.7 kb) and one gene deletion was found in two cases (alpha 3.7 kb/aa) indicates overall prevalence of 36% (4/34) in thalassaemia major. In twenty three cases of thalassaemia intermedia one 3.7 kb deletion was observed in three subjects and triplication of alpha gene was found in four subjects. This furtbher indicates the prevalence of alpha gene (13%) in thalassaemia intermedia patients.



HEALTH CULTURE AMONG THE TRIBAL WORKERS OF TEA PLANTAT-IONS IN WEST BENGAL-A STUDY INCONTINUITY AND CHANGE  

The study has high-lighted the critical role the socio-cultural factors play in shaping the health of the community. It is being observed that a vast majority of the respondents are dissatisfied with the health centres located at their plantation. They complain of inadequacy of facilities and the services given by the health staff. Regarding family planning the awareness among the tribal tea garden workers is very poor. It has been observed that the tribal workers of tea plantation have still retained much of their traditional health living in tea plantation for about one hundred years where modern medical treatment and medicine are made available easily to the tribes. However, it has also been observed that in the plantation where better facilities are easily available the tribal workers have shown keen interest in the use of modern medical treatment. But they still believe in their traditional health culture to a large extent. It is, therefore, evident that if proper and adequate medical facilities are provided to the people, they gradually become inclined to accept them. It is often said that deep traditional cultural roots of the tribal is the barrier for accepting modern medical treatment since they have their own traditional magical and herbal systems for curing illness. If modern medical facilities do not reach in the hand of people easily and if modern medical treatment involve such economic pressure, people hesitate to accept them. What we need now for spread of modern treatment is to improve the machinery of health services. Adequate health services, medical staff including doctor, nurse, midwife etc. and medicine are necessary to improve the attitudes of the people towards modern medcine and treatment.




HUMAN GENETICS
STUDY OF HEALTH EXPENDITURE IN INDIA : A CASE STUDY OF A DISTRICT  

In India, private health care services account for about three fourths of all health care. But information on its utilisation and expenditure is not known in any significant manner. A study was supported at the Foundation for Research in Community Health, Bombay, to explore this important aspect. The only data on health expenditure which is consistently available is the one spent by the central and state Ministries of Health and Family Welfare. Analysis of expenditure has shown to range from 3.2% of total Government expenditure in the early fifties to about 4.3% of Government expenditure at the present. The focus of public health services has over the years shifted to family planning and water supply at the cost of medical care. Thus between 1950-51 and 1984-85 the Government's health expenditure on family planning increased from 0% to 11.4% and on water supply from 1.3% to 23.1% but on medical care it declined from 43.4% to 27.9% of total health expenditure. The private health sector which caters to the latter demand has mushroomed over the years. This is an extremely important finding because it shows that medical care is the weakest link in public health services. In the second part of the study which looked at private household health expenditure in both urban and rural areas, it was found that the average health expenditure borne directly by households is Rs. 182 per capita per year ( in 1987 ), which is 7.6% of per capita total consumption expenditure. This is a substantial amount, especially considering the fact that more than two thirds of the population lives at the subsistence level. A classwise analysis of the data collected in this study shows that with rise in class status morbidity rates increase, use of private health facilities rise, use of public health facilities decline, non utilisation of health facilities decline and per capita health expenditure increases. This means that purchasing power is a crucial factor in health services use and expenditure. Another part of the study has looked into the expenditure and earnings of private medical practitioners in Bombay city. The findings reveal that the average gross earnings of a general practitioner is over Rs. 22,000 per month and their expenditure is about Rs. 4000 per month, giving a net income of Rs. 18000, per month. This study included doctors of allopathy (including licentiates) homeopathy and ayurveda. Finally, the study on corporate sector health benefits shows that the expenditure on medical care expended on employees in the 134 sample companies is Rs. 1339 per employee per year. This expenditure is 2.60% of all emoluments of the employees, 19.33% of all benifits(other than salaries) given to employees and only 0.23% of the companies' sales turnover. On the basis of all these studies it is possible to arrive at a proxy estimate of the total volume of health expenditure in the country. For the year 1989-90, the total health expenditure works out to Rs. 243,500 million which is 6% of GNP. The share of the Ministries of Health is 26% whereas that of households is 60%. Under existing socio-economic conditions this is a highly inequitious situation.



SISTER CHROMATID EXCHANGE AS AN INDICATOR OF CONSTITUTIONALCHROMOSOMAL INSTABILITY IN PRECANCEROUS AND CANCEROUS LESIONS OF CERVIX UTERI  

Sister chromatid exchange (SCE) in the lymphocyte chromosomes is used as an indicator of constitutional chromosomal instability in precancerous and cancerous lesions of cervix uteri. SCE frequency was investigated in 100 cases of precancerous lesions of uterine cervix (41 cases of mild dysplasia, 37 cases of moderate dysplasia and 22 cases of severe dysplasia) and 100 cases of uterine cervix cancer. The SCE frequency was also investigated in 150 age and sex matched controls. The analysis of the data revealed that the frequency of SCE varied from cell to cell and from individual to individual. The frequency of SCEs was found to be 7.26 + 1.52 in mild dysplasia, 8.52 + 1.67 in moderate dysplasias, 8.91 + 1.83 in severe dysplasias and 9.68 + 2.19 in patients with uterine cervix cancer. These values were significantly higher than the SCE values in control women. The average rates of SCEs per individual were found to range between 2.56 - 12.14 in controls, 2.95 - 15.32 in dysplasias and 5.16 - 19.26 in cancer patients. The SCE frequencies among the various groups were, however, found to be heterogenous. To investigate whether chromosomes from patients with precancerous and cancerous lesions of cervix uteri are more susceptible to damage in terms of SCE frequency, culture from lymphocytes of some indivi- dual from precancerous and cancerous lesions of cervix uteri were exposed to mitomycin-C (0.01 ug/ml) together with similar exposure to normal age - sex matched controls. MMC induced SCEs were not significantly different from each other indicating that the lymphocyte chromosomes from precancerous or cancerous lesions of uterine cervix are equally susceptible to damage as compared to normal controls and there exists no differential sensitivity. In the present study, the dysplasia cases progressing to cancer showed a slightly elevated SCE frequencies as compared to non- progressed dysplasia cases. The present study thus suggest that an intimate relationship may exist between constitutional chromosomal instability in precancerous and cancerous lesions of cervix uteri indi- cating that SCEs may serve as useful preclinical biological marker in delineating high risk precancerous lesions progressing to cancer from low risk lesions regressing to normalacy.



MOLECULAR ORGANIZATION AND CLINICAL SIGNIFICANCE OF FOLATE SENSITIVE FRAGILE SITESON HUMAN CHROMOSOMES  

The assessment of folate sensitive fragile sites on human chromosomes was carried out in 100 mentally retarded individuals alongwith 50 normal subjects. An increase of 8.75% of chromosomal damage was observed in mentally retarded group as compared to the normal individuals. Seven fra-Xq27 individuals have been identified with varying degrees of mental retardation, speech deficit, hyperactivity and autistic features. Of the fragile sites observed in mentally retarded individuals and absent in controls, there were 7 fragile sites which have been reported in earlier literature (i.e., 1q21, 7p13, 11q23, 11q24, 17q12, 18q23, Xq26, Xq27) and fragile sites which have not been reported previously. (i.e., 4p26, 4q33, 7q24, 7q34 and 18q23). Folic acid levels have been estimated in 15 individuals with mental retardation. Of these, two cases had levels below the normal range. They were, however, negative for fra-Xq27. Folate level of one patient identified as positive for fra-Xq27, was found to be within the normal range.



SISTER CHROMATID EXCHANGE AS AN INDICATOR OF CONSTITUTIONALCHROMOSOMAL INSTABILITY IN PRECANCEROUS & CANCEROUS LESIONS OF ORAL CAVITY*  

Sister chromatid exchange (SCE) in the lymphocyte chromosome was used as an indicator or constitutional chromosomal instability in precancerous and cancerous lesions of oral cavity. SEC frequency was investigated in 100 cases of oral leukoplakia, 100 case of submucous fibrosis, 100 cases of Palatal keratosis, and 100 cases of squamous cell carcinoma of the oral cavity. Two hundred age and sex matched controls, who has no history of viral infection, hepatitis or any broad spectrum antibiotic therapy at least 4- 6 weeks prior to the collection of blood samples were also investigated. The analysis of the date revealed that the frequency of SCE varied from cell to cell and from individual to individual. The frequency of SCEs (Mean +- S.D) was found to be 8.25 +- 1.74 in oral leukoplakia; 8.91+-1.92 in palatal keratosis; 9.34+-2.21 in submucous fibrosis and 10.23+-2.39 in squamous cell carcinoma of the oral cavity.These values were significantly higher than the SCE values of 5.58+-1.33 observed in normal controls. Combined habits (persons addicated to both tobacco chewing and smoking) induced significantly higher SCEs in lymphocytes as compared to single habits of betel with tobacco chewing or bidi/ cigraette smoking. There was significant correlation between the number of betel leaves with tobacco chewed and the mean SCE frequency and also the number of cigarettes or bidis smoked and the mean SCE frequency in patients with precancerous and cancerous lesions of oral cavity. There was a differential MMC induced SCE induction in pre cancerous and cancerous lesions of oral cavity as compared to normal controls. Stoppage of tobacco usage ( at least for one year)following effective intervention resulted in significant reduction of SCE frequency in patients with pre cancerous lesions of oral cavit. These results indicate that education on tobacco habits should be a feasible and effective method for primary prevention of oral cancer as ceasation of tobacco habits may retard that transformatioin process of normal cells into neoplastic one.



A RETROSPECTIVE & PROSPECTIVE ANALYSIS OF POINT MUTATION OF RAS ONCOGENE IN PATIENTS WITH MYELODYSPLASTIC SYNDROME  

A retrospective and prospective analysis of point mutation of RAS oncogene in patients with myeladysplastic syndrome. The study was carried out on 3 years old bone marrow smear/ metaphase slide preporations of36 myelodysplastic syndrome (MDS) patients and peripheral blood/ bone marrow samples of 19 fresh cases of MDS to investigate the frequency of RAS oncogene mutations (using polymerase chain reaction amplification and differential oligonucleotide hybridization) in order to evaluate the clinical significance of RAS muatations in MDS. Subsequently, clinical information about these patients was also obtained in follow up studies and data were analysed. Among the 36 archival MDS samples studied, 13 were positive for RAS oncogene mutation. Among 19 fresh cases, 6 patients were positive for RAS oncongene mutation. These data, revealed that an increased risk of acute myelogenous leukemia (AML) was found in patients with RAS mutations. Only two patients without RAS mutations. Only two patients without RAS oncogene mutations developed AML. This could be because of the presence of increased bone marrow blasts in these patients. It can thus be on concluded that RAS mutations, although relatively infrequent in MDS, is associated with increased propensity for developing into AML. P. Balakrishna Murthy Department of Toxicology Fredrick institute of plant protection and Toxicology, Padappai




IMMUNOLOGY
CHROMOSOME 22Q11 MICRODELETIONIN PATIENTS WITH SELECTED CONGENITAL HEART DISEASES : STUDY OF PREVALENCE, PHENOTYPEGENOTYPE AND MODE OF INHERITANCE  

2q 11 microdeletion is the most frequently reported interstitial deletion, occurring in 1/4000 live births. It can result in more than 80 different kinds of birth defects and malformations in varyinhg combinations and varying severity, collectively called 22q11 microdeletion syndromes. Typical facial features, velopharyngial insufficiency, hypoparathyroidism, hypocalcemia and thymic aplasia have been described. Upto 75% of patients with this deletion have been reported to have congenital heart defects, mainly, conoventricular defects. Among patients with conoventricular heart defects, the overall prevalence of 22q11 microdeletion is reported to be between 7% to 19%. The detection and diagnosis of the deletion is of vital importance as it has signigicant bearing on the pre and perioperative management during congenital heart surgery, as well as bearing on the pre and perioperative management during congenital heart surgery, as well as for consuneling regarding other maujor long term issues like developmental delay, immunodeficiency, hypocalcemia, learning disabilities, psychological/ psychiatric disorders etc. The aim of this study is to determine the prrevalence of 22q11 microdeletion among patients with conoventricular heart defects in our population, study the spectrum of phenotypic abnormalities and the correlation between the phenotype and genotype and to study the inheritance patterns. This is a 3 years study which will be completed in september 2009. So far 130 consecutive patients (aged less than 2 years), with conoventricular heart defects, have been recruited into the study. Following detailed cardiac evaluation and classification, these patients are evaluated by a Pediatric Geneticist who describes the phenotypic features. Flourescent In- Situ Hybridization (FISH) (using TUPLE critical region probe) is then performed on blood samples to identify the microdeletion. The preliminary results of the study swhow an overall higher prevalence (21%) of 22q11 microdeletion among Indian patients with vonoventricular derects thanwhat is reported in western literature. The study also sheds new light on the phenotypic features whivh are most predictive for the presence of the microdeletion a combination of thin long fingers, micrognathia and bulbous nasal tip present together being in 90.4% agreement with FISH positivity, on multivariate analysis. This new observation can potentially result in a more accurate early clinical precdiction of the genetic defect, which can help immensely in patient management and counseling. The data requires further validation during the remaining one year of the study.



POPULATION SCREENING FOR ANTINUCLEAR ANTIBODY AS A MARKER OF SYSTEMIC LUPUS ERYTHEMATOSUS:A MAJOR EMERGINGHEALTH PROBLEM AMONG YOUNG WOMEN *  

Systemic connective tissue diseases are among the important chronic disease of young women. There are no data on the prevalence of these diseases from India. Therefore, in the present study, attempt was made to find out the prevalence of one of the major systemic connective tissue disease namely systemic lupus erythematosus (SLE). In the study two types of survey methods were used. In the first method the first-step screening was based upon a questionnaire specially prepared for this purpose. The valididy (specificity and sensitivity) was first established under controlled conditions. Two health visitors were trained adequately to be able to use this questionnaire in the field conditions. These persons screened 39,826 persons using this questionnaire. The population studied was from a semi-rural area of Haryana just bordering Delhi. In the second-step all the persons screened as "probable case" were clinically examined and assessed by a specialist in the field of systemic connective tissue diseases. All the persons screened as "definite case" on the second-step screening, were then also tested for the SLE serum market antibody called antinuclear antibody (ANA). Persons showing definite SLE on clinical examination and having ANA in their serum, were then labelled as SLE. Using this method, the questionnaire based survey showed one SLE case out of the 39,826 persons screened, giving a point prevalence of 0.0251/1000 population; or 25 patients per million of the population. In the second method the first step screening was carried out using a new filter-paper technique for performing ANA test. The technique was thoroughtly validated and also cross-checked by an advanced laboratory for performing ANA test in London. It was also validated by testing a "positive control" (a known patient of SLE in a field area namely Pauri-Gharwal in the hills of Western Uttar Pradesh). After, due validation of the filter-paper technique, 51,361 population from semi-rural and urban areas of Ghaziabad, North Delhi; Green Park are of South Delhi; and a small locality of Pauri-Gharwal, were studied. 13 persons were found to be positive for ANA test. These 13 persons were given a detailed clinical checkup by trained specialists in the field of connective tissue diseases. Their ANA was also repeated using the standard technique. Of these 13 persons, only two women were found to be having definite SLE. Another woman had only some of the features of SLE. She was labeled as "probable" SLE. One additional person, another woman, had an undifferentiated connective tissue disease not having all the features of SLE. The rest of the persons had a "false positive" ANA of no clinical relevance. Using this new method of filter-paper ANA test two definite cases of SLE were detected in a population of 51,361 giving a point prevalence of 0.0195/1000 population; or approximately 39 per million of the population. In the final analysis, it can be concluded that the study detected 3 SLE patients in a population survey of 91,088 persons giving a point- prevalence of 0.0109/1000 population or about 33 cases per million of the population. If one "probable SLE" case and one patient with undifferen- tiated connective tissue disease is also included then the figures will rise to 5.5 per 100,000 or 0.055/1000 of the population. Therefore, it can be concluded that although the disease SLE occurs in the population, mostly in young women, at present its prevalence is not alarming.



IN VITRO STUDIES ON THE ACTIVATION OF HUMAN NATURAL KILLER CELLS (NK CELLS) & MONOCYTES WITH CISPLATIN  

The effect of cisplatin on the anti-tumour activities of human NK cells and monocytes was studied. Interaction of cisplatin treated NK cells and monocytes with K562 cells was also studied using the electron microscopy (both TEM & SEM), immunofluorescence (for actin & tubulin) and phase cotnrast microscopy. Mouse monoclonal antibodies against the adhesion or binding molecules or receptors present on NK cells would be raised. Chemiluminescence studies would be carried out on PBML and monocytes on activation and treatment with cisplatin, INF and PMA, and also during their interaction with K562 cells in vitro, with or without cisplatin treatment. The role of calcium ions, microfilaments and microtubules in NK cell/ monocyte - tumour cell interactions in vitro would be studied. Release of tumour cytolytic factors by human monocytes on treatment with cisplatin and their possible separation on FPLC would be studied. Human natural killer (NK) cells and monocytes treated in vitro with cisplatin or rIFN-Y showed enhanced lysis of K562 cells in a time dependent fashion. Cisplatin and rIFN-Y treated monocytes were equally cytotoxic to NK-sensitive (K562) and NK-resistant (Daudi and Raji) cells, whereas NK cells were not rendered cytotoxic against NK-resistant tumor cells. NK and monocyte mediated cytotoxicity against K562 cells was further enhanced when the effector cells were primed with rIFN-Y and then treated with cisplatin. The supernatants collected from cisplatin, LPS, muramyl dipeptide (MDP) or rIFN-Y treated nMNC (with NK activity) showed enhanced release of inter- leukin-1 (IL-1), tumor necrosis factor (TNF) and lysozyme in comparison to untreated nMNC. Treatment of nMNC with cisplatin enhanced the intracellular free calcium levels and protein kinase activity; whereas rIFN-Y treated nMNC showed significant rise in ATP level and protein kinase activity only. Further, cisplatin treated nMNC showed an instantaneous rise in intracellu- lar calcium when NK-sensitive K562 cells were added to the effector cells. Treatment of human monocytes with cisplatin enhanced the expression of membrane bound adhesion molecules, LFA-1 alpha and beta. Addition of anti- LFA-1 alpha and beta antibodies abrogated the monocyte and NK-mediated tumour cell lysis implicating a role of these molecules in the tumoricidal activity of monocytes and NK cells. Lymphokine activated killer (LAK) cells generated in the presence of cisplatin/FK565 along with interleukin-2 (IL-2) showed enhanced binding to tumor cells as compared to LAK cells generated in presence of IL-2 alone. LAK cells showed a reorientation of cellular organelles in the presence of tumor cells. Closer interaction between the LAK cell -target cell and the large number of vesicles in the extracellular space of conjugate pairs may explain the increased lysis of target cells by LAK cells generated in the presence of cisplatin/FK565 along with IL-2 as compared with IL-2 alone.



PRODUCTION AND CHARACTERIZATION OF ANTIBIDIES AGAINST ESTRADIOL- ALBUMIN AND ESTRADIOL- ALBUMIN-DNA CONJUGATES AND ROLE IN PATHOGENESIS OF SLE *  

Study was carried out to see the possible role of the female sex hormone, estradiol, in conjugation with DNA, in the pathogenesis of systemic lupus erythematosus (SLE). Calf thymus DNA fragments (average size 200 bp) were covalently linked to bovine serum albumin and estradiol albumin by treatment with glutaraldehyde. The conuugate was separated from free DNA by gel exclusion chromatography. It was characterised by ultraviolet spectroscopy and thermal denaturation studies. The conjugates induced high titer antibodies which were highly specific towards their respective immunogens. These antibodies did not recognise native DNA (B-form), brominated DNA (Z-form) or poly (rG). poly (dC) (A-form). The estradiol BSA-DNA conjugate showed significant binding with various SLE sera, known to have a high level of anti-native DNA antibodies. The results indicated the recognition of the unique conformation of estradiol- BSA-DNA conjugate by naturally occurring SLE anti-DNA autoantibodies. One of the important findings of this study was the recognition of beta- estradiol by naturally occurring SLE autoantibodies.



THE ROLE OF CYTOKINES IN LOW RESPONSE TO HEPATITIS B VACCINE IN CHRONIC RENAL FAILURE PATIENTS  

The study was carried out in 26 chronic renal failure (CRF) patients (22 male and 4 female,man age of 33 +- 10 yr) and 16 healthy individuals (11 male and 5 females; man age 28.5+-7.7 yr) to investigate the cytokine pathways required for anti HBs antibody production in order to understand the high rate of non responsiveness to the vaccine in CRF patients. The CRF patients received 40 of HBs Ag vaccine at 0,1and 6 months. The relevent cytokines were studied in (i) CRF patients producing protective titres of antibody after vaccination (ii) CRF patients unable to produce prootective antibody titres after vaccination and (iii) individuals showing normal antibody response after vaccination. Man anti HBs antibody titre in normal vaccinated individual was higher (837.3+-236.5 IU/L) as compared to CRF patients (23.43+-236.5 IU/L). Of the 26 vaccinated patients only 13 showed protective anti HBs antibody titres (>=10IU/L) and were referred to as responders while reast of the patients with anti HBs antibody titre less than 10IL/L were non responders.It was found that nonresponder patients had significantly lower levels of IFN-gama in unstimulated cultures, of IL-2 and IL-4 in PHA stimulated cultures and of IL-4 and IFN-gama in HBsAg stimulated cultures suggests that the T cell response in these patients may be skewed to THI phenotype and this is responsible for the low antibody titre



IMMUNOBLOT ANALYSIS OF CYSTICERCUS FASIOLARIS ANTIGEN AND ITS ROLE IN THE DIAGNOSIS OF NEUROCYSTICERCOSIS  

Neurocysticercosis is the commonest parasitic disease of the CNS and commonly presents with seizure disorders. The project aimed at evaluation of Cysticercus fasciolaris, the larval stage of Taenia taeniaeformis, in the immunodiagnosis of human neurocysticereosis. Sera from 50 cases of Neurocysticerosis and 50 healthy as well as diseased controls were evaluated in ELISA using the crude antibodies in case and control sera. Further the sensitivity and specificity of the ELISA was compared using crude extracts of Cysticercus fasciolaris scolex, Cysticercus cellulosae membrane and Cysticercus cellulosae scolex. Sensitivity ranged from 76-94% heighest for membrane sonicate of Cysticercus fasciolaris. Specificity ranged from 78 to 90%. The antigenic profile of all four antigenic fractions was assessed by western bolt with 10 patient sera and two healthy contros. Antihuman IgG or IgM was used as secondary antibody. Several immunoreactive bands were detected between 18-200 KD. Interestingly a prominent band at approximately 66 KD was reactive to both IgG and IgM antibodies in all patient sera tested to memtbrane and scolex sonicates of both Cysticerus fasciolaris and Cysticercus cellulosae. A 41 KD band showed was reactive of IgG antibodies in all cases but reacted to IgM antibody in 6 cases. Further low molecular weight bands at approx. 18 KD were reeactive to IgG but not IgM. The high molecular weight region of >100 KD showed several fineimmunoreactive bands. The 66 KD antigen appears the ideal candidate for analysis in a purified protein based ELISA or Dot blot eassay to eliminate the false positives obtained with the crude extract. Since the immunoreactive bands are common to Cysticercus fasciolaris and Cysticercus cellulosae the C fasciolaris antigen would be good candidate for use in immunoassays since the labroatory development of the larval stages can be carried out in a controlled manner with greater ease and cost effectively in rats.




KALA-AZAR
MYCOBACTERIAL-EPITHELIAL INTERACTION IN INNATE IMMUNE RESPONSE TO TUBRECULOSIS AND ITS ROLE IN TRANSCRIPTIONAL REGULATION OF INDUCIBLE NITRIC IOXIDE SYNTHASE (INOS)  



IMMUNODOMINANT ANTIGENS OF LEISHMANIA DONOVANI  

The study was conducted with the prime objective of isolating an antigen from the crude preparation of whole promastigotes (Leishmania dono- vani) with a view for future exploitation in serodiagnosis and production of monoclonal antibodies. Soluble antigen, prepared from promastigotes isola- ted from a actively growing culture, was fractionated by gel filtration chromatography over a column os Sephadex G200. Three peaks of proteins could be recovered. The antigenic reactivity of different fractions was checked against sera of kala azar patients by immunoelectrophoresis (IEP), rocket immunoelectrophoresis (RIEP) and enzyme-linked immunosorbent assay (ELISA). The first peak was found to be highly reactive in comparison with other peaks. SDS-PAGE and western blot analysis of this antigen revealed a major antigen of promastigotes in the vicinity of 65 to 66 KDa, while a weakly reactive triplet could also be detected in the low molecular weight region.



SYNTHESIS AND BIOLOGICAL EVALUATION OF LEISHMANICIDAL AGENTS  

One of the major requirements of a drug for eliciting leishmanicidal activity is its ability to enter the marcrophage system providing shelter to the amastigotes. The desired molecule chould also be able to interfere with the enzymes system such as superoxide dismutase and glutathione reductase etc. of the parasite to achieve the above biological profile. A series of 5-methyl-4 substituted pyrazoles: 1. 4- substituted dihydroquinolines 2. spiro-naphthalenes 3. pentasubstituted pyrroles 4. and 4-phenyl-1- (b-substituted ethyl) imidazolidin-2-ones 5. and benopyran (3,4-b pyrroles (6) have been designed and synthesised. Most of the compounds synthesised were evaluated against 'Leishmania donovani' in hamsters ('Mesocricetus auretus'). Phytoinvestigation of 'Nyctanthes arbortristis' seeds (F.verbenaceae) have also been carried out and its active principles arbotristocides A-F have been isolated. The results show that new structural leads could be generated. In all 31 compounds were evaluated for their 'in-vitro' leishmanicidal activity. Most of the compounds tested caused >75% inhibition of the amastigotes of 'L.donovani' at a concentration of 100 ug/ml. However, none was able to bring out 100% inhibition of the parasite. A total of 78 compounds were evaluated for their antileishmanicidal activity against 'L.donovani' infection in hamsters. Most of the compounds tested were found to be inactive against 'L.donovani' except compound Nos. 29,30 and 34 which caused 72-95% inhibition of parasitaemia upto day 7 post-treatment. Of these compound No.29 was followed upto day 28 which exhibited 94% activity. Two structural prototypes were designed and synthesised namely substituted pyrazoles and imidazolidones. The in-vitro and in-vivo techniques for evaluating leishmanicidal activity of compounds have been established and a number of compounds have been tested using above test methods. Immunomodulatory activity (prophylactic) studies have been standardised and method to improve drug activity by better drug delivery system has been suggested.



STUDIES ON THE CHARACTERI- ZATION OF IMMUNOREGULATORY FUNCTIONS OF PLASMA MEMBRANE ANTIGEN(S) OF PROMASTIGOTES OF LEISHMANIA DONOVANI  

In the present study, a major 66 kD plasma membrane associated molecule of promastigotes of 'Leishmania donovani' (UR-6) has been isolated and affinity purified. The monospecific antibodies were raised against this antigen. The immunoreactivity of 66 kD molecule was lost upon expsure to heat and treatment with trypsin. The metaperiodate oxidation also reduced its immunoreactivity. The 66 kD molecule of promastigotes of 'L.donovani' was, therefore, glycoprotein in nature. Employing a fluorescent probe, the 66 kD molecule was found to be located on the tip of flagellum and on the kinetoplast of promastigote of 'L.donovani'. The affinity purified 66 kD molecule provded partial protection resulting in about 40% reduction in the liver parasitaemic load as compared to unprotected animals. This study thus highlights that promastigotes of 'L.donovani' utilises 66 kD molecule not only for recognising the ligand attached to macrophages also appears to reduce the parasite burden upon immunostimulation of the host.



IMMUNOLOGICAL CHARACTERIZATIONOF 63KD AND 54KD GLYCOPROTEINSOF L.DONOVANI PROMASTIGOTES AND THEIR EVALUATION IN PROTECTION AGAINST VISCERAL LEISHMANIASIS *  

Study was carried out to characterize the 63 Kd and 54 Kd glycoproteins of the L. donovani promastigotes, determine the antigenicity and immunogenicity of these glycoproteins and to evaluate the role of these glycoprotein antigens in the induction of protective immunity against visceral leishmaniasis in experimental animals. The crude soluble antigen (CSA) of Leishmania donovani promastigotes was shown to contain several proteins in the subunit molecular weight range of about 14 to 100 Kd. Out of these a glycoprotein of 63 Kd was major component followed by another glycoprotein of about 54 Kd. Additional bands in the region of 45, 30-28 and 20-18 Kd were also evident. Sera from kala-azar patients reacted to CSA in the enzyme-linked immunosorbent assay (ELISA) and recognized the 63 Kd component in immunoblots. Some of the sera also recognized 54 Kd, 28 Kd and other bands. Immunization of animals with the CSA or separated 63/54 Kd glycoproteins induced the formation of circulating antibodies against both these antigens detectable by ELISA as well as immunoblotting experiments. Further studies showed that both the glycoproteins had the ability to induce cell-mediated immune response in immunised animals as evaluated by the lymphocyte transformation tests in vitro. Protection experiments carried out in hamsters demonstrated that immunization with 63/54 Kd antigens could induce significant reduction in the parasitic load in vaccinated animals following challenge infection with L. donovani.




LEPROSY
DETECTION OF LEISHMANIA INFECTION IN SANDFLY BY IMMUNOASSAY AND DNA HYBRIDISA-TION METHOD  

Standardisation of DNA-DNA hybridization by dot-blot method using Leishmania donovani promastigotes (UR 6 & GD Strains) has been done. L.D. promastigotes and standard kDNA (both from cultured UR-6 strain) showed positive hybridization spots. Isolation and purification of kDNA from culture of promastigotes (L.donovani) maintained in the laboratory from Kala-azar patients was also performed. For further work it is being stored in the laboratory. On the basis of total protein profile in SDS-polyacrylamide gel electro- phoresis, it was possible to differentiate between the responsive and non-responsive strains of 'L.donovani'. Protein bands of 'L.donovani' and 'P.argentipes' differ. A simple method for crypreservation of 'L.donovani' promastigotes and a medium for its isolation and cultivation without blood have been develop- ed. It was possible to maintain the colony of 'P.argentipes' upto 6th generation. In the biotopes, sandflies (P.argentipes) were found above 1.8m (6 ft.) of the ground level, requiring spray upto ceiling. In a study of population dynamics it was estimated that from 2407 flies collec- ted, a total of 7369 may emerged in a cowshed. Artificial feeding of about 800 sandflies through 6 different membranes were tried for 37 times. In three attempts from chick membrane only, altogether 4 sandflies took artificial feeding mixed with blood and promastigotes. Out of 648 sandflies fed on confirmed Kala-azar patients, promastigotes were found to develop in the gut of only 1 sandfly.



ROLE OF TRANSFER FACTOR IN MANAGEMENT OF LEPROSY  

The ability of transfer factor (TF) to reverse the anergy seen in lepromatous leprosy (LL) patients was investigated. 6 active, bacilli- ferous, anergic, LL patients and 6 inactive, nonbacilliferous, treated but still anergic, LL patients were randomly allocated to test and control arms. TF prepared from healthy donors, reactive in vivo and in vitro to M. leprase, was injected subcutaneously into patients in the test arm thrice a week for 6 months (a total of 1.3x10^10 lymphocyte equivalents). Patients in the control arm recieved the diluent, sterile water for injection as placebo, via the same route. All patients were on concomitant appropriate chemotherapy excluding clofazimine. Before starting the trial TF was shown to be free of nonspecific toxicities, and immunologic activity was demonstrated by the transfer of KLH sensitivity to naive recipients. At the end of the trial, only one active LL patient, who recieved TF, showed rapid bacterial clearance from skin and histopathologic evidence of cell-mediated immune response in the lepromin skin test site and in a skin lesion. All other patients in the test and control arms showed no difference The inconsistent effects of TF preclude use as an immunotherapeutic agent in lepromatous leprosy.



GENETIC STUDIES ON LEPROSY BASED ON HLA AND B CELL TYPING 

Study was carried out with the objectives to investigate if the inheritance of the susceptibility or resistance to leprosy HLA-linked; to determine if there is any association of HLA antigens with different types of leprosy; to investigate families with multiple cases involving parents and/or siblings to establish the sharing of any HLA haplotypes linked with the disease; to investigate the association of HLA antigens with immunity to leprosy among contacts, as judged by the lepromin test and to follow up the above contacts in order to find out whether specific HLA antigens have any bearing on the development of the disease. Study was conducted in 500-600 families to cover about 2000 participants in order to find out the possible association of HLA antigens with the susceptibility or resistance to the disease. Results of the study revealed that none of the HLA-A,-B,-C antigen had any significant correlation with the disease types except in BB group only. In this group HLA-A10 specificity showed a significant association even after correction of the P value. However, the number of BB cases were too small. Unlike earlier reports from the group of deVries results of the study did not show any significant correlation of DR-antigens with any of the disease types. This discrepancy may be due to the inclusion of larger number (408) of families as compared to the study conducted by deVries et al in 1976 and 1980 in which 16 tuberculoid families and 15 tuberculoid patients were selected respectively. Maximum RR values for haplotype frequencies for HLA-A,-B,-C and -DR antigen were noted as follows:- For TT (A26, B8; A29, C2; B22, C2; A30, DR3; B27, DR; C2, DR3); for BT (A25, B21, A2, C1 and A2, C5; B18, C2; A28, DR8; B27, DR4; C2, DR4); for TT+BT (A32, B49; A29, C2; B22, C2; A28, DR8; B27, DR4; C2, DR3); for BB (RR=0 for any haplotype association however, for one haplotypes frequency, i.e. B40, C5 a X2 value of 4.55 (P<.05) was also observed to have a O RR value) and for LL (RR=0 for any haplotype association. However, for three haplotype frequencies i.e. A3, B14; A11, C3 and C7, DR10 having X2 values of 10.90 (P<0.05), 3.92 (P<0.15) and 4.40 (P<0.05) respectively also showed 0 value for RR); in LL frequency of Gc 1-1 was less and Gc 2-1 was more than that of the normals. In LL Gc 1-1 was significantly less and Gc 2-1 was significantly more than those of TT patients.



DEVELOPMENT OF M.LEPRAE SPECIFIC HUMAN T.CELL CLONES  

The study was successful in in-vitro production of T cell lines/clones which recognise the recombinant protein LSR2 as well as the native bacillus. It would appear that even at the level of a T cell line or clone, those T cells that recognise M.leprae are able to proliferate after LSR2 stimulation. Most T cell lines/clones appear to be of CD4 phenotype, indicating that the helper/inducer cells may be selected by the in vitro culture conditions. This appeared to be true for lesional cells also, even though in situ studies had indicated that CD4+ and CD8+ cells are present in BT and ENL lesions. All T cell lines/clones obtained to date showed a/B T cell receptor bearing cells. It had recently been shown that T cells in reactional leprosy lesion had Y/& T cell receptors and it would be relevant to clone lesional cells from more reactional patients. In summary, it is considered possible to address tha question of T cell epitopes at molecular level now that we are able to obtain homogenous T cells and have a recombinant protein whose aminoacid sequence is known. Thus peptides can be synthesised based on amphipathic regions, or as overlapping sequences to define the epitopes required in T cell help. This approach may help in identifying epitopes responsible for immunoregulatory events during the disease process as well as peptides of immunoprophylacti use thus paring the way for second generation vaccines. the study has completed its planned duration.



THE ROLE OF DENDRITIC CELLS INTHE ANTIGEN INDUCED IN VITRO LYMPHOPROLIFERATIVE RESPONSES IN LEPROSY  

Studies were mainly conducted to assess the role of the lymphokine IFN gamma to reverse the energy in LL patients and to act as an adjunct to chemotherapy for the control of the disease. The single most significant finding was the reduction in bacillary load in 2/3 of patients. This reduction indicated not only bacillary killing but a clearance of bacilli from the lesions. For ethical reassons the patients continued to receive multidrug therapy and the lesions injected with the lymphokine were compared to those injected with excipient. MDT is expected to reduce the bacillary index by one log over a one year period. In many patients this was achieved within one month and persisted upto 3 months. Further follow-up upto 12 months showed no difference in the bacillary load of test and control lesions. The delayed type local response indicated that 3 consecutive injections were sufficient. Further, increase even after a test period led to refractoriness. There was no significant difference between 10 and 20 mg dose. 8 patients did not show any response even after 3-5 injections of IFN-r. Immunologically, the injected sites showed a migration of CD4+ helper/inducer subset of T cells into the lesions, these my release more IFN-g locally and perpetuate the macrophage activating functions and induction of MHC class II antigens and IP-IO protein. That lesional cells of chronic granulomas of LL patients can be stimulated in vitro to release free oxygen radicals indicates further that IFN-r may be a functionally useful lymphokine in controlling bacilli in lepromatous leprosy. This is perhaps the first report where macrophages from LL granulomas were assessed for functional ability to release H-2 O-2 and O-2.



ROLE OF NUTRITION ON IMMUNOLOGICAL STATUS OF LEPROSY SPECTRUM  

Nutritional studies on leprosy spectrum including anthropometric measurements, serum albumin, transferrin, vitamin A, vitamin E, serum elements like zinc, calcium, copper, magnesium and iron were determined. Our study has showed that the disease process has no bearing on the anthropometric indices but it has a definite correlation with micro and maccronutrients. Thus we had earlier concluded that leprosy patients had mild to moderate lowering of body measurements, this was due to associated poverty and not disease per se. Data on analysis of micronutrient concentration showed that copper increased from BT to LL end while calcium decreased from TT to LL and magnesium levels increased from TT to LL end. But serum zinc level showed no bearing on the spectrum, though its level decreased in the whole spectrum as compared to normal controls. Serum levels of vitamin A and E progressively increased from LL to BT . The levels of dietdependent protiens in serum samples ( such as serum albumin, transferrin, and prealbumin ) increased from LL to TT end. Serum iron level also increased from LL to TT end. It is tempting to postulate that gliding down by the leprosy spectrum might depend on macro and micro- nutritional markers, however ther is no experiment or clinical evidence to prove this. Growth and development, both physical and psychological of 182 children of leprosy patients were investigated. All belonged to a low socioeconomic status and were born in leprosy colonies to either one or both parents suffering from leprosy. These children were given prophylactic Dapsone monotherapy and administered BCG vaccination. Also 271 control children ( 117 high and 154 low socioeconomic status ) were included for comparison. Body measurements showed that as the boys grew older, body weights, heights and skinfold thickness fell behind in the children of leprosy patients and control children of low economic status as compared to the healthy children of high economic status. Similarly with increasing age, Quetelet indices of the male children of leprosy patients fell behind that of the normal children of the high economic status. Our findings of the body measurements of female children of leprosy patients were found to be similar to that of the male children. As the girls grew older, the height, weight and quetelet Indices also fell behind in the female children of leprosy patients of low economic status, as compared to the normal girls of high socio-economic status. More interest- ingly, the growth of normal female children of low economic status was found to fall behind even to that observed in the female children of leprosy patients. This indicates that the decreased growth velocity of the latter children in relation to the children of high social status was not associated with the illness of their parents, but perhaps due to their economic disparities. The mid upperarm circumference and skinfold thickness of the girls of the high economic status was more than that found in female children of lepromatous patients and normal children of low economic status. The mean haemoglobin levels in boys and girls, normal as well as those of leprosy patients was below 11gm\dl, suggestive of anaemia.



ULTRASTRUCTURAL CHANGES IN EARLY LESIONS OF LEPROSY  

A total of 70 clinicaly early, unclassifiable and doubtful cases of leprosy with disease of less than 12 months duration and with upto five lesions were studied to elicit fine structural changes in early lesions of leprosy, to identify definite criteria for early diagnosis and understand the pathological mechanism involved in the development of early lesions in the leprosy. All patients were subjected to skin biopsy. Light microscopy revealed histologically developed granulomas in 18, indeterminate leprosy in 14 and no lepromatous changes in 38 subjects. Electron microscopy in 25 subjects who had no specific changes on light microscopy, showed nonspecific inflammatory changes in 20 patients. Five patients revealed derangement in the fine structure of the dermal nerve twigs in the form of oedema of the nerve fiber with separation of Schwaan cells and swelling of the axons and splitting of myelin sheath. In addition, fragments of electron dense material which could be of bacillary origin were also found within the Schwann cell cytoplasm. It is concluded that as the electron microscopy of patients with negative histological finding on light microscopy showed specific changes of leprosy in only 20 per cent of subjects, it is not of significant use to confirm the disease.



INTERLEUKIN-1 AND INTERLEUKIN-2 IN LEPROSY PATIENTS AND THEIR KINETICS DURING MULTIDRUG THERAPY  

Aharent cells of peripheral blood mononuclear cells (PBMC) from leprosy patients were stimulated with Phylo Haem Agglutorium (PHA) and Phobal Myristate Acetate (PMA)/M.leprae and the quantity of Interleukin-1 B produced was quantitated by a commercial Elisa kit. PBMC were also stimulated with PHA and PMA/M.leprae and the quantity of the Interleukin-1 B produced was quantitated by a commercial Elisa kit. PBMC were also stimulated with PHA and PMA/M.leprae and the quantity of Interleukin-2 produced was measured by a commercial Elisa kit. Before commencement of therapy LL/BL patients produce significantly lesser quantities of both IL-1 Beta and IL-2 when compared to health control . After 6 and 12 months of multidrug therapy both these levels do not change significantly from their respective pre-treatment level. Before treatment indeterminate and TT/BT patients produce significantly lesser quantities of IL-1 Beta when compared to healthy controls. Like LL/BL patients these levels at the end of 6 months do not change significantly from their respective pretreatment values. Before treatment indeterminate patients produce significantly lesser IL-2 when compared to healthy controls. But TT/BT patients produce IL-2 which is almost same as that of healthy persons. At the end of 6 months of multidrug therapy both Indeterminate and TT/BT patients produce significantly increased quantities of IL-2 when compared to their respective pre-treatment levels. CONCLUSIONS: a) Therefore that a) there is some immunological defect with respect to IL-1 beta production in all the 3 groups of leprosy patients ( in indeterminate, TT/BT and LL/BL). this defect do not change after treatment with multidrugs for 6 months (in indeterminate and TT/BT or 12 months (in LL/BL). b) LL/BL patients also show defective IL-2 production and this defect do not seen to alter after 12 months of multidrug therapy. c) TT/BL patients do not show defectives IL-2 production and after 6 months of multidrug therapy the IL-2 production rises significantly from its pre-treatment level. d) Though the Indeterminate patients show an initial defective IL-2 production, the IL-2 level raises significantly after 6 months of therapy.



TRIALS ON INTRALESIONAL RECOMBINANT INTEREFERON GAMMA IN LEPROSY PATIENTS  

The study was carried out on 66 patients with polar lepromatous leprosy or borderline lepromatous leprosy to find out the efficacy of intraderma recombi- nant interferon gamma (rIFN- ) and to evaluate the duration, dose and multipli- city of injections required to induce improvement in clinical and histological features and bacillary clearance. The cellular infiltrates were also studied in the biopsies using immunological markers. After completion of interferon treatment i.e. 4-6 days later, the patients were given multidrug therapy (MDT). The MDT was continued throughout the follow up period. Patients in reaction were excluded from the study. rIFN-y in 100 ul of excipient was injected intrader- mally into well characetised lepromatous lesions. Contralateral lesions, clinically similar in terms of infiltration and erythema were injected with a similar volume of excipient only as control. rIFN-y proved to have potent effects on the lepromatous dermal lesions. Ithad multiple effects, inducing inflammatory responses akin to delayed type hypersensitivity reaction associated with cell mediated immunity. The leproma- tous skin was found to be capable of mounting an immuno-inflammatory response. There was no defect in migration of cells nor an irreversible abnormality in themicroenvironment of the bacilliferous granuloma. The studies involving histology,immunochemistry and ultrastructure gave credannce to the fact that multiple cellular and molecular events were initiated by rIFN- . The skin induration was due to epidermal thickening resul- ting from keratinocyte proliferation and infiltration of T cells and young mono-cytes into the dermis. The predominant lymphocyte was CD4+. Major histocompa- tibility complex (MHC) class 2 antigensnot only increased on macrophages but they were newly induced on hitherto nega-tive keratinocytes. In addition, IP 10,a specific protein induced by IFN-y was also seen in the skin. Repeated injec- tions of rIFN-y resulted in a distinct reduction at 3 weeks of the cutaneous load of organism from 4+to5+to1+to3+. Thedisposal of bacilli occurred faster in the diffused site compared to nodular sites. The immune induced elimination wasrapid and more effective than that observed by anti-leprosy drugs alone. It was associated with activation of macrophages. The study thus indicated that rIFN-y is a powerful biological therapeutic for leprosy. Both improvement of local immunity as well as bacillary elimina- tion were achieved in two thirds of the patients. Further systemic studies are required to establish the power of this agent as an adjunct to MDT in leprosy.



ROLE OF REACTIVE OXYGEN SPECI-ES IN KILLING OF M.LEPRAE IN CELLS OF LEPROSY PATIENTS & POSSIBILITY OF THEIR MODULATI-ON IN DIFICIENT CELLS OF HOST* 

The role of reactive oxygen intermediates in killing of phagocytosed M.leprae by the macrophages of normal healthy individuals and leprosy patients and the causes that were responsible for poor ROI in patients were studied. Experimental materials were macrophages obtained as in vitro culture from the peripheral blood of human individuals. They included healthy normals, bacteriologically positive leprosy patients (B+L), bacteriologically negative long term treated patients (B-L) and tuber- culoid leprosy patients (TT). The fully mature phagocytic macrophages cultured for 7 days were challenged with live or heat killed armadillo derived M.leprae and the H2O2 and O2 released were estimated. The viability of M. leprae phagocytosed by the macrophages was determined by determining the ability of fluorescin diacetate binding to the bacteria while inside the cells. The number of green fluorescin bacterial is the proportion viable among the total acid fast bacteria. In normal macrophages, bacilli with a viability of 56% after phagocytosing was reduced to 21%; whereas in all the other types of macrophages there was no reduction in viability after phagocytosis. The phagocytosed M.leprae were recovered and their ability to grow in the foot pad of mice was determined along with those cells treated with scavengers of superoxide, the superoxide dismutase and that of hydrogen peroxide - catalase. It was clear along with production of O2, viability was lost and when O2 was removed by SOD and H2O2 by by catalase, viability was not reduced. To confirm the role of reactive oxygen intermediates (ROI) in the killing of M. leprae in the cells of normal individuals and inability in the cells of leprosy patients, experiments were carried out in presence scavengers of ROI like superoxide dismutase, catalase, sodium benzoate or thiourea. The data clearly indicated that if ROI production is blocked, the killing of the bacilli inside the phagocytes of normal individuals was blocked. In another set of experiments with phagocytes obtained from leprosy patients, when they were challenged with live M. leprae in in vitro cultures, phagocytosed bacteria were not killed. But if the cells have been exposed to an immunomodulator, it was found that activated super- natant from such DOC exposed cultures were able to modify the macrophage to recognise M. leprae and produce ROI and also kill the M. leprae. This again was blocked by the scavengers. Further experiments were carried out to test whether the early transient viability of M. leprae in the paucibacillary leprosy were also due to failure of the bacilli being killed by the reactive oxygen intermediates. This is important since paucibacillary patients (tuberculoid leprosy type) are endowed with ability to eliminate bacteria through cell mediated immunity, just like normal healthy resi- stant individuals. Yet the immune mechanism in paucibacillary cases is abnormal and this could be due to an unusual type of CMI initiated by a particular set of antigens, unlike the resistant normals. Further, data clearly indicated that macrophages from paucibacillary individuals are unable to recognise M. leprae and produce ROI nor kill M. leprae. The macrophages of lepromatous leprosy patients were found to have unusual features regarding levels of these 3 surface markers as compare to the normal resistant individuals. However, on exposure to the DCC stimulated supernatant, the surface markers were modified to the levels closer to normal macrophages. This could indicate a normalization of the defective macrophages, which then could recognise M. leprae and phagocytose and kill them through ROI. However, such an explanation may not be true for tuberculoid leprosy patients since they do not show the pre-existing surface changes. The only possibility under this condition that DCC as an antigen stimulates the macrophages and T-cells of paucibacillary patients quite differently from that of whole M. leprae to which also the macro- phages and able to respond. In separate studies using antibody coated M. leprae and DCC it was demonstrated that the macrophages of the In separate studies using antibody coated M.leprae and DCC it was demons- trated that the macrophages of the paucibacillary patients appear to have different binding/recognition sites for M.leprae and DCC. This then again indicated a surface disposition in paucibacillary macrophages, perhaps, different from normal resistant individuals. It appears that the basic defect of preexisting membrane perturbations both in the lepromatous and tuberculoid leprosy patients may make the macrophages unable to recognise M.leprae and kill the phagocytosed bacilli through ROI. It was demonstrated that macrophages from normal individuals are able to phagocytose M.leprae and kill them through the production of reactive oxygen intermediates like H2O2, O2 and OH. In leprosy patients such an event does not take place. However, an immunomodulator which has vaccine potential is able to activate the leucocytes of leprosy patients and the culture supernatant of such a process is in turn able to activate the the macrophages to kill M. leprae through reactive oxygen intermediates. The mechanism of activation by the culture supernatant appears to modify the surface properties of the macrophages of the leprosy patients so as to normalise the preexisting defects. Such normalization leads to recogni- tion of M. leprae and their killing through ROI system.



STRATEGY FOR A POSSIBLE NEW VACCINE AGAINST LEPROSY-USE OFRECOMBINANT GENE PRODUCT  

Monoclonal antibodies to delipidified cell components (DCC) of M.leprae were obtained through hybridoma technique and two monoclonals with affinity to 38kDa and 65kDa proteins of DCC have been identified. Other two did not pick up any specific components in the Western blot. One monoclonal helped distinguishing this 65kDa cloned protein as different from classical hsp 65kDa of M.leprae. The monoclonals were not specific but showed reactivity to some other mycobacteria. The mono- clonals can pick up antigens in the sera of lepromatous leprosy patients by ELISA as distinguishable from normal or tuberculoid leprosy sera.



STUDIES ON THE ACID-FAST NOCARDIOFORM CHEMOAUTOTROPHIC BACTERIA ISOLATED FROM HUMAN &ANIMAL TISSUES INFECTED WITH LEPROSY BACILLUS*  

The aims and objective of study the to study the acid fast nocardioform chemoautotrophic bacteria isolated from human and animal tissues infected with leprosy bacilluso. Tissue specimens were obtained form 27 patients attending the department of leprology, School of Tropical Medicine, Calcutta and also from 5 patients attending the leprosy unit of the department of Dermatology, Calcutta Medical college. All these cases showed active clinical disease, and as far as could be known, did not have any antileprosy treatment. Of these 32 cases, biopsies were obtained from 12 patients and slit-skin specimens were collected form the rest. The biopsy homogenates were preserved at -10 centigrade for further work leprosy tissues followed by feshly isolated cultures forn BL/LL cases were subjected to different tests for identity with the leprosy bacillus. These comprised Gram stain, Z-N stain, degree of acid fastness, pyridine extractability of acid fastness, abilities to attack/ utilise collagen, gelatin, liquid paraffin, urea as well as presence of DOPA-oxidase and catalase. It was observed that all the 32 isolates were weakly acid fast, possessed DOPA- sxidase, catalase and gelatinase, and grew luxuriantly in presence of urea or paraffin as the sole source of carbon. Several other studies are now being carried out to find out the similarities of chemoautotrophic nocrodoform (CAN) bacteria with leprosy bacillus. In vitro antibacterial effect of rifampicin, norfloxacin, ofloxacin and other on CAN bacteria was under taken.It was observed that the antibiotics/ chemotharapeutic drugs finally selected are given in tis included inclusion of some new and exclusion of several; 15 CAN isolates could be tested. Ome prospective antileprosy drugs were taken up first for in vitro susceptibity tests on CAN bacteria which could be equivalent of the leprosy bacillus. For this purpose, each drug was incorporated in GMA and also in fluid GM at concentration of 0,1,5,10,20 and 50 mue gram/ ml, taken in McCartney bottles. The bottles were inoculated with small inocula (ca 10000 bacteria), incuibated at 28 centigrade and observed for appearance of growth first after 2 weeks and then twice a week for a period of 3-6 months. Results are given ofloxacin was found to be considrably effective, equalled or closely followed by rioampicin; a minimal antimicrobial activity was noted with norflo norfloxacin. Results of studies wit Na stibogluconate ( a pentavalent antimony compound), urea stibamine, clofazinine, DDS, INH and other quinolones and others are given. This is the first time that an antibiotic sensitivity of in vitro equivalents of leprosy bacillus had been raperted on the basis of conventional antibiotic sensitivity test system.



ROLE OF MICROSCOPIC NEUROLYSISIN LEPROSY  

Surgical nerve deompression (neurolysis) alone or combined with intralesional (perineural) corticosteroid therapy for treatment of nerve involvement in leprosy were evaluated in 61 leprosy patients. Patients studied presented with features of neuritis viz pain, tenderness, palpability and sensory or motor deficit in the ulner nerve territroy of less than six months duration and without atrophy of hand muscles and contracture or deformity of hand. Children below 10 years of age were not included because of difficulty in evaluation of nerve damage. All patients were on anti leprosy drug during the study. Patients were studied in two groups. Group A patients (33) were subjected to neurolysis while Group B patients (28) were subjected to neurolysis plus intralesional (perineural) corticosteroid injection. Nerve function was evaluated prior to neurolysis and at the end 3rd week, 3rd month and 1 year following neurolysis. Patients in Group A, possibly due to perineural corticosteroid. Relief from pain and tenderness was seen in all patients of both groups, but it occured earlier in Group B. Recovery of motor and sensory function also occured earlier in Group B. Recovery was better when the duration of nerve involvement was short (< 3 months>), nerve function deficit less and in patients with paucibacillary leprosy. patients with short segment of nerve involvement with minimal thickening had better recovery. One patient (Group A) with deterioration at one year had fibrous adhesions around the nerve and thickening of nerve was more marked. The results on a small show that in patients on specific drug treatment, progressive nerve damage could be prevented by neurolysis. The beneficial effect of surgery could be accentuated with local corticosteroids at suitable intervals. Combined treatment of neurolysis with perineural corticosteroid injections is recommended when nerve damage is early. However, this requires confirmation on a large sample.



FUNCTIONAL VALUE OF NERVE DECOMPRESSION IN LEPROSY PATIENTS  

Functional value of nerve decompression in leprosy patients. The nerves of 34 patients of leprosy with nerve involvement ranging from 22 days to 7 years were subjected to nerve decompression to assess its benefit in terms of symptomatic releif and improvement in functions/sensitivity of the afected part ( foot and hand). Due to difficulties in evaluation of nerve damage and improvement, children below 10 years of age were excluded from the study. Twenty five nerves were subjected to nerve decompression alone under local anaesthesia, while the other 25 nerves were treated with intralesional conticosteroid injection intraoperatively, followed by 2 injections at weekly intervals postoperatively. Postoperative recovery of function was assessed at the end of 6 weeks. Sweat prints of the affected part ie palm/foot were taken preoperatively and 6 weeks postoperatively to assess the enhancement of sweat secretion. Neurological involvement was observed more ofter in the tuberculoid leprosy patients (88.23%) and least in the lepromatous patients (5.9%). Loss of sweat secretion and nerve thickening was seen in all patients. Nerve pain and tenderness were seen ( in 84% and 94% patients respectively) in the majority. Improvement in nerve function was observed in most of the nerves subjected to nerve decompression. The nerves subjected to intralesinal steroid injection had better improvement in nerve function. Deterioration or no change in nerve function was observed in nerves with a long duration of nerve involvement and presence of nerve abscesses. Lesser the duration of nerve involvement better was the recovery following either nerve decompression alone or when combined with intralesional steroids. The best results were obtained when nerve involvement was of less than 6 months duration. Marked relief in pain was observed in all nerves subjected to nerve decompression. Enhancement in sweat secretion from the preoperative status was noticed in both the groups which was more marked in patients given steroids. However, the amount of sweat secretion did not reach normal level in either group during the study.



DOMINANT ROLE OF LIPOPROTEINS IN FOAM - CELL FORMATION AND NERVE DAMAGE IN LEPROSY  

Objective of the project were to study the role of Lipoproteins in nerve damage and foam cell formation in leprosy; to demonstrate that the degenerating and regenerating peripheral nerves in leprosy possess the components of cholesterol transfer mechanism for membrane biogenesis; to trace the cellular association of Apo-E, Apo-A1, Lipoprotein(a) and LDL receptors in various types of leprosy; to study the effect of potential anti leprosy drugs, vaccines or multidrug therapy on these lipoproteins. The development of foam cells is one of the key events in leprosy and the origin of lipids in foam cells has been attributed to LDL and VLDL. Extra cellular lipids in foam cells are thought to be in a dynamic state taking up and degrading cholesterol. The important role of macrophages in the pathogenesis of leprosy to phagocytose bacterial cells has been known but the process of foam cell formation invivo and its possible role in leprosy remains to be elucidated. The circulating levels of Lp(a) a genetic variant of LDL and LDL receptors in lepromatous leprosy are found to be elevated as compared to the Tuberculoids and Controls. These observations implicate the role of LDL receptors and Lp(a) in foam cell formation in leprosy. These receptors have been characterized in terms of their physico-chemical properties to find out if these have undergone any significant alterations due to bacterial infection or is it that onlu the number of binding sites have changed. A double antibody equilibrium RIA specific for Apo-E was standardized in our laboratory and used to estimate the the circulating levels of Apo-E in leprosy patients. These levels were significantly raised and the Apo-E receptors in nerve and skin biopsies showed an increased amout of binding sites in Tuberculoid group as compared to the other groups. The focussing pattern of the delipidated serum by an IEF shows bands in a p1 range from 7-8 in the lepromatous and from 5-7 in the Tuberculoid group.



LEPROSY VACCINE TRIAL  

Objective of the project were to study the role of Lipoproteins in nerve damage and foam cell formation in leprosy; to demonstrate that the degenerating and regenerating peripheral nerves in leprosy possess the components of cholesterol transfer mechanism for membrane biogenesis; to trace the cellular association of Apo-E, Apo-A1, Lipoprotein(a) and LDL receptors in various types of leprosy; to study the effect of potential anti leprosy drugs, vaccines or multidrug therapy on these lipoproteins. The development of foam cells is one of the key events in leprosy and the origin of lipids in foam cells has been attributed to LDL and VLDL. Extra cellular lipids in foam cells are thought to be in a dynamic state taking up and degrading cholesterol. The important role of macrophages in the pathogenesis of leprosy to phagocytose bacterial cells has been known but the process of foam cell formation invivo and its possible role in leprosy remains to be elucidated. The circulating levels of Lp(a) a genetic variant of LDL and LDL receptors in lepromatous leprosy are found to be elevated as compared to the Tuberculoids and Controls. These observations implicate the role of LDL receptors and Lp(a) in foam cell formation in leprosy. These receptors have been characterized in terms of their physico-chemical properties to find out if these have undergone any significant alterations due to bacterial infection or is it that onlu the number of binding sites have changed. A double antibody equilibrium RIA specific for Apo-E was standardized in our laboratory and used to estimate the the circulating levels of Apo-E in leprosy patients. These levels were significantly raised and the Apo-E receptors in nerve and skin biopsies showed an increased amout of binding sites in Tuberculoid group as compared to the other groups. The focussing pattern of the delipidated serum by an IEF shows bands in a p1 range from 7-8 in the lepromatous and from 5-7 in the Tuberculoid group.



INTEGRATION OF NATIONAL LEPROSY ERADICATION PROGRAMME ACTIVITIES WITH PHC SERVICES FOLLOWING MULTIDRUG TREATMENT IMPLEMENTATION  

The main objective of the study was to implement NLEP activities through Primary Health Care (PHC) staff in areas where MDT was implementd through vertical approach for 5 years or more and study its implications and impact. To achieve this it was planned to access the training requirements of Primary Health Care staff for implementation of activities NLEP, develop a training manual and train PHC staff. To compare and evaluate the NLEP activities when implemented through vertical set up and PHC set up side by side; to find out the impact of NLEP integration on other programmes being implemented through PHC namely family welfare, expanded programme on immunization, maternal and child health (MHC) etc. Continued implementation of conventional NLEP strategies under the existing circumstances of reduced disease burden after implementation of MDT and static status of new case detectin of leprosy is not cost effective. Integration of the NLEP with the PHC system is promising alternative. To study implementation of NLEP activities through PHC staff in areas where MDT was implemented through vertical appraoch for 5 years or more and study its implications and impact. CLTRI Chengalpattu organized a pilot study on integration of NLEP with PHC in two districts of Andhra Pradesh State during 1997-1999. Population covered under the study project was 16,39,307(2,39, 141 study and 14,00,163 control group). The results the study at indicate that prevalence in the study and control area after the implementation of integratin is 2.70 and 1.91 per 10000 respectively. The difference is statistically significant at 95% C.I. level ( p= 0.00001). NCDR is 5.71 and 4.16 per 10000 person years. The difference is statistically not significant (p=0.49). Case holding in stud and control area is 66.3% and 96.9% respectively. This difference is statistically significant at 95% confidence interval (p=0.0001). No difference is observed in the objective performance of the other programmes by the MPHWs in the area. The integration of vertical NLEP activities with horizontal approach of PHC system is feasible, if general health care providers are properly motivated, adequately trained and given the responsibility of implementation. Problems like individual technical incompetency, lack of supervisory and monitoring skills, no referral services, poor record maintenance and other administrative issues were encountered. Such problems can be dealt by providing a specialized component of services which means provision of training facilities, technical supervision and referral services at intermediate and middle level of planning and evaluation.



EPIDEMIOLOGY OF DISABILITY IN MULTI DRUG THERAPY PROJECT DISTRICT  

The major objective of this project were to study the magnitude of the problem of disability before the onset of MDT project and after the completion of the project; to study the deformity rate in the newly detected cases; to study the extent of disability that can be directly related to disease process per se. and that due to inadequate patient care in relation to anaesthesia and muscular weakness. A total of 2547 cases (860 MB, 1687 PB) released from September 1985 to September 1995. Only 2015 subjects (79.1%) could be contacted 612 subjects were MB type. The others had either left the place or died. Out of 2015 cases 113 cses (5.6%) were wrongly diagnosed and 84 (4.2%) cases had not completted MDT. It is the convention not to report grade I disability but in the present study all the the grades I, II and III while evaluating grading. It was observed that grade I disability rate had decreased significantly after MDT in the patients studied. In upper extremities grade I disability rate fell from 3.9% to 2.4% and similary in lower extremities from 3.1% to 2.0%. But it was not due to reversal to normalcy but their patients progressed unfortunately to serve deformities i.e. grade II and III. If only grade II and III were considered a ddefinite increase in disability rate was observed form 6.1% to 7.6% in upper extremities. Siilarly an increase form 4.1% to 5.1% in lower extremities was also obtained. It is worth mentioning that under National Leprosy Eradication Programme the protocol for disability grading enlists and records grades II & III only. The relsuts of present study also highlight disability rate to be 5 to 7 times more common in a multibacillary (MB) cases, if compared wih disability rates in Paucibacillary cases (PB). It is quite evident that deformity has progressed during treatment which could have been reduced if proper care and monthly examination of patient was done in actual practice and in the right ernest. We had observed that the most of the medical officers working under National Leprosy Eradicaition programme examine patients rarely. Similarly para medical wokers (PMWs) are not quite competent enough to recognise the severity of deformity status. Hence, the negligence increases the agony of the patients that is highly in desirable. It means that the drug distributin points are true to their name only where drugs are distributed. Clinical status of disease and complication are rarely and seldomly looked for, hence the peril of the patients continues. Most of the cases with disability had a long duration of disease. Half (51%) of them had disease for a duration of over 5 years before they started MDT, one fourth (24.5%) had the disease for 3 to 5 years. This observation confirms the earlier reports are and it also highlights that if we pick up a patient within 6 months of development of disease that disability could practically disappear from the scene. The reductin in thhe incidence of disabilities can be explained by the efficacy of drugs, tjhe reduction in the duration of the disease, the reductions in incidence of lepra reacitons and by operational factors such as; Improvement in early detection and management of cases. Only visible physical disabilities have been considered here. Temporary physical disabilities before and during treatment, reactions and complication, and the inconvenience of sustaining long duration treatment, should all be considered in order to estimate the impact. Moreover, among all infections and it is difficult to assess the individual social and psychological impact it causes. Estimates based on informations collected from the field through control programmes are often quensioned. It is clear that the sensitivity, specificity and completeness of informations collected should be evaluated. On the other hand, applying rates collected from scientific studies conducted in limited places to a theoretical ' populatino at risk' of leprosyy is also likely to lead to over estimates. Moreover, information on the incidence of disabilities with out intervention and before, during and after treatment is very limited. Hence, it is expected that combinations of methods will give some idea of the complexity of the problem, and will stimulate further work on the collection of reliable data relating to incidence of disabilities.



ROLE OF LSR/15 KD RECOMBINANT PROTEIN OF M. LEPRAE IN REACTIONAL STATES OF LEPROSY  

Aims and the objective of the study waer to evaluate LSR/A15 recombinant protein as predictor/ marker of reactional states in leprosy using conventional ELISA; To characterise and identify B cell epitopes of LSR/A15 that are recognised by the reactional patients; to evaluate LSR/A15 antigen stimulated T cell functions in reactional patients using lymphoproliferative and cytokine with PBMC. Both T and B Cell responses of leprosy patients to LSR/A15 antigen of M.leprae was undertaken on 311 subjects, 20 of whom were healthy contacts in a prospective study with 6 monthly follow up over>3 years. The lymphoproliferative and antibody responses were evaluated with a view to identifying the T cell and B cell epitopes of the LST/A15 as recognised by the leprosy patients during the course of stable as well as reactional disease. Overlapping 10-15 mer peptides as well as end to end peptides with and without C G residues at the termini were used to dissect the epitope recognition by sera and PBMC of leprosy patients. Moreover the antibody response of patients was monitored at 6 monthly intervals. Cytokine release and expression was also examined. In general the epitopes recognised by different ethic populations varied for both T and B cell responses. It was evident that P2 and P3 peptides were recognised by sera from ENL reaction patients but not by those with stable disease. Antibodies to the peptides and LSR decreased during multidrug therapy to negligible levels by 12 to 24 months in the majority of patients. Significantly, 15 subjects continued to show these antibodies and develope ENL reaction by 6 to 12 months. Thus monitoring of antibody titres would have clinical and prognostic significance in ENL reactions. P2 and P3 peptides have predctor role for ENL but not for reversal reactions. IgG subclsaaes also showed differences between stable and reaction patients with predominance of IgG3 and low levels of IgG4 to the peptides in ENL reactions. T cell epitope recognition showed 4 patterns, multi epitope recognition, restricted epitope recognition, single epitope recognition and no epitope recognition. Apparently anergic lepromatous leprosy subjects showed T cell responses with peptides as well as LSR. In general more patitents recognised P2, P3 and LA8 peptides. Cytokine pattern showed the presence of both IFN gamma and IL4 in majority of stable disease patients <40% lepromatous and >50% tuberculoid patients showed only IL4 and IFN gamma respectively. Interestingly during ENL, reactions in lepromatous patients, IL 4 was not detected and INF gamma emerged as the major T cell cytokine. It would appear that dysergulation of IL4 may be the major precipitating factor for ENL reactions.




MALARIA
AN INVESTIGATION OF LEPROSY AFFECTED BEGGARS AS A HIDDEN RESERVOIR FOR TRANSMISSION OF LEPROSY  

Aims and objectives with which the scheme was started were to systematically screen clinically and bacteriologically all leprosy affected beggars; to describe the background of those berrars; to examine the family members if any, living with the beggars; to treat those positive with MDT; to develop a continuous maintenance and evaluation system through community organizations for recurrence/ relapse such baggar populations. A pilot study was on 193 beggars in and around Vellore Town, the Head Quarters of Vellore District in Tamil Nadu. Most of them had begged in the same place on most of the occasions. Of 193 beggars, 58 had leprosy with previous history of treatment from leprosy treatment centres. None of them were on any treatment during screening ( All the patients were released from treatment Mono/MDT according to their statement). Out of the 58 leprosy affected persons, 10 (18%) patients showed skin smear positivity with signs of clinical activity they were motivated to attend treatment centres for MDT. The study reinforces the need to screen beggars regularly. Those who turn out to be "positive" should be motivated to continue/ start treatment, guiding them to various centres available. Their close contacts must also be screened frequently, educated to carry out self examination for patches and report to repective centres if they have any suspicion.



DEVELOPMENT OF A SYNTHETIC PEPTIDE VACCINE AGAINST THE SPOROZOITES OF PLASMODIUM FALCIPARUM AND PLASMODIUM VIVAX  

The 'P.falciparum' and 'P.vivax' circumsporozerte (CS) proteins con- tains repeat sequences that are mostly conserved in various strains. These repeat sequences are thought to be the target of protective immunity. The repeat sequences of 'P.falciparum' and 'P.vivax' CS proteins viz (NANP) 4 and (GDRADGQPA)2 have been synthesised by solution phase technique. These were characterized by physico - chemical means. Antipeptide antibodies were generated in rabbits using water soluble adjuvant, tuftsin. It has been already established that tuftsina tetra- peptide, helps in antigen presentation to various cells of the immune system especially to macrophages and T cells. The antibody titres are comparable with that generated by Freunds' adjuvant. Hence in vaccine trials, use of tuftsin, as an adjuvant cannot be ignored. Moreever it is permissible to humans, is non-toxic and non-pyrogenic. Further, during the competitive EIA procedure, a minimum structure of three repeats of NANP and two repeats of GDRADGQPA are found to be essential for immunological reactivity. These peptides have been tested as test antigens for detecting anti-CS antibodies to 'P.falciparum' and 'P.vivax' infection in endemic area, where mixed infection is prevalent (i.e. in Ghaziabad district). The results show age dependent increase in antibody levels and the titre is dependent on the parasite density in a particular village. This is the first reported study using defined peptide sequences of P.falciparum' and 'P.vivax' to detect anti CS antibodies, that can discriminate mixed infection. Furthermore, the present work highlights that the Indian strain had more or less conserved sequences within the repeat region.




MATERNAL AND CHILD HEALTH
DETECTION OF MALARIAL INFECTION (SPOROZOITE STAGE) IN VECTOR MOSQUITOES USING MONOSPECIFIC ANTIBODIES  

Detection of material infection (sporozasite stage) in vector mosquitos using monospecific antibodies: The study was carried out in four malaria endemic areas of West Bengal viz. (i) distric Jalpaiguri, (ii) Ajodhya hills of district Purulia, (iii) selected villages in district South 24 Parganas and (iv) selected areas in calcutta city with the objective of collecting information regarding vectors of malaria, their vectorial potential and their relationship to disease. The vecotr species encountered in district Jalpaiguri were Anophelese. An.culicifices, An.minimus and An.fluviatilis. Survey in Ajodhya hills revealed the presence of An.subpictos, An.culicifacies, An annularis, An. maculotus and An.fluviatilis. The anopheline fauna of district South 24-Parganas consisted of 16 species of which six were vectors of malaria viz. An.ennularis, An.fluviatilis, An.subpictus, an.culicifacies, An.fluviatilis and An.sudndaicus. The Culcutta city harboured An.stephersi and very less number of An.subpictus also. ELISA on mosquito-triturates of vector speices of anophelines (An.annularis, An.fluviatislis, An.subpictus and An.maculatus) for the detection of Plasmodium falciparum circum-sporozoite-protein (CSP) using pf-monoclonal and pf-monospeicfic antibodies and thei8r corresponding controls did not reveal the presence of P.falciparum sporozoitas, where as only 3.28% of 3200 An.culicifacies showed the presence of sporozoites An.stephens from Calcutta city also found to harbour P.falciparum sporozoites. Results of blood meal analysis of An.culifacies, An.annularis and An.stephesi revealed that 98.28% An.culicifacies and cent percent An.annularis from catle sheds were boviphilic, where as, 87.5% and 10.22% An.culicifacies collected from human blood respectively. On the other hand 89.79% and 7.4% of An.annularis from human dwellings showed the presence of bovine and human blood respectively. Amongst An.stephensi collected from catle shed 98.4% were positive for bovine blood, 1.03% for human blood, while An.stephensi collectedfrom human dwellings 13.04% had fed on human, 77.17% on bovine blood and 9.78% samples showed the presence of both bovine and human blood in their guts.



COMPREHENSIVE STUDY ON MCH CARE  

The Indian Council of Medical Research (ICMR) initiated an eight centre study in 1985 to improve the coverage and quality of MCH services at the PHC level within the existing norms of the primary health care delivery system. The study was undertaken in the states of Uttar Pradesh, Madhya Pradesh, Rajasthan, Haryana, Maharashtra and Gujarat. A "Comparative MCH Care Pack- age" of intervention was developed adopting the approach of identification and management of high risk factors in the pregnant women and their off- springs. The following strategies were used for implementing the comprehen- sive MCH care package - (i) Re-orientation training of medical and paramedi- cal functionaries to improve their technical/supervisory skills for common high risk factors in pregnant mothers and newborn (ii) Community education to increase their awareness about the high risk factors and utilization of MCH services (iii) Development of better data recording system for monitor- ing and evaluation of MCH services (iv) Developing a feasible referral system for the care of "at risk" mothers and newborn. The collaborating centres had the ICMR's research team representing the disciplines of Obstet- rics and Gynaecology, Paediatrics and Community Medicine as Principal Inves- tigators/Co-investigators as well as the District Health Officer represent- ing the state health authority in the study areas. Periodic meetings at regular intervals were held to ensure proper coopeation and coordinati between the ICMR's research team and the District/State Health Authorities. The Primary Health Centres (PHCs) were identified for the study according to the selection criteria laid down by the ICMR such as (i) No other active intervention programme should be ongoing in the study area and (ii) The infant mortality rate (IMR) should be similar to that of the respective state. The situation analysis which lasted for six months revealed that the population covered by each of the eight PHCs was variable, ranging between 80,000 to 1,69,000 except in the state of Maharashtra where the Government of India's norm of 30,000 population per PHC had already been implemented. Except for the equipment and vaccines under the Universal Immunisation Programme (UIP), the availability of otehr facilities in terms of physical space, other general supplies and equipment including the drugs were poor at the PHC level. In general, the availability of manpower was satisfactory as per norms. The provision of MCH services was limited to providing the Iron- Folic Acid (IFA) tablets and Tetanus Toxoid (TT) immunization. There was no concept of detection of high risk factors by Auxiliary Nurse Midwives (ANMs) in pregnant women/newborns or for their better care by having a suitable referral system. The quality of care was poor during antenatal, intranatal (including the use of safe delivery kits) and postnatal periods. The data recording was poor and reflected gross underreporting of births, deaths a other vital statistics. The gaps which were identified during the situation analysis were bridged in terms of strengthening infrastructural facilties with the help of state health authorities and also by the ICMR's research team as required during the preparatory phase. The other activities during the preparatory phase included the preparation of training manuals, health educaitonal materials and development of systematic data recording forms such as MCH cards and registers. A baseline survey was also done during the preparatory phase to obtain the household information and MCH/FP practice in the study area as well as to determine the denominators of pregnancy and vital rates. The otehr important activities during the preparatory phase included the initia- tion of re-orientation training programmes for health functionaries, commu- nity education activities, improving the record forms and data feedback system and establishing a referral system. The package of interventions for the comprehensive MCH care utilizing the high-risk approach strategy was initially implemented at the one third area of the PHCs in the 30,000 popu- lation for a period of two and half years (30 months). Thereafter, these interventions were expanded to the remainder of the two thirds of the PHC study area. As a result of above interventions, there was a significant improvement the quality and coverage of MCH services. For example, there was a progres- sive increase of registration of pregnant women during the intervention period - 2282 women in 1988, 5202 women in 1989 and 5423 women in 1990. The majority of pregnant women registered, about 77 percent, were between 19-28 years of age. The average incidence of pregnancies in women having less than 18 years of age was 6 percent, except a higher incidence of 14 percent observed in the PHC at Maharashtra. About 20 per cent of registered women were primiparous and 17 percent were para 4 and above. Apart from the progressive increase in the registration during the intervention phase, there was an encouraging trend of increase in early registration of pregnant women who were less than 19 weeks of pregnancy. Besides increase in number of home visits by the health functionaries, the coverage of pregnant women also showed an increase for IFA tablets distribution from 53 percent to 71 percent and for TT immunization from 61 percent to 82 percent. While majority of 73 percent of deliveries were conducted at home, it was heartening to note that there was an overall increase in the use of Trained Birth Attendants (TBTAs) by the pregnant women during the intervention period. The birth weight recording which was non-existent before, showed a significant increase of its recording by spring balance in 69 percent of newborns. A decreasing trend of the babies being born of low birth weighi less than 2499 gms. was also observed during this period. Apart from an icnrease in the postnatal visits from 68 percent to 75 percent by the health functionaries, visits by them within 24 hours also showed an increase from 39 percent to 45 percent. Earlier, there was no practice of identification of commonly known risk factors in rpegnant women and newborns by the health functionaries nor any referral system existed for their better care. As a result of training progrmames,,, the health workers reported an overall incidence of one or the other of high risk factors in pregnant women to around 35 percent during the intervention phase. The prevalence of commonly known high risk factors was greater in women who were either in the ages of less than 18 years of over 30 years or who had an interpregnancy interval of less than 18 months or who had a previous bad obstetric history. Amongst the pregnant women,,, the incidence of multi-gravida (>5) was 15 percent and about 9 percent of the women had previous bad obstetric history. Amongst the newborns, the risk factor of low birth weight i.e., less than 2499 gms. birth weight was present in 12 percetn and early delivery at less than 37 weeks of gestation was seen in 6 percent. It was observed that amongst those pregnant women in whom one or the other risk factors were present, their pregnancy outcome was adversely affected as indicated by the higher incidence of low birth weig babies. It was encouraging to note that no difference in the provision of various types of MCH services was rpacticed by the health functionaries during the antenatal period to the pregnant women who had either no risk factor or who had one or more of the high risk factors. However, as a result of training progrmames, these health workers provided greater concern and intensity of care to the pregnant women in whom the presence of one or more of the high risk factors were identified. For example, the pregnant women who had the high risk factors were registered earlier i.e., at less than 20 weeks gesta- tion. These women had more hom visits by the health functionaries during the antenatal period and a significantly higher percentage of them were seen by the medical doctors. A greater awareness was observed amongst the health functionaries for the referrals since 18 percent of women having risk fac- tors were referred by them for better care as comapred to 7 percent of referrals for pregnant women having no risk factor. As a result of communi- ty education programmes, the community awareness of the utilisation of referral services for high risk pregnant women was greater. For example, 53 percent of high risk women had utilized the referral services as compared to 45 percent of women who had no risk factor. An important finding was that the greater weightage for the utilization of referral services was given the pregnant women to the age factor. For example, the pregnant women having the ages of less than 18 years or over 30 years had availed 65 per- cent and 50 percent of referrals respectively. Another important stimulus for the utilisation of referrals was the presence of clinically apparent symptoms of oedema, eclampsia or convulsions since 67 percent of the preg- nant women having one of these symptoms had utilized the referrals. It was important to note that 75 percent of referrals were made to the subcentre (SC) level. Furthermore, the outcome of pregnancy was better amongst the risk women who had utilzied the referral services, since incidence of low birth weight babies was 26 percent as compared to 35 percent in women who did not avail the referrals. In summary, the comprehensive MCH care package of interventions utilizing the high risk approach resulted in a significant improvement in the coverage and uqality of MCH care for pregnant women and newborns at the PHC level within the norms of existing primary health care delviery system in the country. The data also indicates that while there was a general improvement in the MCH services, better care and referral services could also be provid- ed to the rpegnant women having onr or the other commonly known high risk factors. Furthermore, there was an increase in utilization of MCH services by the pregnant women. The experience gained and the lessons learnt fr the present study can be suitably applied to the Child Survival and Safe Motherhood (CSSM) initiative being udnertaken in India and other devleoping countries.




MEDICINAL PLANTS
ASSESSMENT OF ANTIOXIDANT STATUS IN PREGNANCY INDUCED HYPERTENSION  

The present study was conducted with the notion that elevated free oxygen radical productiom can induce the pregnancy induced hypertension due to damaging effect on endothelial cells found in PIH. The aim of this study was elevate the blood levels of oxidative stress assessed by measuring malondialdehyde, antioxidant enzymes and antioxidant factors in women with PIH comparing these levels to healthy pregnant women and non pregnant healthy women. Total 190 women were recruited, 10 were non pregnant healthy women, 56 were normal pregnant women, 124 women with pregnancy induced hypertension ( PIH) which were again classified into two categories; 77 women with sever PIH ( blood pressure = 171/114) and 47 with mild PIH (blood pressure = 138/100). Average period of gestation were about 36 weeks. Oxadative stress was assessed by measuring MDA levels in blood, were significantly higher in severe PIH women. Antioxidant enzymes viz; superoxide dismutase and glutathione peroxidase activity in blood were significantly decreased in pregnancy induced hypertensive women than normal pregnent women as compared to non pregnant women indicaiting an adaptational phenomenon to combat oxidative stress associated with pregnancy. However, catalase activity in blood was not significantly altered among PIH groups, while it was significantly higher in non pregnant women as compared to PIH women. This shows that the neutralization of free radicals by calalase in pregnancy is hampered. These findings suggested that pregnancy itself may have deleterious effect on catalase activity. Antioxidant factors like reduced glutathione levels were significantly decreased in normal pregnant women, women with MPIH as well as SPIH and as compared to non pregnant women. In contrast, vitamin E levels which act as antioxidant were significantly elevated in pregnancy induced hypertension as compared to that of normotensive pregnant women. Vitamin C leves were higher in pregnant women compared to non pregnent women and further increased significabntly in women with mild PIH. The increased vitamin E in SPIH and vitamin C in MPIH could be associated with increased utilization of reduced glutathione. A significant positive correlation was observed bteween MDA and catalase activity in severe PIH and a negative correlation with GSH as well as vitamin C with respect to increased blood pressure. It appears that this imbalance between oxidant antioxidant is the effect of disease and not the dausative factor and hence unnecessary supplementation of exogenous antioxidants in pregnancy may actually be not helpful for combating PIH, as any excess of antioxidant in pregnancy may it self affect the fetus at time.



STUDIES ON THE ANTICANCER ACTIVITIES OF CROTUS SATIVUS AND SARACA ASOKA  

Study was carried out to evaluate the potential anticancer activity of Crocus sativus and Saraca asoca flowers by in vitro cytotoxic studies using various cell lines like DLA, S-180, Hela, Vero etc. Both the extracts inhi- bited the growth of ascites tumours and solid tumours experimentally induced in mice. Oral administration of the extracts reduced the size of 20-methyl cholanthrene induced fibrosarcomas in mice while the same extract prevented growth of papillomas induced by 7,12 dimethyl benz (a) anthrecene in mice, besides delaying the onset of papilloma formation significantly. The extract of saffron was found to enhance the therapeutic efficacy of cyclo- phosphamide treated mice when given in combination. Many of the side effects of cyclophosphamide treatment like leucopenia, fall in haemoglobin levels and increase in enzyme levels were effectively modulated by combined admini- stration of saffron extract. Studies showed the pharmacological properties of the extracts as anti- tumour chemopreventive and a chemoprotective agent.



FILARICIDAL POTENTIAL OF SOME PLANT SUBSTANCES  

Filaricidal potential of some plant substance. Rhizome extract from Zingiber officinale, essential oils from the leaves of Anthocephalus morindaefo and Xanthium strumarium and solamargine from Solanum viarum were tested in vivo on Setaria cervi transplanted in albino rats, Acanthocheilonema vitae in multimamate rate and Dirofilaria immitis in parish dogs for their antifilarial activity. DEC was used as a reference standard. None of the plant substances showed any toxic effect when given by oral route (500 mg/kg/d for 10 days for solamargine, 400 mg/kg/d for 10 days for A.morindaefo was not toxic by subcutaneous route at 60 mg/kg/d for 10 days. Z.officinale at 200mg/kg/d (total 8 g/kg in 40 days), A.morindaefo at 200 mg/kg/d (total 9 g/kg in 45 days), and at 45 mg/kg/d by subcutaneous route (total 675 mg/k in 15 days), X.strumarium at 200mg/kg/d (total 830 mg/kg in 5days), DEC at 200 mg/kg/d (total 1 g/kg/d in 5 days) and 111 combination of solamargine and rhizome extract of solarum varium at 166 mg/kg/d ( total 830 mg/kg in 5 days) showed varying degrees of microfilaricidal activity. The combination drug (Solamargine + rhizome extract) at a relatively low dose produced a sustained antifilarial effect and was, therefore, most promising. A.morindaefo also showed macrofilaricidal action eliminating 70-80% adult worms. The subeutaneous route was ferrnd to be more effective compared to orale reite.




MENTAL HEALTH
INVESTIGATIONS ON THE ANTICANCER ACTIVITY OF COMPOUNDS ISOLATED FROM GARLIC 

Sulphur containing compounds viz Diallyl disulphide (DADS), Diallyl sulphide (DAS), Allyl methyl sulphide (AMS) and S- allyl cysteine sulphoxide (SACS) present in garlic were studied for their immunomodulatory, anticancer and radioprotective acitvities in Babb/c mice. Intrapentonecell administration of sulphur compounds was shown to stiumulate the haemopoetic system as seen from the incrreased total count bonemmarrow leneocyte cellularity and alpha esterase positive cells. Treatment with sulhur compounds antibody increased the curculating titre and number of antibody producing cells indicating the stimulation of humoral immune response. The cell mediated immune responses in normal as well as tumour bearing animals were alsoenhanced by the administration of sulphur compounds enhanced by the administration of sulphur compounds as seen from the increased NK cell and antibody dependent cellular cytotoxic activities. Sulphur compounds enhanced the production of cytokines such as IL-2, IFN-8 and GM-CSF in normal animals. The lowered levels of these cytokines in the cyclophosphamide treated animals were brought back by the treatment with these compounds. Sulphur compounds exhibited tumour reducing activity. Administration of these compounds could significantly inhibit the solid tumour development induced by DLA cells in mice. Similarly these was a significant inibition of sarcoma development induced by 20 methyl cholanthrene. Further, the sulphur compounds showed a significant radio protective effect. DADS. Protected the haemopoietic system of radition exposed animals as seen from the enhanced bone marrow cellularity and alpha esterase positive cells. Sulphur compounds also could significantly reduce the chromosomal damage induced by radiation exposure. The results are indicative of the usefulness of sulfur containing compounds from garlic in the prevention and management of cancer.




MICROBIOLOGY
A STUDY OF THE EVALUATION OF THE EFFECTIVENESS OF BRIEF INPATIENT FAMILY INTERVENTION VERSUS OUTPATIENT INTERVENTIONFOR MENTALLY RETARDED CHILDREN 

Evaluation of the effectiveness of brief in patient family intervention vs. out patient intervention for mentally retarded children. A prospective study was carried out on mentally retarded childrento evaluate the effectiveness of brief in patient and out patient family focussed interventions (developed in house) in terms of their impact on the childs development and behaviour, and family adaptation (preceived stress and coping). The components of the interventin included medical measures, family oreientation, general parenting measures, and parent training. Outcome variables studied included child variables (intellectual/ adaptive functioning and presence and serverity of problem behavviour) and family variables (self report and observer rated stress in families and level of family adaptation). A total of 157 mentally retarded children of either sex (75 in the in patient group and 82 in the out patient group) from low socio economic group (both rural and urban) and with moderate to profound degree of mental retardatin were recruited for the study. Children who had already undergone major interrvention, those with sever hearing or vision impairment which was more disabling than the mental retardation and children with progressive neurologic disorders were excluded from the study. Thirty three (44%) children/ families is the inpatient and 47(57%) in the outpatient group could commplete one year of follow up. Improvements or gains were noted on all outcome measures to a varying extent in both the groups. The gain in the child adaptive functioning was more pronouned in the inpatient group (10 points) as compared to the outpatient group (4 points). Problem behaviour came down by 55-60%, family stress reduced by 27-35% and familyadaptation was improved by 30% in both the groups. Maximum gains occurred during the first 3-6 months of follow up and subsequently remained stable over the remainder of the follow up period. There were no major effects of age, sex, and residence of outcome. It is therefore concluded that it is possible to formulate and test effective family focussed interventions in mental retardation which are suited for Indian conditions and such improvenments in both children and their families.



PULMONARY ASPERGILLOSIS AND ITS SEROLOGICAL STUDIES  

The study was carried out to find out incidence of Aspergillus species in the sputum of normal healthy persons. The incidence of antibodies in normal healthy persons was also determined. The incidence of aspergillosis in bronchial asthmatics and in allergic pulmonary infections was studied. Estimation of immunoglobulin levels including IgE in Pulmonary aspergillosis and in allergic pulmonary infections was done. The sputum samples were collected from patients admitted to KMCH, Manipal with chronic respiratory tract infections like Pulmonary tuberculosis, bronchial asthma, chronic obstructive pulmonary disease (COPD) suspected allergic bronchopulmonary aspergillosis (ABPA), bronchiectasis and reactive airways disease. Sputum samples collected on 4-6 consecutive early morning samples were examined by direct microscopy with KOH mounts, Gram's and Z.N. stain methods. The specimen were subjected for concentration method by using N-acetyl cystine hydrochloride and after concentration the specimens were inoculated into SGA, LJ media, blood agar (aerobic and anaerobic) RCM, BHI agar. The serum samples from clinical cases and normal healty persons were sub- jected to serological investigations. Antigen for serological tests was pre- pared; Aspergillus Hyper Immune serum in rabbit were raised; Gel diffusion tests were done; Countercurrent immunoelectrophoresis was done; Immunoglobulin estimation of IgG, IgA and IgM was done by Radial Immunodiffusion using Tripartigen plates. Estimation of IgE as per the methods Total IgE by RIST and specific IgE by RAST technique. Elisa test was carried out; Skin tests-Antigen for skin test was procured from V.P. Chest Institute Delhi and the tests done on Volar aspect of skin of fore- arm. I.D. The reading of skin tests was done as per the instruction provided with antigens. Out of 825 cases studied, 127 cases (15.3%) grown Aspergillus in sputum repeatedly. A fumigatus in 92 cases (11.15%) A. niger in 27 cases (3.2%) and A. flavus in 8 cases (9%). A total of 160 (19.3%) cases showed superpositi- vity by CIEP. CIEP is more sensitive than gel diffusion technique. Out of 406 normal healthy persons, none of the sputum samples, yielded Aspergillus repeatedly. Gel diffusion and CIEP were positive in 5 cases (1.2%). ELISA was positive in 3 cases (1.4%) out of 202 cases. Out of 151 cases of immunoglobulin estimation, IgG was raised in 115 cases (76%). IgM in 110 cases (72.8%) and IgA in 84 cases (55.6%). Out of 130 cases of IgE estima- tion, 106 cases (81.5%) positive of PRIST and 53 cases (40.7%) positive for RAST. None of 50 cases of normal healthy persons showed any positive PRIST or RAST. Out of 213 cases of Tested for ELISA, 145 (68%) were positive for Elisa. Only one out of 100 normal healthy persons was ELISA positive. Out of 117 cases skin tests, 46 cases (39.3%) were positive for Aspergillus antigens. Seropositivity was more common above the age of 50 years, in males than females.



PATHOGENIC FUNGAL ISOLATIONS FROM RESPIRATORY TRACT OF PATIENTS WITH CHRONIC LUNG DISEASE AND MALIGNANCIES  

A total of 669 patients with chronic lung diseases and malignancies with suspected fungal lung infections were investigated. The patients with haematological malignancies, bronchogenic carcinoma, pulmonary tuberculosis pneumonia, lung abscess with pneumothorax, chronic bronchitis with bronchiectasis, aspergilous lung diseases and allergy, comprising bronchial asthma, allergic bronchopulmonary aspergillosis (ABPA), aspergilloma and miscellancous cases with chronic obstructive airways disease, secondaries in lungs, pulmonary eosinophilia, cavitary disease of lungs, diabetes mellitus, chronic lung infiltrates and P.U.O were selected for the study. Specimens for culture collected were sputum and throat swab on three consecutive days alongwith bronchial aspirate, tracheal secretions and pleural fluid whenever needed. Serological tests performed for demonstra- tion of antibody were (i) Immunodiffusion test for antibody against Candida, Aspergillus and Histoplasma (ii) Counter Current Immunoelectrophoresis for Candida and Aspergillus (iii) whole cell agglutination for Candida and Cryptococcus (iv) slide latex agglutination for cryptococcal antigen detec- tion (v) Enzygnost IgE monoclonal kit test (Behring) for total IgE estima- tion (vi) Phadebas kits and Phadozyme (R) kits and avidin-biotin ELISA for specific IgE against Aspergillus fumigatus and (vii) skin test against aspergillin in patients with bronchial asthma, ABPA and aspergilloma cases for demonstration of type I, type III and type IV hypersensitivity. Out of 51 cases with haematological malignancies, 19 patients were found to be positive for significant growth of Candida albicans, one for C.tropicalis and 3 for Aspergillus flavus Serology was positive and correla- ted in 13 patients with significant growth of Candida and in all patients for Aspergillus. Of the 6 cases with bronchogenic carcinoma, 2 were positive with significant growth of C.tropicalis and one for C.albicans. Serology was also positive in two cases. Of the 110 cases with pulmonary tuberculo- sis 53 patients showed significant growth C.albicans (5), C.tropicalis (1) C.krusei (1) C. guilliermondii (1) A flavus (1) and A.fumigatus (1) Serology performed in 26 patients showed higher titres of Candida agglutinins in 11 out of 12 cases showing significant culture. Strong precipitin bands were seen in one patient with significant growth of A.fumigatus on culture. Significant culture of C.albicans (11 patients). C.tropicalis (7 patients), C.parapsilosis (1 case) and A.flavus (1 case) were found amongst 85 cases of pneumonia. Serology done in 25 patients confirmed significant growth of Candida in 14 patients and A.flavus in 1 patient. Out of 58 cases with chronic bronchitis bronchiectasis, lung abscess/pneumothorax, significant growth or Candida was seen in 25 cases and Aspegillus. flavus in 2. Serology performed in 14 cases, confirmed significant growth of Candida in 4 and Aspergillus in 2 patients. Amongst 220 cases with miscellaneous disorders, significant cultures of candida were seen in 102 patients, A. flavus in 2, A. fumigatus in 2 and C.neoformans in 1 patient. Serological investigation was available in 60 patients. It confirmed significant cultures of candida in 22 patients, A.fumigatus and in one patient Cryptococcosis. Of the 19 bronchial Asthma cases all were negative for Aspergillus on culture and ID test. Type I hypersensitivity was positive in 7 patients who also showed raised total IgE in serum. Specific IgE was best detected by RIA and and ELISA test. Twenty three cases of ABPA showed positivity by cultures for Aspergillus Spp. in 9 patients (A. fumigatus-4, A. flavus-2, A.niger-1, A.fumigatus+A. flavus+A.niger-2). Type I hypersensitivity was observed in 18 and type III in 3. Total IgE was raised in 17 out of 18 patients (94.4%), specific IgE was observed by RIA in 6(33.3%), ELISA in 9(50%) and AB-ELISA in 7(50.3%) patients. Amongst 6 patients with Aspergilloma, Aspergillus culture was positive in all (A.fumigatus-3 and A.species-3) Serology by ID was positive in all patients. Type I hypersensitivity and raised total IgE was observed in 3 patients although specific IgE was negative by all tests. Significant fungal isolation was noticed in 38% patients with testing of single sample, as compared with 61.5% with 2-4 specimens and 77.3% with 5 or more samples. Thus 5 or more samples of consecutive days helped in detecting more cases of respiratory fungal infections.



GARDNERELLA VAGINALIS IN THE ETIOLOGY OF BACTERIAL VAGINOSIS OR NON SPECIFIC VAGINOSIS  

A total of 807 cases were studied and 259 characterised as nonspecific vaginotos if the vaginal discharge fulfilled at last three of four criteria suggested by Memsel et al. Swabs were processed for isolation of various aerobes and anaerobes. 53.7% of NSV cases revealed that G. vaginalis is the most common organism (p>.001) associated with NSV either as a sole pathogen or in conjuction with anaerobes. Bacteroides and peptostreptococci species are the common anaerobes associated with G. vaginalis. 68.3% of NSV cases revealed plymicrobial etiology. Columbia agar base with 5% human blood nalidixic acid and gentamycin support good growth of G. vaginalis. Colonisation of the organisms in the vagina during different gestational stages of pregnancy had no ill effect of its outcome i.e. is not a maternal hazard. Of the 70 infertility cases 24% were colonised with G. vaginalis but the number is small to draw a definite conclusion. Biotyping of G. vaginalis as suggested by piot et al is an easily adoptable procedure and our findings of biotype 1 and 5 in greater frequency correlated well with other workers. It also suggests that there is no significant difference in the geographical distribution of the different biotypes. Adhesive properties of some of the isolate were studied with the haemagglutination reaction of G. vaginalis isolates from clue cell positive and negative cases with human, sheep and chick RBCs. It showed on correla- tion with the clue cell phenomenon. Hence clue cell formation may be due to other factors. Tissue culture study may throw light on this and the pathogenesis.



A CLINICO-MICROBIOLOGICAL AND CYTOLOGICAL STUDY OF NON-SPECIFIC VAGINITIS  

This study was undertaken to determine the prevalence of Non-specific vaginitis (NSV) and some associated microorganisms in women attending the GOPD Gyane out Patients's Department and Family Welfare Clinic of LNJPN Hospital, New Delhi. Eight hundred and two (802) women were included in the study, 544 as symptomatic cases of vaginitis and 258 asymptomatic controls. Trichomonas vaginitis was found in 5.5 percent and Candidal vaginitis in 6.9 percent of the cases. NSV was found in 50 percent of the symptomatic women, but also among 18.9 percent of asymptomatic women enrolled initially as controls. M.hominis was found to be significantly associated with NSV among CuT users (P<0.01) while G.vaginalis was significantly associated with NSV among non-CuT users (P<0.001). NSV and culture positivity for G.vaginalis were associated significantly with presence of excessive vagninal discharge, itching and malodour (P<0.001). A positive amine test and predominance of gram negative or gram variable coccobacilli in vaginal fluid gram's smear were found to correlate very well with NSV and G.vaginalis infection. Among the epidemiological factors studied, a duration of less than or equal to 6 years of married life was significantly associated with presence of G.vaginalis (P<0.01). NSV was more common among women with a parity of more than 2, in their postovulatory stage and were more than 22 years old (P<0.01). G.vaginalis isolates were biotyped on the basis of hippurate hydrolysis, production of lipase and beta-galactosidase (ONPG). The most prevalent biotypes were 5,2 and 1. However, no correlation would be made between any particular biotypes and NSV. Cervical cytology was possible in 441 subjects only out of the total study population and inflammation was present in 350 subjects. A statistically significant association was found between inflammation and presence of G.vaginalis o nn culture and NSV. Since the initial study did not reveal any correlation between clinical disease and epidemiological characterization of G.vaginalis by biotyping, further characterization of G.vaginalis isolates was carried out by antigenic analysis of whole cell proteins by sodium dodecyl-polyacrylamide gel electrophoresis (SDS-PAGE). The antigenic profile of G.vaginalis was compared with that of other vaginal isolates that may resemble G.vaginalis on the basis of morphology, culture and biochemical characteristics. Any variation in the antigenic profiles amongst G.vaginalis strains in respect of clinical source and biotype was also looked for. Majority of the strains showed presence of 25-30 protein bands (approximate molecular weight 17kD-20 kD) with an average of 28 bands, with prominent bands which migrated to a distance corresponding to a molecular mass of 90,74,53,51,49 and 41 kD. Visual inspection of gels showed similar patterns in all the strains of G.vaginalis and the similarity calculated by dice coefficient ranged from 73.1 - 100% (average 88.9%). G.vaginalis strains recovered from different clinical groups (NSV, Non NSV and Healthy) or belonging to different biotypes did not differ with regard to their SDS-PAGE antigenic profile or pattern. The antigenic profile of vaginal diphtheroids and unclassified coryneforms organism (U.C.O.) was quite distinct from that of G.vaginalis, the vaginal diphtheroids showed presence of 12-14 protein bands with a wide and prominent band at 140 kD and majority of U.C.O.'s showed presence of 2-3 faint protein bands only. One vaginal isolate that was initially identified as an U.C.O., was subsequently identified as G.vaginalis on the basis of its antigenic similarity to the other G.vaginalis isolates. In an attempt to develop a serotyping scheme, antisera to six vaginal isolates of G.vaginalis were raised in rabbits, however, only one of the antisera showed presence of precipitating antibodies against its immunising strain.



ISOLATION OF CANDIDA FROM HUMAN INFECTIONS, DIFFERENTIATION OF SPECIES BASED ON SUSCEPTIBILITY TESTS AND ITS COMPARISON TO CONVENTIONAL METHODS  

Study was carried out to isolate Candida from various human infections and to evolve a scheme for species identification based on sensitivity to dyes and chemicals and compare it to conventional techniques. A total of 1031 specimens were collected from various types of infections and Candida species was isolated from 433/1031 speciments. All the 433 strains were first speciated by conventional methods and then by disc diffusion tests. The chemicals/dyes used in the disc diffusion test were Brilliant green, Janus green Cyclohexamide, Fast green, Rhodamine 6G, ethidium bromide, Triphenyl tetrazolium chloride, and sensitivities were coded in that order. Majority of strains of C.albicans had the code 1200560, C.tropicalis 1230567 C.krusei 1000060, C.guillermondi 1230560, C.parapsilosis 1000507, C.stellatoideae 1000500 and C.pseudotropicalis 1000567. In the case of C.stellatoideae and C.pseudotropicalis all strains of each species gave a single code but the number of strains tested was small. All other species gave more than one profile with majority of strains being represented by one code ie., majority of strains of C.albicans had the code 1200560 but some strains had profiles which deviated slightly from typical strains. The only code which represented more than one species was 1200567 which was seen in 15% of the strains of C. albicans and 4% strains of C. krusei. This was the only instance of profile overlap and these strains could be readily differentiated by other properties such as pellicle formation in liquid media by C. krusei. The disc diffusion test appears to be reliable method of speciating candida isolates from clinical specimens, because it is simple, inexpensive and gives reliable results in 24 hours.



MENINGOCOCCAL MENINGITIS IN AND AROUND CHANDIGARH  

A total of 10 hospitalised patients from May 89 to Sept. 90 with bacteriologically proven meningococcal meningitis were enrolled in the study from neighbouring states of Haryana, Himachal Pradesh, Punjab, Chandigarh and in Panchkula (near Chandigarh). Out of these, 2 patients expired. For antigen detection, latex agglutination and CIEP was performed and was positive in 9 out of 10 patients on CSF, blood and urine. A total of 40 contacts of these patients were also examined for carriage of meningococci. It was found that only 2 out of 40 contacts (not related) were positive for meningococci. A total of 70 sera from subjects vaccinated already with meningo A+C vaccine Biomerex were collected. Antibody response against N.meningitidis A in 25 hospitalized cases of meningococcal disease, 71 persons in close contact with the above cases and 95 (50 individuals vaccinated with A+C vaccine and 45 "old" vaccinated) were studied. During the acute stage of the disease, haemagglutination antibodies could be detected in 32% and IgG antibodies by ELISA in 16% of samples. During the convalescent period both IHA and ELISA showed significant rise of antibody in 80% of the samples. In the 31 contacts of the patients antibody was detected in 58% and 38% by IHA & ELISA respectively. In the vaccinees, specific Haemagglutination antibody was detected by IHA in 56%, 82%, 78%, 74% of the pre, one month, three months and six months post vaccination respectively. ELISA test could demonstrate antibodies in 2%, 80%, 78 & 66% of the four respective groups. On the basis of the study it is concluded that the active serological response to meningococcus group A does occur in patients, contacts and following vaccination. Individuals should be followed for longer periods in this part of the country for the efficacy of the vaccine. Meningococcal vaccines manufactured by other concerns should also be studied & checked for the serological response they evoke in set up.



OUTER MEMBRANE COMPONENTS OF BACTEROIDES FRAGILIS CONTRIBUTING TO PATHOGENICITY  

Outer membrane proteins were prepared from Bacteroides fragilis, growing the organisms in brain heart infusion broth supplemented with growth promoting factors. SDS-PAGE of antigens prepared from organisms cultured have been similar polypeptide patterns in the two strains tested. Seventeen to twenty polypeptide bands in the range of 14 to 116 kD were seen in all outer membrane preparations. Polypeptide bands corresponding to 31.40kD, and 58kD were present in antigen prepared from organisms grown in culture conditions like brain heart infusion broth as such, when supplemented with cholesterol, cystine and methionine separately and all together. 21.5 kD band was present in antigen prepared from organism grown in culture conditions like BHI supplemented with cystine and cholesterol i.e. 7th and methionine and cholesterol. Antigens prepared from culture were used in immunoblotting. Various immunodominent bands were detected i.e. 116 kD, 66 kD and 42 kD in antigen preparations when sera from mice infected with B.fragilis were tested. These immunodominant antigens will be further evaluated as an immunogen for a possible candidate vaccine against 'B.fragilis' infections.



PREVALENCE OF GENITAL CHLAMYDIAL INFECTION IN SYMPTOMATIC AND ASYMPTOMATIC WOMEN  

The study was carried out with the objective of screening the preva- lence of chlamydia trachomatis infection in genital tracts of symptomatic and asymptomatic women. Symptomatic group constituted 125 women attending S.T.D. out patients dept. and 375 women attending gynaecology out patient dept(OPD) asymptoma- tic group consisted 250 each of apparently healthy women attending antenatal O.P.D. and family planning O.P.D. Endocervical specimens were collected from these women and screened for C. trachomatis by enzyme immuno assay. The results revealed that C. trachomatis is prevalent in symptomatic women attending S.T.D. and Gynaecology O.P.D. The O.P.D. as compared to the women attending gynaecology O.P.D. The prevalance of C.trachomatis is low in asymptomatic women. However group of women with Cu 'T' in situ showed a high incidence of chlamydial infection. C.trachomatis infection was observed to be more in young women (<20 years). Data also revealed that C.Trachomatis infection is associated more with pelvic inflammatory disease than with any other clinical condition.



STUDIES ON PLASMID ASSOCIATED VIRULENCE PHENOTYPES IN SALMONELLA TYPHIMURIUM  

The study was carried out on 126 multiple drug resistant strains of salmonella typhimurium obtained from the National salmonella Phage Typing Centre, New Delhi (collected from different geographical regions of India), to define potential virulence factors viz. enterotoxin production haemagglutination properties, and adhesive and invasive traits. The plasmid profiles, phage types and antimicrobial resistance patterns of S. typhi- murium strains and the association between plasmids and some of the virulence factors were also studied. All the strains produced mannose resistant haemagglutinin and bound to HeLa cells to varying degrees. These strains also potentially invaded HeLa cell monolayers. Plasmids did not influence the adhesive properties of S. typhimurium as revealed by triparental mating studies. Plasmid positive wild type strains and its cured clones (without plasmid) more or less equally adhered to HeLa cells. Mannose resistant haemagglutinin (MRHA-B) encoded on non-autotransmissible plasmids. Hence it is possible that adhesive and invasive traits in S. typhimurium may be partially controlled by chromosomal and plasmid genes. Enterotoxigenicity of S. typhimurium was tested in two biological assay system viz. rabbit ileal loop test and skin permeability test. Enterotoxigenicity was observed in 30.5 per cent strains with 17.8 per cen showing high enterotoxigenic activity in culture supernatant. The most common R-patterns were ACKTSSxTp followed by ACKSSxTp and ACTSxTp. Prevalent phage types were 99,36 and 32. Most strains (117) were untypable. Plasmids were of different molecular weights viz small molecular weights: 5.4-1.8 Md, large molecular weight: 114.3-73.5 Md and intermediate molecular weight: 35.8-22 Md Different plasmid patterns corresponded to a specific R-pattern while the converse was not seen. Phage untypable strains could be grouped into different plasmid profiles. Plasmid pattern determi- nation could be used as an epidemiological tool in a large geographic region as the plasmid profile of a given clone was zone specific.



DEVELOPMENT OF NON-CULTURE TECHNIQUES FOR RAPID IDENTIFICATION OF GROUP A STREPTOCOCCAL INFECTIONS  

Studies were carried out to compare the effect of different modes of administration of human tetanus immunoglobulins in tetanus (intramuscular, intrathecal and combined), decrease the morbidity and mortality in patients suffering from tetanus, shorten the hospital stay of the tetanus patients and assess the efficacy of human tetanus immunoglobulins (HTIG) in treatment of tetanus patients. 90 patients aged 15-65 years admitted to the adult tetanus ward of Nehru Hospital, Post Graduate Institute of Medical Education and Research, Chandigarh, between December 1990 and December, 1992 were divided into three therapeutic grades (mild -I moderate - II, severe - III) of 30 patients each. The three grades were further divided into three groups (I, II & III) depending upon route of administration of human tetanus immunoglobulin. The sub group I received intramuscular 30 IU kg-1 body weight of human tetanus immunoglobulin, group II received 500 IU of human tetanus immunoglobulin intrathecally and group III received 30 IU kg-1 body weight and 500 IU intrathecally of human tetanus immunoglobulin. The other treatment intramuscularly included sedatives, non depolariser muscle relaxants and artificial respiration. survival of patients in the present series was 81.11%. Patients who received combined intramuscular and intrathecal human tetanus immunoglobulins showed statistically significant higher survival (100%) as compared to either mode of administration alone. Hospital stay of these patients was relatively less as compared to either mode alone. Patients with incubation period of 7 days and more and patients administered those with period of onset more than 24 hours had better survival. Most common cause of death was severe chest and respiratory tract infection followed by peripheral circulatory failure and autonomic imbalance. Maximum mortality was observed during first 8 days after admission to hospital (64.7%). Incidence of tetanus was more in rural population (93.33%) and in patients who sustained trauma (58.88%).



EFFECT OF MEDICAL TYPE LASERS ON BIOLOGICAL,BIOCHEMICAL AND GENETICAL CHARACTERISTICS OF STAPH.AUREUS,KLEBSIELLA SPP AND ENTEROTOXIGENIC ESCH.COLI* 

Nitrogen laser showed no obserable effect on the colony morphology, viable count and bio-chemical characters of Esch. coli. There was an observable decrease in the number of colony forming units after Nitrogen-dye laser exposure. Carbohydrates were not fermented by the organism after Nitrogen-dye laser exposure for 70 mins. Fermentation of Dulcitol was negative after Nitrogen-dye laser exposure for 40 mins. Indole production was affected after the exposure for 60 mins. Amino acid decarboxylation; Orni thine decarboxylation became negative after the laser exposure and Arginine decarboxylation became positive after exposure. He-Neon laser showed no observable effect on biological, biochemical characters studied. There was no effect on the antibiotic susceptibility pattern of Esch. coli after the exposure of Nitrogen-dye and Helium-neon laser, whereas the organism, which was sensitive to sulphadiazine became resistant after Nitrogen laser exposure. When compared to control and Nitrogen Laser treated cells, the intensity of the plasmid DNA band have been decreased in Nitrogen dye and Helium-Neo laser treated cells. There was no effect on the toxin production of enterotoxigenic Esch. coli after the exposure of Nitrogen, Nitrogen-dye and Helium-Neon laser. Coagulase production of Staphylococcus aureus was inhibited after 40 minutes exposure of Nitrogen Laser. Manitol fermentation by Staphylococcus aureus became negative after 35 minutes exposure of Nitrogen laser but became positive after 40% 45 minutes of exposure. Since we have to complete the project within one year we could not take up the study of Argon and CO2 laser treated cells.



THE STUDY OF DTH RESPONSE IN ENTERIC FEVER USING OUTER MEMBRANE PROTEINS OF S.TYPHI AS THE ELICITING AGENTS  

Immune response specific to outer membrane proteins (OMPs) of Salmonella typhi was studied in vitro in 30 bacteriologically proved cases of typhoid fever, and follow-up after 3 months alongwith 15 age and sex matched normal healthy controls. Cell mediated immunity (CMI) against sy. typhi was studied by leucocyte migration inhibition (LMI), lymphocyte transformation test (LTT), production interleukin-1 (IL-1 alpha) and IL-2, production of leukotriene B4 (LTB4) and LTC4. Enumeration of percent and absolute peripheral lymphocyte sub-populations was performed using flourescein-isothiocyanate (FITC) conjugated mouse monoclonal antibody. Results showed that S.typhi induced specific CMI response in typhoid, both in acute phase and on follow up. Lipopolysaccharide present in OMP prepaations induced only transient cellular response, that too in acute phase of typhoid. Transient non-specific suppression of CMI was observed in acute phase. OMP specific antibodies were detected in all typhoid sera in acute phase as well as on follow up.



EVALUATION OF SEROLOGICAL PROCEDURES FOR ANTIGEN AND ANTIBODY DETECTION IN INVASIVECANDIDIASIS IN PATIENTS WITH AND WITHOUT IMMUNODEFFICIENCY* 

The study was carried out for evaluation of various serological procedures for detection of mannan and cytoplasmic antigens as also antibodies for diagnosis of invasive candidiasis. Mannan and cytoplasmic antigens were prepared from the blastosporesand mycelial phases of Candida albicans serotype A strain ATCC-1065 manintained on Sabourauds dextrose agar. Cytoplasmic antigens were purified from mannan fraction by affinity chromatography. Antisera were raised in rabbits against these antigens for antigen detection by latex agglutination test (by coating latex particles with hyperimmune IgG fraction) and countercurrent immunoelectro-phoresis (CIEP). Cytoplasmic antigen fractions were also screened for immunodominant antigens by immunoblot procedure using sera from five patients with histopathologically proven invasive candidiasis. A 47 kDa protein was foundto be the most immunodominant fraction. One hundred patients (confirmed invasive candidiasis-29, suspected invasive candidiasis-30, superficial candidal colonication and without any deep seated candida infection-41) were screened for antigen and antibody. Amongst different candida antibody detection procedures (like immunodiffusion test, CIEP, whole cell agglutination, and immunoblotting assay), immunoblot test for detection of antibody against 47 kDa protein fraction was found to be the most ideal procedure with a sensitivity of 79.3 per cent, specificity of 85.4 per cent, positive predictive value of 79.3 per cent, negative predictive value of 85.4 per cent, and efficacy of 82.9 per cent. This procedure could detect antibody in patients with immunodeficiency. Amongst antigen detection procedures, latex agglutination test for detection of mannan or cytoplasmic antigen was found to be better than CIEP. However, this procedure requires to be evaluated in patients with other fungal infections. Candida specific precipitating antibody detection by gel diffusion or CIEP was of high specificity but lacked sensitivity. Candida whole cell agglutination for detection of antibody was found to be a good sensitive (75.9%)procedure but the specificity was very low (31.7%). There was no significant difference observed in results using either of the phases of Candida - blastospores and mycelial. It may be concluded that detection of specific 47 kDa fraction antigen in serum seems to be a better procedure compared to the antigen detection tests in use currently.



CERVICO-VAGINAL FLORA IN PREGNANCY AND ITS RELATION WITH PERINATAL OUTCOME  

Cervico-veginal flora in pregnancy and its relation with perinatal outcome: The study was carried out to determine the prevalence of Chlamydia trachomatix, Mycoplasma hominis, Ureaplasma urealyticum, Gardnerella vaginalis, Neiseria gonorrhoeae and other aerobic and anerobic organisms in pregnant women and the effect of these on perinatal outcome and maternal morbidity. An effort was also made to evaluate the effect of treatment on perinatal outcome and maternal morbidity. Initially 200 apparently healthy pregnant women attending the antenatal clinic were screened twice for crevical and vaginal flora, once in the second trimester and once in the late third trimester. These women were followed up intrapartum and postpartum. Women with history of recent antibiotic use, obstetrical complications like APH, toxaemia, gestational diabetes, multiple gestation, etc. and those with severe anaemia, diabetes, chronic hypertension, renal diseases, etc. were excluded. The women(116) who were positive for any pathogenic organism formed the study group while those (84) with no evidence of pathogenic organisms served as controls. In order to evaluate the effect of treatment on perinatal outcome, 59 women of a group of 92 screened in the later part of the study were treated. For candida the treatment comprised cotrimoxazole pessaries for the women and candid onitment for their huubands; erythromycin stearate 500 mg 6 hourly for one week was given to both wife and husband for the other pathogens. Initially C, trachomatis, M. Hominis, U. urealyticum, and G. vaginalis were detected in 5.0, 8.5, 28.5 and 15.5 percent women respectively. The prevalence of Candida sp and Gruop B streptococci was found to be 18.0 and 0.5 percent. Lactobacilli alone were present in 42.0 percent, streptofaecal is in 1.5 mobiluncus in 0.5 and Staphylococcus epidermidis in 9 percent of women. Anaerobic organisms were detected in 4.5 percent, staphylococcus diphtheroids and acinetobacter in 1.0 percent each and Escherichia coli in 0.5 percent of women. Sixty eight (58.62%) women had single pathogen while 48 (41.38%) had multiple organisms in various combinations. The incidence of premature rupture of membrane (PROM) was slightly higher in the study group (10 out of 116 : 8.62%) as compared to control (5 out of 84; 5.24%). The incidence of preterm labour was significantaly more in study group compared to control. There was no significant difference in the mean birhtweight of neonates in patients compared to control. Evidence of vertical transmission of infection ie neonates being colonized wht the same organism as the mother, was seen in 18.96 percent of the cases (22 of the 116). Clinical manifestations of neonatal infection in the form of oral thrush was significant difference was seen with conjunctivitis, pneumonia and septicaemia. Post partum fever occurred in 7.5 percent (9 out of 116) women in the study group and 5.95 percent in the control group, but this difference was not significant. Treatment resulted in the lowering of incidence of PROM, preterm labour, incidence of low birth weight, oral thrush in neonates and postpartum fever in mothers.



CHARACTERISATION OF ANTIGENS OF H. PYLORI AND SPECIFIC SEROLOGICAL RESPONSE IN ASYMPTOMATIC COLONISATION AND INFECTIVE UPPER GI DISEASES *  

Characterization of the antigens of Helicobacter pylori and the specific serological response in asymptomatic colonization and infective upper gastrointestinal diseases. The study carried out on endoscopic gastric biopsies of patients with various gastrointestinal disorders to isolate, characterze immunologically and stdy the strain variation in antigenic structure of Helicobacter pylori. A total of 555 endoscopic gastric antral biospy samples from 504 consecutive patients with dyspeptic symptoms were studied for their H. pylori status. Smear microscopy, rapid urease test and culture isolation revealed 46,43 and 23 percent positivity respectively for H. pylori. SDS-PAGE protein analysis of 20 H. pylori clinical isolates consistently having nine major protein bands (molecular weights 90, 65, 60, 48, 35, 31, 28 and 21 kDa). There were detectable strain variations limited to the less prominant bands at 48-68 (1 band), 35-42 (3-4 bands) and 21-28 (3 bands) kDa regions. Western blot enzyme immunoasay (WB-EIA) of electrophoretically separated proteins of clinical isolattes of H. pylori and the reference strain using hyperimmune rabbit serum showed major common immunoreactive components at 21, 28, 42,48, 65, 70 and 116-120 kDa zones. Some minor immunoreactive bands which showed strain variations were limited to 26-32 (1-2 bands), 30-40 (4-5 bands) and > 120 kDa (1-2 bands). WB-EIA with serum samples from patients and controls showed four reactive bands in the molecular weight range of 45-65 kDa in all subjects irrespective of their H. pylori staus or histopathological severity of the lesion. In conrast 4-5 immunoreactive bands between 21 and 45 dDa were found only in patients with positive H. pylori status and evidence of active gastritis. A total of 120 family members of 40 dyspeptic patients (20 eachh age and sex matched H. pylori positive and H. pylori negative) were screened for seropositivity for H. pylori using an ELISA, developed in house and validated. wenty nine of the 69(42%) family members of the positive and 20 of 51(39%) of the negative group were found seropositive for H. pylori antibodies; with no significant difference between two groups. Seropositivity increased with age from 34 percent at < 20 years of age to > 80 percent in the 6th decade. No family clustering could be found on the basis of seropositivity. There was a strong correlation of seropositivity with upper gastrointestinal symptoms in the family members of the H. pylori positive group.



CHARACTERIZATION AND EVALUATION OF HYDATID ANTIGENSHAVING DIAGNOSTIC POTENTIAL IN EARLY ASYMPTOMATIC HYDATIDOSIS  

The study was carried out for identification and characterization of puri- fied antigens from hydatid cyst and for identification of immunoreactive hydatidfluid antigenic fractions by Western blotting which could ultimately be used forthe early diagnosis of hydatidosis and to study the kinetics of immune response in experimentally infected animal model. Hydatid cyst fluid collected from hydatid cysts of naturally infected sheepwas processed for the preparation of crude soluble hydatid fluid antigen by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). SDS-PAGEanalysis revealed multiple protein bands of molecular weight ranging from 8 to 200 kDa. Of these, most demonstrable protein fractions occurred as a thick band in the range of 66-90 kDa. Western blot analysis of hydatid cyst fluid with serum samples from patientsof hydatidosis, neurocysticercosis, amoebiasis, intestinal helminthic infection and normal healthy individuals indicated the presence of multiple immunoreactiveantigenic components ranging from 8 to 200 kDa. Of these, 8 to 45 kDa antigenicfranctions were found to be specific for the diagnosis of human hydatidosis. Sensitivity and specificity of enzyme linked-immunosorbent assay (ELISA) for thedetection of specific hydatid antibodies in optimally diluted (1:1280) serum samples of patients clinically suspected to have hydatidosis was found to be 81.8 and 100 per cent respectively. Experimental infection could be established in mice by intraperitoneal inoculation of 2000 protoscolices of hydatid cysts obtained from naturally infected sheep. Kinetics of immune response to the crude soluble extract anti- gen revealed positive response by the second month post infection which persis- ted till the sixth month post infection (follow up for six months). Crude soluble extract antigen was found to be more suitable than the purified anti- genic fraction for assessing immune response in early hydatidosis.



MOLECULAR FINGER PRINTING OF BACTERIA CAUSING NOSOCOMIAL INFECTIONS: ITS APPLICATION TO TRACE SOURCE OF INFECTION  

Plasmid fingerprinting was applied for tracing the source of nosocomial infection with plasmid containing bacteria. In addition, bacterial chromosomal fingerprinting after restriction endonuclease digestion was done to determine the relationship between isolates of bacteria causing nosocomial infection but not containing plasmids. Forty seven strains of Klebsiella pneumoniae were subjected to klebocin typing by scrape and streak method using 6 indicator strains. All the strains were found to be positive to klebocin thereby offering no discrimination. By klebocin extraction method these 47 strains could be grouped into 11 distinct klebocin types. However, on repeated testing only 35 strains gave similar pattern indicating a reproducibility of 74.5 per cent. Three major categories of klebocin types predominated even in epidemiologically unrelated strains making discrimination difficult. The two outbreaks (August 1994, June 1995) investigated were caused by a klebocin type designated 444, a group indi- cating resistance to klebocin produced by all six producer strains and essentia-lly untypable by klebocin typing. Whole cell protein analysis (WCPA) by SDS-PAGEcould subdivide the 7 strains from the first outbreak into 5 groups while the 3 strains from the second outbreak were found to be homogenous. Ribotyping of thesame isolates indicated 5 groups in the first outbreak and 2 groups in the second outbreak were found to be homogenous. Ribotyping of the same isolates indicated 5 groups in the first outbreak and 2 groups in the second outbreak were found to be homogenous. Ribotyping of the same isolates indicated 5 groups in the first outbreak and 2 groups in the second. Ribotyping had better discrimiantion and indicated that these outbreaks were actually pseudo-outbreaksand not common source outbreaks as was implied by the use of klebocin typing. Forty two strains of Pseudomonas aeruginosa isolated from bronchoalveolar lavage from intubated patients between December 1993 to June 1994 were also ana-lysed. Pyocin typing of these strains by scrape and streak method using 22 indicator strans revealed excellent discrimination in 40 strains. But on repea-ted testing the group designation changed indicating that the system had low reproducibility. SDS-PAGE of whole cell protein profile indicated presence of 45 protein bands of different molecular weights, individual isolates had 37 to 42 prootein bands ranging from 340 to 143 kDa. On the basis of Dice Index ofsimilarity the strains could be grouped into 20 types. Since all strains could be typed the system had adequate typability. Similar results were obtained on repeated testing indicating good reproducibility. Ribotyping of these isolatescould group them into 18 groups; the major group comprised 8 isolates. It is thus clear, that pyocin typing had excellent discrimination but poor to no reproducibility while klebocin typing had adequate reproducibility but notsufficient discimination. Therefore, these methods cannot be of use for reliable fingerprinting of isolates leading to tracing of the source of infection in an outbreak. Whole cell protein analysis by SDS-PAGE offered a relaible, reproducible and fairly discriminating method for identification of clones of bacteria. Plasmid analysis was not found to be a very discriminatory or sensitive indicator for fingerprinting. But ribotyping, using a DIG labelled 18 mer oli- gonucleotide probe complementary to a conserved region of the 16 S rRNA gene showed good results. The probe was stable and hybridized with DNA extracted from a wide variety of bacteria. Since the rrn operon is present in multiple copies in the DNA of each bacteria, a number of bands would light up making determination of the clonal nature of the isolates easy. Discrimination could be improved by selection of more than one restriction endonuclease for cutting the bacterial DNA.



PREVALENCE OF MYCOPLASMA PNEUMONIAE IN LOWER RESPIRATORTRACT INFECTIONS IN CHILDREN AND ELDERLY  

Prevalence of Mycoplasma pneumoniae in acute lower respiratory tract infections. The study was carried out in 249 patients (192 children <10 yr. of age, 43 adults between 18-30 Yr, and 14 elderly >60 Yr) with acute lower respiratory tract infections and 55 normal healthy individuals (matched for age) to find out the age related prevalence of Mycoplasma pneumoniae and correlate the clinical findings with M.pneumoniae to identify the risk factors. M.pneumoniae was found to be an important respiratory pathogen in patients of all ages; the prevalence being 34.89%(67/192) in children,46.5%(20/43) in adults and 14.28%(2/14) in the elderly. Coexistence of M.pneumoniae with other bacterial pathogens (both Gram positive and negative) was frequently seen in all age groups; 17.07,48.9 and 14.28% in children, adults and the elderly, respectively. A past history of parents having had upper and lower respiratory tract infections was identified as a risk factor in the children whose parents had at least 1-3 episodes of upper and lower respiratory tract infections per year. The high incidence of M.pneumoniae in patients of all age groups thus emphasizes the need for examination of specimens of this pathogen in all patients with acute lower respiratory tract infections. For the complete diagnosis of M.pneumoniae infection culture/antigen detection must be supplemented with serological examination so as to plan appropriate treatment strategies for the management of acute lower respiratory tract infections in these patients. Since Mycoplasma infection was seen along with other bacterial p pathogens, it is suggested that the therapy should be directed towards both pyogenic and a typical pathogens for a good clinical and therapeutic response.



SERODIAGNOSIS OF INVASIVE ASPERGILLOSIS  

The study was carried out to identify and purify immunodominant antigens from circulating antigens of Aspergillus fumigatus present in invasive aspergilliosis as also to evaluate the purified immunodominant antigens in antibody and antigen detection tests for the diagnosis of invasive aspergiolosis in experimentally infected mice and in patients suffering from invasive aspergillosis. Crude antigens of the two stains used in the study ( standard strain Aspergillus fumigatus ATCC 13077 and clinical isolate A.fumigatus MCC LS 77,0010) were prepared and rabbit hyperimmune sera raised against the respective crude antigens (CF-73, MYC-73 and C-10) were used for detection of circulating antigens. Acute invasive aspergillosis was established in cyclophosphamide treated adult Swiss albino mice After infection 100% mortality was observed in mice within four days of infection. After the establishment of experimental animal model of acute invasive aspergillosis circulating anitgens were detected by Western blotting. Prominant protein bands were found at 18 kDa positioin using anti CF-73 antibody.



ROLE OF BACTERIA IN THE PATHOGENESIS OF JUVENILE IDIOPATHIC ARTHRITIS  

Enteric bacteria are known to trigger reactive arthritis in HLA B 27 positive individual juvenile idiopathic arthritis (JIA) an immuno inflammatory arthritis of childhood has an association with HLA B 27. Thus we looked for role of enteric bacteria in patients with JIA. Synovial fluid was obtained from 28 patients with JIA and 10 patients with proliferation assay was done using crude lyaste of enteric bacteria Salmonella typhimurium, Yersinia enterocolitica, Shigella flexneri and Camphylabacter jejuni as antigen. Escherichia coli crude lysate was used as a control antigen. HLA B 27 typing was done by PCR using sequence speicific primers.homing of gut educated T cells by tricolor flow cytometry. To see the presence of bacterial DNA16 Sribosomal gene PCR and bacterial species speicific PCR and bacterial species specific PCR was done. Twenty three out of 28 patients were HLA B 27 positive. Synovial fluid cells from 14 of the 28 patients showed antigen specific response, 6 to Salmonella four to Campylobacter two each to Shigella, Yersinia. One of the 10 SF from patients with RA showed response against salmonella antigen. Expression of CD 103 was found to be higher on synovial fluid lymphocyte as compared to peripheral blood lymphocytes in paitents with JIA. Expression of CD 103 was higher on CD8+ T cells as compared to CD4+ T cells. Bacterial DNA was present in majority of samples in the synovial compartment as well as in peripheral blood. Bacterial species specific PCR was begative for all samples except one sample. In one patient with JIA sample were positive for Salmonella typhimurium DNA. To complete, the data indicate that enteric bacteria play a role in disease exacerbation in patients with JIA. These patients show evidence of selective homing of gut-primed cells in the synovial compartment. Our Our findings of specific cellular immune responses against enteric bacteria in a proportiion of patients with JIA suggests that despite absence of history of a symptomatic enteric infection , these patients have a disease resembling chronic ReA. Thus a proportion of patients with JIA may have a forme fruste of chronicReA.



DEVELOPMENT OF FLUORESCENT LABELED POLYCLONAL ANTIBODY REAGENTS FOR SPECIFIC IDENTIFICATION OF ASPERGILLUS CANDIDA AND CRYPTOCOCCUS IN PARAFFIN EMBEDDED TISSUE SECTIONS  

Accurate identification of Genus/species of the fungi in the tissue section subjected to histopathological examination is important for the institution of speicfic therapy. Tissue forms of several fungi have similar appearance. Sometimes it is differentiate the fungi accurately in the tissue sections based on the morphological forms. So the present study was undertaken with the objective to develop fluorescent antibody reagents to differentiate some morphologically closely resembling fungi in the tissue sections such as Fusarium, Aspergillus and Pseudallescheria boydii and similarly betewwn yeasts like Candida and Cryptococcus. Antiserum was successfully raised against Aspergillus funigatus, Aspergillus niger, Aspergillus, Fusarium oxysporum,C. albicans, C. krusei, C.tropicalis, Cryptococcus neoformans and Pseudallescheria boydii. The cross reacting antibodies present in the antiserum was removed by adsorption with the cross reacting antigen/ antigens. However it was not possible to remove cross reaction between species under the Candida or Aspergillus. Initally all antiserum were evaluated by the indirect Immunofluorescence (IIF) test. Adsorbed pooled Aspergillus antiserum and Fusarium antiserum specifically identified the respective fungi in the mice tissue sections by IIF test. Both Cryptococcus neoformans and candida antiserum sepcifically recognized the respective yeasts in the mice tissue sections by IIF test. Direct immunofluorescent (DIF) reagents were prepared by purification and labeling the specific antiserum with the fluorescent isothiocyanate (FITC). Labeling was unsucessful of P. boydii antigen. The specific reagents were tested with the mice tissue sections containing specific fungi. The reagents prepared against C. albicans, Aspergillus, Cryptococcus neoformans and Fusarium oxysporum specirfically identified the respective fungi from all the mice tissue sections tested indicating that these reagents may be valuable in differentiating the fungi in the tissue sections of the patients. C. albicans, Aspergillus and C. neoformans reagents were also tested with the culture and/or smear proven tissue sections from patients sample contining the specific fungi. All these reagents gave the fluorescence ranging from 3+ to 4+ with the tissue sections of patients tested indicating that these reagents can specifically identify the fungi in the tissue sections processed for the histopathological examination. Though Fusarium reagents gave positive results with the tissue sections of mice origin, we could not test it on human tissue section due to non availability of such confirmed case in histopathology department. We now propose to evaluate these reagents in other centers. At the time of institution of this project, it was decided that Prof. S. Dutta Gupta of Histopathology Department, All india Institute of Medical Sceinces would evaluate the reagents in his pool of specimens.



AEROBIC AND ANAEROBIC BACTERIOLOGICAL PROFILE OF DIABETIC FOOT ULCERS WITH SPECIAL REFERENCE TO METHICILLIN RESISTANT STAPHYLOCOCCUS AUREUS  

The aim of the study was to determine the microbiological profile and antibiotic susceptibility patterns of organisms isolated from diabetic foot ulcers of different Wagners grade. Potential risk factors for infected ulcers with methicillin resistant Staphylococcus aureus (MRSA) and outcome infections were also studied. Pus sample for bacterial cultures were collected from 100 patients admitted with diabetic foot infections over a period 3 years. Sixty-two had co-existing (62%) osteomyelits. Staphylococcal isolates were tested for susceptibility to oxacillin by screen angar method, disk diffusion and mec A based PCR and susceptibility to vancomycin by screen agar. Gram negative bacilli were testedc for extended spectrum beta lactamse (ESBL) by double disk diffusion. Potential risk factors for MRSA- positive samples were explored. A total of 224 pathogens were isolated ; 187(89.5%) aerobes and anaerobes 37(16.5%). Gram-negative aerobes were most frequently isolated (48%) followed by gram-positive aerobes and anaerobes (32.2% and 10.2%, respectively). Methicillin- resistance and ESBL production was noted in 60.5% and 41.6% of bacterial isolates. MRSA status is not associated with patient demographic characteristics, ulcer type, ulcer duration, ulcer size or duration of hospital stay. The factors significantly associated with MRSA positive status were presence of hypertension (p= 0.001), neuropathy (p=0.003) and esteomyelitis (p=0.02). Significantly more number of patients with MRSA infections required amputation (p=0.002). It is concluded that infection with MRSA is common in diabetic foot ulcers and is associated with increased requirment for amputations. These results can guide appropriate empirical antibiotic therapy in these patients and reduce the risk of complications




NEUROLOGICAL SCIENCES
PREVALENCE OF M.PNEUMONIAE INFECTION IN PATIENTS OF ACUTEEXACERBATION OF CHRONIC OBSTRUCTIVE PULMONARY DISEASE  

Eighty percent of the cases of acute exacerbation of chronic obstructive pulmanary disease (AECOPD) have an effective etiology; atypical bacteria including Mycoplasma pneumoniae account for 5 - 10% of these. However, some studies could not find evidence of association of M. pneumoniae with episodes of AECOPD at a referral hospital in Delhi. Sputum samples and throat swabs were collected from a total of hundred patients of AECOPd attending Vallabhbhai Patel Chest Institute and thirty five healthy control subjects. The samples were investigated for the presence of aerobic bacterial pathogens and Mycoplasma pneumoniae. Diagnosis of infection with Mycoplasma pneumoniae was based on culture serology, PI antigen detection and PCR for the PI antigen detection and PCR for the PI gene. Bacterial etiology could be established in 16 patients. The organism isolated most frequently was Pseudomonas from 4, Klebsiella sp. from 2; and Acinetobacter sp. and Moraxella catarrhalis from one case each. In three of these cases, the M. pneumoniae infection. However, one of the cases which was cultrue positive for Pseudomonas sp, also had m. pneumoniae specific 1gG titer>= 100 U. This patient, thus probably and a mixed infection of Pseudomonas sp and M. pneumoniae. Of the100 patients studied,39 were IgG positive. Ten of these showed boderline positivity.Of the thirty patients followed up, 5 of showed a four fold rise of antibiodies in 4 weeks. Amongst the 18 samples positive with GPA, three samples had a titers>= 1;320. The IgM ELISA assay in our study was positive in 4 serum samples. IgA antibodies were seen in 10 patients. None of the 8 borderline positive patients which followed up showed a significant change in IgA titer. Four patients had both positive IgG and positive Iga titers, indicative of a primary . pneumoniae infection. P1 antigen was deteced in 3 samples. Two of these patients had positive IgG and IgA responses, but no IgM responses. PCR for the P1 adhesin gene was negative in all the samples studied. Teh prevalence of M. pneumoniae infection in our study population was significantly higher than that in the control group. To conclude, on the basis of derology and antigen assay, at least 17% of the 100 patients of the exacerbation of COPD showed evidence of M. pneumoniae infection. Mycoplasma pneumoniae, therefore, emerged as an important etiological agent of AECOPD in our study population.



EXPRESSION AND BIOCHEMICAL REGULATION OF CYCLIC NUCLEOTIDES IN RAT BRAIN AND THEIR RELATION TO NEURONAL FUNCTION  

The study was carried out to understand the biochemical mechanisms which regulate the cyclic nucleotide metabolism in rat brain and physio - logical importance of this regulation. The effects of factors modulating adenylate cyclase which are not related to neurotransmitters, were studied. The possible influence of biogenic amines like dopamine (DA), serotonin (5-HT) norepinephrine(NE) and also acetylcholine (ACh) and other agents on adenylate cyclase (AC) and phosphodiesterase (PDE) in the rat brain was also evaluated. Dopamine NE and 5-HT stimulated rat brain AC activity A dose dependent stimulation of AC by DA (with 1-1000 muM) and NE (with 25-75muM) was observed with DA being more potent. Serotonin stimulated rat brain AC activity by 60 per cent at 10 muM, which increased to 100-160 per cent in presence of NaCl. But the dose dependent stimulation of AC by 5-HT was not as sensitive as by DA and at higher concentrations(more than 10 muM) inhibited AC. The presence of acetylcholine inhibited AC activity. Higher concentra- tion of ACh inhibited the DA sensitive AC and this inhibition reversed by increasing DA, thereby showing a competitive inhibtion of AC by ACh. It is thus probable that the action of biogenic amines in central nervous system (CNS) may be mediated (atleast in part), by cAMP formed in the post synapt receptor sites. Atropine and scopolamine both muscarinic receptor antagonists prevented the inhibition of AC by ACh without affecting basal activity. The action of atropine on DA sensitive Ac could be reversed by increasing the concentra- tion of DA. The stimulation of AC was effectively blocked by antipsychotic drugs known to block DA receptors. Phosphodiesterase (PDE) showed two Km(Kinetic constant) values; a high Km 0f 0.5x 10^-3 M and a low Km of 3.8x10-6M. Theophylline was found to be a most potent inhibitor of PDE with a inhibitor constant (Ki) of 1x10-4M. Both dibutyryl cAMP and isobutyl methyl xanthine (IBMX) also inhibited PDE with Ki of 1.2x10-6M and 2x10-4M respectively. Copper and zinc inhibited PDE whereas in absence of Mg both Ca and Li could activate PDE. Antipsy- chotic drugs like fluphenazine, mepazine being more potent. The relative potency (in decreasing order) being mepazine>trifluoperazine>phenothiazine> fluphenazine>haloperidol. It was observed that both calmodulin and calcium were required for the activation of PDE. Both Ca and calmodulin were found to stimulate Ca sensitive ATPase in synaptosomal membranes. The activation by calmodulin was dose dependent and 5 muM CaCl2 was optimal concentration for maximal activity of Ca ATPase in-synaptosomal membranes. It was concluded that a neurotransmitter sensitive AC is present in rat brain ; DA sensitive being more potent than NE and 5-HT sensitive AC. DA receptor appeared to be associated with AC in rat brain. It was established that ACh was a most potent competitive inhibitor of DA sensitive AC. DA and ACh act synergistically on AC. It appeared from the study that AC system may be a useful in vitro model for study of the functional role of DA and ACh receptors and the pharmacology of certain drugs.



CERTAIN BASIC IMMUNE FUNCTIONSIN INTRA-CRANIAL TUMOUR PATIENTS.  

Certain basic immune functins in intracranial tumour patients. In order to elucidate the role of neuralmodulation of immune response in patients of intracranial tumours, immune parameters were studied before and 30 days after treatment in these patients. Normal individuals matched for age, sex and socio-ecomomic status served as controls. Patients with benign tumours were treated by surgery whereas those with malignant tumours were treated by surgery whereas those with malignant tumouurs were treated by surgery, radiotherapy and chemotherapy. It was found that patients with benign and malignant tumours had higher total leukocyte count before treatment which decreased significantly after treatment. Lymphocyte percentage showed a reduction but absolute monocyte count and monocyte percentage showed an increase in both benign and malignant tumour patients. There was an elevation in neutrophil functions as evidenced by increased phagocytic index, avidity index, NBT (Nitroblue tetrazolium) reduction and SIC (serum soluble immune complex) index in brain tumour patients. As compared to controls total T cell and T4 counts were reduced in both benign and malignant tumour patients before and after treatment whereas there was an increase in T8 counts. Serum IgA and IgG levels showed a decrease and IgM level an increase. Preoperatively altered immune parameters like lymphocyte percentage, monocyte percentage, absolute T lymphocyte count, T8 count and IgA levels returned to base- line levels after treatment in benign tumour patients. The study confirmed the altered innate, cell mediated and humoral immunity in brain tumour patients before and after treatment. Most of the immune changes observed were related to cell mediated immunity ad were immunosupperssive. Though the pitutary region tumours are benign, patients with these tumours showed either marked increase or decrease in the levels of immune parameters studied than observed in those with tumours in other areas. The immunomodulatory effects of malignant tumours were more marked than those of benign tumours.




NUCLEAR MEDICINE
A STUDY OF THE NATURAL HISTORYAND SEIZURE OUTCOME IN PATIENTS WITH SOLITARY CYSTICERCUS GRANULOMAS AND SEIZURES  

Study of the natural history and serizure outcome in patients with solitary cyticercus granuloma and seizure. Aprosepective study was carried out in 183 patients with seizures to determine the natural history of solitary cerebal cyticercus granuloma (SCG) and seizure outcome after the resolution of the granuloma and simultaneous or early withdrawal (2 to 3 months after resolution) of antiepileptic drags. Only those patients who fulfilled all the diagnostic criteria (clinical and computerised tomographic for SCG) were included in the study. One hundred and twenty nine (70.5%) pateints presented with patial seizures with or without secondary generalization, 53 (29%) with generalized seizure and 1(0.5%) with partial complex seizure. Of these 78.1% patients had 5 or less seizures, 16.9% had 6-10 and 4.9%>10 seizures at initial presentation. Most of the patients had their initial CT scan within one month of their first ictus. Majority (93%) of patients could be managed with a single antiepileptic drug (monotherapy). Breakthrough seizures occurred in 30(16.4%) patients who needed increased dose of the drug or an additional drug. ELISA for cysticercus antibodies in serum was of limited value as it was found to eb negative in a majority of patients. The CT lesions were typically between 5-15 mm in maximal dimension and were usually associated with mild to moderate oedema. The parietal love was the most favoured site of lodgement of the granuloma. SCG was found to have a varied natural history and the granulomas resolved spontaneously though at different rate in individual patients. Kaplan meier analysis of lesion resolution in the present study showed that at 3 months after first CT scna, 96.7% patients had persistent granulomas (though smaller in size in nearly 40% patients ), at 6 moths 75.5% patients and even after 12 months 46.5% patients had persistent granulomas. The shortest period recorded for the resolution of lesion was 64 days after the initial CT scan whereas the longenst duration of persistent lesion seen in the CT scan was 429 days. Antiepileptic drugs were withdrawn within 2-3 months, after resolution of granuloma was seen on the CT scan. O the 60 patients who could be followed after withdrawal of the drugs ( median follow up period 10 months), 57 were symptom free, and recurrence of seizures was noted in 3 patients at 3 to 10 months after withdrawal of drugs. It can be concluded thath SCG, a common cause of seizures in Indian patients, is a spontaneously resolving lesion with a variable rate of involution ranging from weeks to a years or more. The first follow up CT scan can be delayed for up to 6 months decrease the number of scan examinations. Patients with SCG generally have a bengin seizure prognosis aqnd the antiepileptic drugs can be withdrawn after resolution of granuloma. However, a longer follow up is necessary to arrive at a definite conclusion regarding the longterm seizure outcome in these patients.




NUTRITION
EFFECT OF LOW LEVEL PRENATAL AND NEONATAL IRRADIATION ON THE ADULT BRAIN DEVELOPMENT AND REPRODUCTIVE FUNCTION OF MOUSE  

Effect of low level prenatal and neonatal irradiation on the adult brain development and reproductive function is mouse. The study was carried out on inbred Swiss albino mice to evaluate the effect of gamma irradiation at selected critical developmental stages during gestation on the adult neurophsiology and to see, if the neurophysiological effects of prenatal irradiation persist to later part of life. Efforts were also made to find out if prenatal irradiation would impair the reproductive performance of mouse. Pregnant mice were whole body irradiated with 0.25,0.35 or 0.5 Gy of gamma radiation on days 11.5, 12.5 (late organogenesis days), 14.5 or 17.5 (foetal days) post coitus (pc) and allowed to complete gestation and parturate. Behavioural and haematological changes were studied in the adults at 6, 12 and 18 months of age. Reproductive performance was tested at 6-8 months of age after mating the irradiated F1 males with normal females of the same age. For neonatal exposure, pups were whole body exposed to 0.5 Gy of gamma radiation on days 4, 5 or 6 post-partum. Adult behaviour was not affected by prenatal irradiation with 0.25 Gy. Exposure to 0.5 Gy on days 11.5, 12.5 or 14.5 pc produced significant changes in the exploratory, locomotor and learning and memory performances at 6 months of age. The changes were more pronounced after irradiation on the late organogenesis days (11.5 and 12.5 pc) than on the foetal day (14.5 pc). Normal brain function was restored by 12 months in all animals, except those exposed on 11.5 day pc which the impaired locomotor activity and learning performance of the young adult mice. Animals born to mice irradiated on day 17.5 pc werre more resistant to these effects. The peripheral RBC counts and haemoglobin showed significant decrease at 6 months of age when the pregnant mouse was exposed to 0.5 Gy on the foetal gestation 914.5 or 17.5 pc) days, but not during the organogenesis period. Post-partum exposure did not have any significant effect on the adult behaviour or haematological parameters. None of the hippocampal biogenic amines (noradrenaline, dpamine, 5-hydroxytryptomine and its metabolite -5-hydrooxyindolacetic acid) studied showed any significant change after any level of exposure on any of the gestation days. The reproductive performance of the F1 males exposed to different doses of irradiation did not show any significant change from normal. It is clear from the study that the late organogenesis period, especially day 11.5 pc is a particularly sensitive phase in braain development and long lasting behavioural changes can be produced in the adult by exposure at this stage to doses below 1 Gy.



STUDIES ON CHOLESTEROL AND OXIDIZED LIPIDS FROM DAIRY ANDEGG PRODUCTS ON LIPID METABOLISM RELATED TO CARDIOVASCULAR DISEASES  

This study is about dairy products such as milk and ghee contain saturated lipids and cholesterol which are considered to be atherogenic in nature. However an earlier studies indicated that feeding rats a diet containing dairy lipids exhibited hypocholesterolemic effect (1). Studies conducted under similar conditions with diets containing egg yolk lipids did not show any benefical effects in modulating serum lipid profile in rats. These studies were conducted in normal diet. However cholesterol enriched diet are known to elevate serum cholesterol levels and the influence of diary lipids under hypercholesterolemic conditions is known. To evaluate this, the present investigation was undertaken to study the influence of diary lipids and egg yolk lipids in rats rendered hypercholesterolemic by feeding a diet enriched in cholesterol. The hypocholesterolemic properties of dairy fat cross examined when rats were rendered hypercholesterolemic by feeding a diet containing. Supplementing the diet with cholesterol at 0.5% level incrased serum cholesterol level cholesterol level by 40% compared to rats fed diet without cholesterol. Rats fed diet containing milk or ghee lipids had 9-11% lower levels of serum total cholesterol compared to control rats fed with cholesterol enriched diet but devoid of milk lipids. The present experimental observation indicates that milk lipids, either in normal cholesterolemic cnditions or in hypercholesterolemic condition will lower serum cholesterol concentration in rats. It is tharefore concluded that diary lipids can beneficailly modulate serum and tissue lipids and hence it is not a cause for concern in terms of increasing risk factors for cordiovascular diseases.



DEVELOPMENT OF NUTRACEUTICALS CONTAINING ANTIOXIDANTS AND FLAVONOIDS FOR THE PREVENTION OF CORONARY HEART DISEASES  



LONG TERM MANAGEMENT OF RETINAL AND RENAL COMPLICATIONS IN EXPERIMENTAL DIABETES BY VANADATE AND ANTIDIABETIC COMPOUNDS  

Combined Antidiabetic effect of Sodium orthovanadate and other antidiabetic compounds such as Trigonella seed powder and Momordica fruit extract a new propective therapy: Hyperglycemia druing diabetes results in majority of the metabolic derangements and clinical complications that contributes towards the morbidity and mortality of diabetic patients, the latter may be prevented or reversed with an effective control of increased blood glucose. Repeated insulin administration controls the short term diabetic symptoms, but it fails to prevent the serious vascular and other complications of diabetes. Moreover, exogenous insulin treatment fails to provide a regulated glycemic conditions in association with variable dietary intake and variable physical activity in Type 1 diabetes. Episodes of severe hypoglycemia leading to a deleterious cerebral impact are common during tinsulin administratioin (30). Other drugs available also have similar problems in addition to their toxic side effects. Therefore, appropriate antidiabetic compounds without or less toxic effects are required to primarily control the blood glucose, which can subsequently, effectively reverse the altered biochemical and physiological changes during diabetes. The insulin mimetic action of vanadate has been explored since the last decade in various diabetic model systems including IDDM and NIDDM juman subjects (31). Trigonella seed powder and Momordica charantia fruits are well known antidiabetic agents and have been shown to rejuvenate and Beta cells in the islets of Langerhans, thus increasing the capacity of insulin secretin in type 1 diabetes (32). The insulinotropic property of 4 hydroxyisoleucine, and amino acid extracted from trigonella is also suggestive of insulin secretion modulation in its therapeutic action (33). Similarly insulin like peptide (polypeptide-P) has been isolated from Momordica fruits and has been shown to act like insulin (34). However, no detailed study is available to establish whether the plant extracts follow similar signaling biochemical routes such as as those taken by insulin and vanadate. Being a natural product and a part of regular diet with multitude of antidiabetic effects, Trigonella seeds and Momordica fruits can possibly be used as insulin replacement or adjuvant in the management of diabetes. Vanadium has a poor therapeutic index inspite of its significant biological potential and well known insulin mimetic mechanism (13). However ligands, when complexed with vanadium, potentiate its insulinomimetic acitivity, both in-vivo and in-vitro. Many perspective involving biochemistry and bioinorganic chemistry of vanadium and its complexed with several types of ligands have been proposed as useful for treating diabetes mellitus in experimental diabetic animals. It has been shown by Shinde et al, 2001(35) that chronic treatment with an organic complex of vanadium such as bis(maltolato) oxovanadium (IV) (BMOV) was effective in improving glucose and lipid homeostasis. Hence, the attempt and emphasis on the use of vanadium complexes in treatment of diabetes melliuts is emerging as a new concept. Earlier works from our laboratory and in the present work an attempt has been made to prevent the toxic effects of vanadium by reducing the dose of vanadium administered (0.2 mg/ml) and combining its administration with that of plant products for the treatment of the diabetic animals. This combined treatment of vanadium with plant products was observed to be more beneficial with respect to biochemical stablization as well as improvement in the physiological parameters of experimental diabetic animals, besides establishing normal glucose and lipid homeostasis. It can be suggested that there may be some in-vivo formation of organometallic compounds by sodium orthovanadate with organic compounds, may be active constituents, made available by plant extracts that are responsible for bringing the improved management of blood glucose level and at the same time also reducing the toxicity of sodium orthovanadate. The findings of the present work suggest the effectiveness of the combined therapy of vanadate and plant products on the control of glucose homeostasis and lipid metabolism druing experimental diabetes and can be sonsidered as a better alternative for the amelioration of diabetes. In the present study diabetes was produced by ginving alloxan injection to the rats, all diabetic rats showed severe hyperglycemia, hyperphagia, polydipsia, glycosuria and severe body weight lose. After giving treatment with insulin, trigonella, Momordica, higher dose of vanadate alone and lower dose of vanadate with trigonella and momordica restored high blood glucose level to the normal values. Combined treatments were found to be much more effective in normalizing the diabetic complications in short and long term diabetes. However, further investigation compounds formed in-vivo, to find the active constituents of these plant products and to work out their exact molecular mechanism in combination with sodium orthovanadate.




ONCOLOGY
BIOLOGICAL EFFICACY OF ANTIHYPERTENSIVE PEPTIDES FROMCEREALS AND PULSES COMMON TO INDIAN DIET  

Several chemical compounds like captopril, Analapril etc are employed as antihypertensive drugs, which are known ACE inhibitors. The most common adverse effect associated with ACE inhibitor is a presistent non productive cough. Hence there have been attempts to develop ACE inhibitors sans the side effects. Recently several ACE inhibitory peptides have been isolated from plant foods. Enzymatic digensts of various food materials, including casein, zein, gelatin, sake, soyabean fermented milk, sardine muscle, tuna muscle, dried salted fish, fish sauce and onion. These peptides are reported to inhibit the ACE both in vitro and in vivo studies. However limited reports exist regarding isolation and employment of ACE inhibitory peptides from cereals and pulses. Hence this project mainly aims at Our earlier studies have also shown that chymotryptic digested fractions of Sorghum storage protein, alpha-kafirin, possess strong ACE inhibitory activity. We characterized the inhibition from a kinetic point of view. We determined the IC50 of the potent fractions were found to be 24.3 muegram/ml to 1.3 muegram/ml while the IC50 of Captopril was 0.0067 mueM. The inhibition by teh fractons was found to be competitive and uncompetitive with the substrate. Our results offer a biochemical mode of inhibitino by the hydrolytic fractons were aslo found to inhibit rat tissue ACE (in vitro)to a significant extent. The results also suggest the potential mechanism that might occur in vivo, to act as a potential antihypertensive agent. Studies were conducted to measure the basal angiotensin converting enzyme (ACE) activity in rat tissues. Comparative evaluation of the enzyme activities was carried out in different tissues of rat of different age groups of rats. Heighest ACE activity was seen in lung of adult rats, the major site of its, synthesis. However circulating ACE activity, in serum, was minimal in rats of all the age groups. Cadmium chloride (CdCl2), a well established inducer of hypertension in animal models was found to alter ACE activity in different rat tissues to varying extent. CdCl2 induced a significant increase in ACE acitvity in the rat lung, while it decreased the enzyme activity in serum. Pre treatment with Cap;topril, a known ACE inhibitor was found to reserve the Cd induced alterations in tissue ACE activity. Further in vitro studies reveled tha serum, small intestine and brain ACE were more susceptible to inhibition by captopril. The above studies have clearly yielded steps and procedures for studies employing ACE for its deploymen in evaluation of ACE inhibitory compounds. isolation and further studies on ACE inhibitory activities of peptides from cereals and pulses common to Indian diet in perticular such sorghum, ragi and black gram dal. In our preliminary studies were prepared and evaluated protein hydrolysates from sorghum and rgai flour. Both total proteins as well as storage proteins ( prolamins- the alcohol soluble proteins) were subject to proteolysis with selected emzymes viz. trypsin, chymotrypsin, pronase, papain etc. The digests obtained were analyzed by SDS-PAGE and those showing maximum digestions were subjected to further studies. Trypsin and chymotrypsin digests of sorghum total prolamins and pronaase digest of ragi tatal prolamins were found to yield peptide fractions possessing signicant ACE inhibitory activities (50% and 62% respectively)



IMMUNOCYTOCHEMICAL LOCALISATION AND BIOCHEMICAL ESTIMATION OF PEPTIDE HORMONE BINDING CAPACITY OF BREAST TISSUE  

Immunocytochemical localization and biochemical estimation of peetide hormone binding capacity of breast tissue. The study was carried out to examine immunohistochemically detectable prolctin (PRL), follicular stimulating hormone (FSH) and leutinising hormone (LH) binding sites in various breast lesions using a highly sensitive modified dinitrophenyl hapten sandwich staining procedure and biochemically estimating PRL binding in particulate membrane preparations of breast tissue. One hundred and seven formalin fixed paraffin embeded breast tissue sections were immunostained for localisation of prolactin binding sites in benign and malignant breast lesions. Of these, 35 and 28 sections were also stained for FSH and LH binding sites respectively. Eighty tow percent of breast carcinoma gave an intracellular positive reaction in tissue sections for prolactin binding sites but the positivity was 72 percent in fine needle aspiration smears. In an attempt to biochemically quantitate prolactin receptors, both purified and crude plasma membranes of tissues from benign and malignant lesions, were prepared. Breast carcinoma showed more consistent staining as compared to benign breast lesions. Also, prolactin receptors could be located biochemically in malignant breast tissue membrane preparations. Prolactin binding sites were present in greater proportion in peri or post menopausal breast cancer patients. It is thus concluded that the more consistent immunostaining in breast carcinoma as compared to benign breast tissues as well as presence of PRL receptor in crude membrane of brest carcinoma tissue suggest the role of PRL in mammary tumourigenesis. The results of immunocytochemistry on FNAC being ecoomical, quick and nonoperative procedure, couls be more easily used for study of hormone binding sites in breast lesions.



GENERATION OF MONOCLONAL ANTIBODIES TO ESTROGEN RECEPTOR AND THEIR APPLICATIONIN THE STUDY OF HUMAN BREAST CANCERS  

The study was carried out to isolate estrogen receptor (ER) protein from ER human breast cancer cell lines (like MCF-7) and also from ER human breast tumours and generate monoclonal antibodies to the purified ER protein. Immunohistochemical studies were carried out on human breast cancer tissues and fine needle aspiration biopsy (FNAB) specimens using monoclonal anti- bodies specific to ER. Estrogen receptor protein was isolated from MCF-7 cell lines and breast tumour tissues by pulverization of the material snap frozen in liquid nitorgen, polytron homogenization, high speed centrifuga- tion. The ER was characterized by Scatchard analysis and sucrose density gradient centrifugation. SDS PAGE identified the ER as a single discrete protein with a molecular weight of approximately 66-67 k Da. Three monoclonal antibodies (MAbs) were generated by hybridization of immune spleen cells of BALB/c mice immunized with ER, with Sp2/O myeloma cells using 50 per cent PEG as fusing agent. The positive clones were screened by ELISA and selected by PAP technique using ER positive frozen breast tumour tissue sections. These MAbs, designated as ER E5, ERE10 and ERD7 were found to recognize a cytosolic ER. These MAbs were purified by ammonium sulphate precipitation and protein A sepharose chromatography Specificity of these MABs to ER was ascertained by dot blot analysis, western immunoblot, immunoprecipitation, radioimmuno uptake studies and immunofluorescence study. Analysis of large number of normal breast tissues, benign and malignant breast lesions of different histological types, breast lesions of different histological types, breast FNAB specimens and other ER tissues of carcinomas of the ovaries and endometrium by ABC and PAP immunocyto- chemistry revealed that these ER MAbs exhibited specific staining of cytosolic ER. Most of the tumour cells were strongly positive in the case of infiltrating ductal carcinomas and mucoid adenocarcinomas. The ER values of 50 malignant breast tumour cytosols determined by DCC assay and by enzyme immunoassay (EIA) using MAb ER E5 were found to correlate. For 15 typical breast tumours with ER values in the range of 3.5-270fmol/mg protein, the staining paterns were proportionate to ER values. It was concluded that MAbs ER E5, ERE10 and ERD7 might serve as a valuable diagno- stic tools. Besides, these may be highly useful in understanding the molecular mechanism by which estrogens stimulate cell proliferation.



INVESTIGATION OF DOPPLER FLOWMETRY IN CANCER CHEEK  

The study was carried out for investigating the blood flow patterns and rates in facial artery in patients with cancer cheek and age and sex matched patients with leukoplakia cheek, individuals addicted to tobacco chewing and smoking separately and normal healthy subjects to correlate the pattern of blood flow to (i) histopathological grading of cancer cheek, (ii) responsee to chemotherapy,(iii) occurance of cervical lymph node metastasis and (iv) survival. There was abnormality in blood flow in the facial artery in chronic tobacco chewers in the form of partial obstruction. In cancer cheek also similar findings were noted. In addition the doppler study revealed formation of collatrals in cancer cheek. Khaini was found to be the most common form of addition in 71.42%. The risk ratio (Odd's ratio) due to Khaini chewing was 1.036, to smomking 1.088, and addition of alcohol increased the risk to 1.097. Night quid keeping habit had odd's ratio of 3.73 (P<0.001). Buccal vestibular mucosa was found to be the commonest site of cancer (54.46%). Ulceration and pain were prominent clinical findings in cancer cases. There was severe nutritional derangement in cancer patients (P<0.001). DNA content was significantly raised in cancer patients in comparison to tobacco addict and normal controls.



A STUDY ON THE FREQUENCY AND NATURE OF PRE-NEOPLASTIC HEPATOCELLULAR LESIONS IN HUMAN ANIMAL LIVER  

The study was carried out to determine the frequency of hepatocytic of preneoplastic nature in livers of population groups with different incidences of hepatocellular carcinoma and Wister strain albino rats rhesus monkeys exposed to different hepatocarcinogenic agents. Morphological and cytochemical profiles of preneoplastic cells were also studied. Rats were treated with dimethylnitrosamine(single dose of .4 mg/100 g bodywt. intraperitoneally 24 h after partial hepatectomyl) or acetylaminofluorene (3 doses of 3 mg/100 g body wt. i.p. at 24 h intervals followed by partial hepatectomy 24 h after the last dose). A total of 5 rats and 4 monkeys were treated with aflatoxin b1. Qualitatively and quantitatively the sequence of preneoplastic and neoplastic events were similar in rats irrespective of the carcinogen used. Structurally altered hepatocytes occured usually as small foci which later evolved into larger areas as nodules of altered liver cells. In monkeys treated with aflatoxin B1 changes were mild and the lesions few. Histochemical localization of enzymes of carbohydrate metabolism viz glycogen synthetase glycogen phosphorylase or glucose-6 phsphate dehydrogenase revealed no significant change in these enzymes. No significant changes were also noticed with the other enzymes. The acidophilic cells showed a variable pattern of staining for various enzyme It appears that chemical carcinogenesis induces multifocal hepatic glycogenosis which may be related to development of multicentric tumours. It is probable that metabolic disturbances leading to hepatocellular glycogenosis might be related to the neoplastic transformation of the hepatocytes. Liver sections prepared from livers obtained from medico-legal autopsies of 114 cases (55 from Madras and 59 from New Delhi) were studied for the presence of altered cell foci were observed in 19 (34.5%) cases from Madras compared to 14 (23.7%) in NEw Delhi. The commonest cell type was the clear (glycogen rich) cell and some of these foci had acidophil cells admixed. The number of foci with acidophil cells were more in livers obtained from Madras compared to New Delhi.



A STUDY ON THE ROLE OF ELLAGICACID, A DIETARY PHENOL,IN CHEMICAL CARCINOGENESIS IN MICE  

The study was carried out on NMRI inbred mice to evaluate the effect of ellagic acid (EA) on hepatic and pulmonary phase I and phase II drug metaboliz- ing enzymes and antioxidant enzymes as also on lipid peroxidation in the liver and lung. The role of EA in modulating the binding of chemical carcinogens, 3H-benzo(a) pyrene and 14C-diethyl nitrosoamine with DNA in vivo and in vitro and its effect on binding of 3H-benzo(a)pyrene and 14C-diethyl nitrosoamine with DNA,mediated through peroxidative cooxidation of the carcinogens in vitro were also studied. The effects of EA feeding at doses of 3,6 and 12 ug/ml drinking water for 8 weeks to male and female mice, on phase I and phase II drug metabolising enzymes were similar. EA did not change the activity of arylhydro-carbon hydroxylase (AHH) in vivo and in vitro. Feeding of EA to male mice for 8weeks significantly decreased the hepatic cytochrome P-450, cytochrome b5 and cytochrome c-reedutase; the strongest effect was found at the dose of 12 ug/ml. On the other hand, AHH and cytochrome c-reductase activities in lungs increased on EA feeding. Hepatic UDP-glucoronyl transferase (UDPGT), glutathione-S-trans-ferase (GST) and reduced glutathione (GSH) were significantly enhanced on EA feeding, and the effects appeared to be EA dose dependent. However, in lungs UDPGT and GSH levels were increased without any change in the GST activity. GSTinhibition curve, obtained at different concentrations of EA in the 10,000 g hepatic supernatant using 1-chloro-2, 4 dinitrobenzene (CDNB) assubstrate suggested two types of isoenzymes of GST (EA-binding and EA-non- binding). Kinetic studies revealed that EA inhibited GST activity non-competi- tively with respect to CDNB as a substrate. EA feeding at a dose of 12 ug/ml inhibited the percentage inducibility of hepatic AHH by phenobarbitol from 25 to 6.5 per cent and the percentage induci- bility of AHH by methylcholanthrene was decreased from 173 to 154 per cent. In the lungs, the percentage inducibility of AHH remained unaltered. Addition of EA to liver microsomes of mice resulted in a steady increase ininhibition of NADPH-dependent lipid peroxidation upto 2mM concentration. The maximum of 70 per cent inhibition of ascorbate-dependent lipid peroxidation was achieved at ImM concentration of EA. EA was found to be a stronger antioxidant than the known strong antioxidants like BHA, BHT, toco- pherol and retinyl acetate. But the alizarin, quercetin, myricetin and purourogallin were found to be stronger inhibitors of in vitro lipid peroxida- tion compared to EA. Feeding of EA significantly increased the levels of redu- ced glutathione and glutathione reductase in the lvier and lungs of male and female mice. However, there were no changes in the activities of catalase and superoxide dismutse. On the other hand, microsomes from liver and lungs of EA fed animals showed significantly suppressed NADPH- and ascorbate-dependent lipid peroxidation. EA feeding at a dose of 12 ug/ml drinking water for 8 weeks to mice resul- ted in significant inhibition of 3H-benzo(a)pyrene binding to lung DNA. In in vitro studies EA inhibited the binding of 3H-benzo(a)pyrene and 14C-NDEA with DNA, mediated by NADPH, ascorbate and linoleic acid-hematin systems. It is thus concluded that EA not only act as an antioxidant but also enhances the GSH-dependent protective mechanisms. The overall consequences of these antioxidant activities might be beneficial and account for the anticarci- nogenic activity of EA.



SELENIUM IN TREATMENT OF CANCER IN ANIMAL MODEL SYSTEM AND ITS MECHANISM OF INHIBITION OF CARCINOGENESIS  

The study was carried out in inbred Sprague-Dawley male rats to find out the optimum dose and mechanism by which selenium functions as an anticarcinogenic agent and to correlate the anticarcinogenic potential of selenium to the activities of oxidative and nonoxidative enzymes associated with the metabolism of carcinogens. Among the three selenium compounds studied selenocystine, selenomethionine and sodium selenite, selenomethio- nine (8 ppm in drinking water) was selected because it was least cytotoxic and had maximum efficacy in reducing preneoplastic hepatic foci. It was found that the inhibitory effects of selenomethionine were primarily exerted on the initiation phase of the hepatocarcinogenic process. However, a continuous long-term exposure would confer a greater degree of protection. Selenomethionine increased the cytochrome P-450 levels and the activities of TPNH-cytochrome c reductase and arylhydrocarbon hydroxylase. It caused a decrease in the activities of UDP-glucoronyl transferase and glutathione-s-transferase. Concentrations of selenium were found to be significantly less in hepatomas/nodules than the surrounding areas in both selenium supplemented and non-supplemented groups of carcinogen treated rats. The concentration of selenium in the carcinogen treated livers was greater than that of carcinogen controls. It is concluded from the present findings that the inhibitory effects of selenomethionine were primarily exerted on the initiation phase of the hepatocarcinogenic process. However, continuous long-term exposure to selenomethionine could confer a greater degree of protection preventing lipid peroxidation and cellular protein damage.



SEARCH FOR ONCOGENIC VIRUSES ASSOCIATED WITH HUMAN PREMALIGNANT AND MALIGNANT CONDITION  

The study was carried out to screen cervical smears for human papilloma- virus (HPV) using in situ hybridization and detection of viral genes in premalignant and malignant cervical issues by dot-blot and Southern blot techniques. Tissue samples from uterine cervical cancers (135) and normal cervix (50) studied immunohistochemically by peroxidase-antiperoxidase staining revealed HPVstructural antigens in 73 (54.07%) of the cervical cancer samples and 4 (8%) normal cervix samples. The antigen distribution was focal and uniform or in diffuse patches. Both dot-blot and Southern blot hybridization analyses were employed for detection of HPV-16 and HPV-18 DNA sequences in the DNA samples extracted from normal uterine crvix tissue, cervical cancer tissue and cervical dysplasia samples. In dot-blot hybridization, 44 of 80 (55.5%) cervical cancer samples, 6 of 21 (28.6%) cervical dysplasia samples and 3 of 30 (10.0%) normal samples were positive for HPV-16 DNA. But only 3 of 50 (6%) cervix cancer samples and none of the dysplasia and normal samples showed the presence of HPV-18 DNA. By Southern blot hybridization, HPV-16 DNA was detected in 47 of 80 (58.7%) DNA samples from cervical cancer, 7 of 20 (35.0%) dysplasia samples and 3 of 30 (10.0%) normal samples. HPV-18 DNA was detected in 3 of 65 (4.6%) cervical cancer samples. Characteristic Bam H1 (8kb) and Pst I (2.8 kb, 1.9 kb, 1.6 kb, 1.0 kb and 0.5 kb) bands were observed in the autoradiographs of HPV-16 DNA positive samples. The results suggest that there is an association between HPV-16 and cervical cancer in women from Kerala. employed for detection of HPV-16 and HPV-18 DNA sequences in the DNA samples extracted from normal uterine cervix tissue, cervical cancer tissue and cervicaldysplasia samples. In dot-blot hybridization, 44 of 80 (55.5%) cervical cancer samples, 6 of 21 (28.6%) cervical dysplasia samples and 3 of 30(10.0%) normal samples were positive for HPV-16 DNA. But only 3 of 50 (6%) cervix cancer samples and none of the dysplasia and normal samples showed the presence of HPV-18 DNA. By Southern blot hybridization, HPV-16 DNA was detected in 47 of 80 (58.7%)DNA samples from cervical cancer, 7 of 20 (35.0%) dysplasia samples and 3of 30 (10.0%) normal samples. HPV-18 DNA was detected in 3 of 65 (4.6%)



RECEPTORS,GROWTH FACTORS AND ONCOGENES IN HUMAN BREAST CANCER: A MULTIPARAMETRIC STUDY  

A prospective study was carried out on 150 breast cancer patients to evaluate the potential prognostic significance of various growth factors. The markers investigated were serum epidermal growth factor (EGF) and its receptor (EGFR), C erb B2 oncoprotein and insulin like growth factor-1 receptor (IGF-1R). Twenty one per cent patients had stage II and the rest had advanced (stage III and IV) disease. Majority of the patients had histological grade II and III tumours (28 and 65% respectively). Of the 150 patients, 58 node positive patients showed C erb B2 expression as compared to 18 node negative patients. The overall survival of patients was significantly unfavourable for patients with node positive, C erb B2 positive breast tumours. There was a significant difference in mean levels of EGF and overall survival in stage II and advanced breast cancer. There was no difference in overall survival in EGFR negative and positive patietns. A total of 21 patients (14%) had tumours positive for IGF-1R and the overall survival was significantly better in them when compared to IGF-1R negative patients. In the univariate analysis, significant differences in overall survival was not observed for patients with C erb B2 negativity, EGF < 1.0 ng/ml serum and EGFR negativity than those with C erb B2 positivity, EGF>1.0 ng/ml serum and EGFR positivity. On the other hand, the difference in the overall survival in two subgroups of IGF-1R was statistically significant. IGF-1R was found to be an independent predictor of short- term prognosis in advanced breast cancers. The multivariate analysis in patients with advanced breast cancers showed that patients who were C erb B2 positive, EGFR positive and IGF-1R negative and had EGF>1.0 ng/ml had worse prognosis as compared to those who were C erb B2 negative, EGFR negative, IGF-1R positive and had EGF<1.0 ng/ml. Based on these observations, stage II patients could be grouped as high-risk (C erb B2 positive, EGFR positive and IGF-1R negative) and low-risk (C erb B2 negative, EGFR negative and IGF-1R positive) groups. It is thus concluded that the presence of IGF-1R in tumours tissue indicates a good prognosis and over expression of C erb B2 and EGFR with absence of IGF-1R is suggestive of a worse prognosis.



IMMUNOLOGICAL STUDIES IN PATIENTS WITH CERVICAL LESIONS(BENIGN AND MALIGNANT) USING RECOMBINANT HUMAN PAPILLOMA- VIRUS FUSION PROTEINS  

The study was carried out to determine the humoral and cellular immune response against human papillomavirus (HPV) 16 in patients with benign and malignant lesions of the uterine cervix and to correlate the response with the clinical status of the patients. Serum samples from 13 hisitopathologically confirmed patients with carci- noma cervix and 9 healthy asymptomatic women were analysed for detection of antibody response to HPV16E7 and L1 fusion proteins in a Western blot assay. Antibodies to HPV16E7 fusion protein were detected in 9 (69.2%) patients and 1 (11.1%) healthy woman, whereas all the 13 patients and 9 healthy women revealed the presence of antibodies to HPV16L1 fusion protein. ELISA was carried out with the synthetic peptides of two B cell epitopes of HPV 16E7 gene product for detecting the presence of antibodies in 30 patients cytopathologically confirmed to have cervical intraepithelial neoplasia (CIN)-I,21 patients of CIN-II, 13 patients of CIN-III, 30 patients of histopathologi- cally confirmed carcinoma cervix and 30 healthy asymptomatic women. Two synthe-tic peptides of HPV16E7 comprising the amino acid sequences QAEPDRAHYNIVTF (a,a. 44-57) and QLNDSSEEEDEI (a.a.27-38) were designated as peptides PI and PIIrespectively. Thirteen (43.3%), 4 (19%), 7 (53.8%) and 8 (26.6%) patients with CIN-I, CIN-II, CIIN-III and carcinoma cervix respectively had antibodies to PI peptide whereas 9 (30%), 16 (76%), 6 (46.1%) and 18 (60%) patients with CIN-I, CIN-II, CIN-III and carcinoma cervix respectively had anti-bodies to PII peptides. None of the healthy women had antibodies to PI and PII peptides of HPV16E7. Lymphoproliferative assays (LPA) were carried out with synthetic peptide (EPDRAHYNIVTFCC: a.a. 46-59) of HPV16E7 T cell epitope for detection of T cell responses. Two (6.6%) of the 30 controls and 19 (63.3%), 9 (42.8%), 4 (30.7%) and 11 (36.6%) patients with CIN-I, CIN-II, CIN-III and carcinoma cervix respectively showed positive resposne to T cell epitope. No correlation could be established between the severity of the disease and T cell responses. The study thus suggests that peptides PI and PII are the two major immunodominant B cell epitopes of the HPV16E7 and PII is more immuno- dominant cmpared to PI. However, more studies need to be carried out to see whether antibody detection to these two peptides can be used to monitor the prognosiis and biological progression of patients with CIN. DNA samples from biopsy material from 30 carciinoma cervix patients were tested for the presence of HPV DNA in dot blot hybridization experiments. Twenty three (76.6%) DNA samples were positive for HPV DNA under low stringency conditons and 10 (43.4%) of these 23 samples showed the presence of HPV 16 DNA. ELISA showed the presence of antibodies to HPV 16E7 in 7 (70%) of the 10 patients withHPV16DNA, 6 (46.1%) of the 13 patients with HPV DNA and 6 (85.7%) of the 7 patients with no evidence of HPV DNA by dot blot hybridi-zation. LPA reactivity to HPV 16E7T cell epitope was observed in 3 of 10 (30%)HPV16 positive, 7 of 13 (53.8%) HPV16 negative but other HPV positive and 1 of 7 (14.3%) HPV negative cervical cancer patients. These findings suggest that LPA reactivity to HPV16E7 T cell epitope was depressed in most HPV 16 positive cervical cancer patients.



ROLE OF CYTOSTRUCTURAL ALTERATIONS IN CERVICAL CARCINOGENESIS  

The study was carried out to find out the role of alterations in various cytoskeletal proteins during the process of tumour progression in uterine cervix. The cytoskeletal proteins analysed included cytokeratins (CKs) 1,10,11,13,14,16,18 and 19 as well as the terminal differentiation protein involucrin. Tissue samples from a total of 180 subjects were studied. This included 60 non-malignant cervical tissue samples, 70 invasive carcinoma samplesand 50 premalignant samples classified as cervical intraepithadal neoplasia - CIN I (nald dyspasia), CIN II (moderate dysplasia) and CIN III (severe dysplasia). Non-malignant samples were obtained from patients undergoing hystereotomy. In non-malignant cervical epthelium CKs 13,14,18 and 19 were expressed in the basal cells while CKs 1,10 and 11 in the spinal cells. CKs 18 and 19 were expressed intesely in endocervical cells. In precancerous lesions CIN I, II and III), there was no expression of CKs 13,16,1,10 and 11 in thebasal cells. CK 19 showed intense expression in basal cells of CIN I and no expression in CIN II and III. CK 14 was found to be intesely expressed in CIN III. In invasive carcinomas, 60 per cent or more of malignant cells were positive for CKs 19,18,14 and 13, showing that expression of these CKs are maintained during tumour progression from non- malignant to invasive carcinoma.CKs 1,10,11,13 and 16, however, did not show intense expression in the majority of patients analysed. Involucrin showed intense expression in the upper spinal layers of normal and inflammatory squamous epithelium. In contrast, lesions with CIN I showed a mild staining pattern in the spinal layers (both lower and upper), whereas CIN II showed mild expression in the upper spinal layer CIN III lesions did not express involucrin. In invasive carcinoma expression of involucrin was found tobe negative in 67 out of 70 samples analysed. It may thus be concluded that analysis of expression of CKs and involucrin provides a very useful complement to the established histologic procedures for understanding tissue pathology as well as various histogenetic pathways involved in tumour development.



RESPONSE OF SARCOMA-180 TO PLUMBAGO ROSEA (CHITRAKA), RADIATION AND HYPERTHERMIA  

Tumour growth inhibiting and radiosensitizing effects of the alcoholic rootextract of Plumbago rosea, gamma radiation and hyperthermia were studied in experimental tumours in mice. Response to treatment was assessed on the basis of tumour regression, colume doubling time, growth delay and tumour free survi- val. Sarcoma-180 tumours were produced in Balb/c mice by intradermal injection of 5x10 viable sarcoma-180 cells. Ehrlich ascites carcinomas were propagated byintraperitoneal injection of tumour cells into young adult Swiss albino mice. The alcoholic root extract of P. rosea was effective in inhibiting exponen-tially growing sarcoma-180 solid tumours. A dose of 75 mg/kg intraperitoneally for 10 days was found to be safe and effective in increasing tumour cure and growth delay. Plumbago extract (75 mg/kg) had an additive effect with hyperthermia (43 degree C for 30 min) but a subadditive effect with gamma radiation (10 Gy) in effecting tumour cure and long-term sur- vival oof mice bearing sarcoma-180. Tumour cure and volume doubling time and growth delay of partially responding tumours significantly increased after com- bined treatment of the extract, radiation and hyperthermia above that produced by bimodality treatments. Even where the extract individually was not very effective as an anti tumour agent, it acted as a good radiosensitizzer increasing the radiosensiti- vity of Ehrlich ascites carcinomas. It produced a synergistic effect in increasing the tumour inhibition and animal survival from 10 to 50 per cent. The active component plumbagin, a napthoquinone derivative from P. rosea was effective in inhibiting the growth of Ehrlich ascites carcinoma in mice and thiseffect could be enhanced by combining the drug with radiation. A total dose of 9 mg/kg, given in 3 fractions of 3 mg/kg each, was found to be optimum for plumbagin when used alone or as an adjuvant to radiotherapy. Plumbagin in combination with radiation (7.5 Gy) significantly incrased the induction of micronuclei in Ehrlich ascites tumour cells in vivo as compared to that induced by single modality treatments. The bimodality treatment produced synergistic incrase in micronuclei count at all time points studied (24 to 72h). Plumbagin treatment resulted in an immediate increase in the S-phase population of cells. Combination of plumbagin with radiation induced reproductive death ofproliferating cells and G2-M block. The increase in micronuclei from 24 to 72 hafter combination treatment probably reflected the delayed expression of the cytogenetic damage when the affected cells enter division after G2-M block. This could contribute to the higher tumour response and tumour free survival after combination treatment as compared to individual modality treatments. The study this indicated that P.rosea could be a promising drug for human cancer therapy, it has a remarkable radiosensittising effect and requires detailed investigation.



CLINICAL PATHOLOGICAL FEATURESAND PROGNOSTIC SIGNIFICANCE OF TUMOUR MARKERS IN TONGUE CANCER *  

A prospective study was carried out on 90 patients with squamous cell carcinoma of the tongue and 5 with leukoplakia, and 5 autopsy specimens (as controls)to evaluate the prognostic significance of various tumour markers in tongue cancer. The determinants studied were prolactin (PRL) and bioactive prolactin in blood samples and prolactin mRNA, cathepsin D mRNA and membrane bound epidermal growth factor receptor (EGFR) in primary tumours. Hyperprolactinaemia was observed in 31 of the 90 (34%) patients with advanced tongue cancer. PRL levels were significantly higher in tobacco chewers/smokers. PRL levels decreased in patients who responded to various treatment modalities compared to pretherapeutic levels. On the other hand it increased with disease progression. Estimation of bioactive PRL using Nb2 lymphoma cell lines revealed that of the 90 patients, good correlationbetween Nb2 bioassay and immunoradiometric assay was observed in 80 per cent patients with hyperprolactinaemia. The bioactive PRL levels were significantly lower in 50 per cent patients than the immunoradiometric PRL levels. All the 5 (100%) autopsied tongue specimens showed the presence of cellularRNA while this was seen only in 3 of 5 (60%) patients with precancerous lesions and 40 of the 90 (44%) patients with tongue cancer. PRL mRNA was detected in only 10 of 40 (25%) patients with tongue carcinomas but in none of the autopsiedtongue specimens or patients with precancerous lesion. Cathepsin D mRNA was expressed in all 5 (100%) autopsied tongue specimens, none of the patients with precancerous lesions and all 40 patients (100%) with tongue carcinomas who were studied. Out of 90 patients, 83 (92%) had EGFR positive tumours and 7 (8%) EGFR negative tumours. It is thus concluded that presence of PRL mRNA confirms its ectopic produc-tion by tongue tumours. This ectopically produced prolactin might be acting as one of the major growth promoters via autocrine/paracrine mechanism. Immuno- reactive prolactin is a better marker than bioactive prolactin for monitoring disease course. High levels of EGFR is an unfavourable prognosis. Overexpression of cathepsin D mRNA is probably an indicator of tumour aggressiveness. Hyperprolactinaemia clearly differentiates patients with aggressive tumours.



DETECTION AND ASSESSMENT OF MOLECULAR MARKERS ASSOCIATED WITH MULTI-DRUG RESISTANCE IN HIGH GRADE NON HODGKINS LYMPHOMA *  

Lymph node biopsies and fine needle aspirates from 109 patients with high grade non-Hodgkins lymphoma (NHL) were analysed to study the expression of mdr gene and mdr gene product-P-glycoprotein in high grade NHL and correlation of the findings with the clinical manifestations and course of the disease. Normallymph nodes dissected during head and neck surgery were used as control. Patients with severe disease at presentation, those with a second malignancy or coming after treatment at another hospital, and those over the age of 70 years were excluded from the study. The patients were clinically followed up for periods ranging from 7 to 34 months after chemotherapy or until the recurrence of the disease. Of the 109 patients, 73 remained disease free during the follow up period and 36 patients showed residual or recurrent disease. Patients with residual orrecurrent disease underwent a second lymph node biopsy analysis. None of the control lymph nodes and only 5 of the 73 (7%) patients who remained disease freeduring the follow up period showed the presence of P-glycoprotein. However, 26 of the 36 patients (72%) with recurrent/residual disease expressed P-glyco- protein suggesting a correlation between the expression of P-glycoprotein and recurrence of disease. In situ hybridization using a specific C-DNA probe to the mdr gene gave a positive reaction in 2 of the 5 disease free patients who showed expression of P-glycoprotein but in none of the 68 disease free patients with no expression of P-glycoprotein. Twenty five of the 36 patients with residual/recurrent disease showed the expression of mdr gene. Western blot analysis showed 30 of the 36 samples from these patients to be positive for P-glycoprotein, while Southern blot analysis for mdr gene showed positive reactions in all the 36 samples. None of the samples from 42 of the 73 disease free patients gave positive Western blot or Southern blot reaction. Repeat biopsies from all the 36 patients with residual/recurrent disease gave positive Western blot and Southern blot reactions. Correlation analysis showed significant relationship between prognosis and the detection of P-glycoprotein by immunocytochemistry and Western blot and mdr gene expression by Southern blot. P-glycoprotein analysis therefore is a prognostic indicator for high grade NHL provided it is accompanied by Western blot and Southern blot analysis to rule out false negative.



EXPRESSION OF C-ERB-B2 ONCOPROTEIN AS A PROGNOSTIC INDICATED IN BREAST CARCINOMA  

Expression of c-erb B-2 oncoprotein as a prognostic indicator in breast carcinoma. The study was carried out on tissue from 50 patients with histologically identified carcinomas of the breast of evaluate newer prognostic factors viz. AgNOR ( Silver binding nuclear organising regions), amplification and expression of proto onogene c-er B-2, and vimetin expression along with estrogen and progesterone receptors (ER and PR). A total of 27 (54%) neoplasms showed positively for c-erb B-2 oncoprotein with variable degrees of staining; staining for vimentin were noted in 21 (42%) of the neoplasms. Twenty (40%) and 11(22%) of the neoplasms were positive for ER and PR respectively, while 5 (10%) were positive for both. The mean AgNOR count for malignant neoplasms was 10.46 with a range from 5.1 to 15.10. AgNORs in these lesions frequently exhibited large clusters, small dots and small clusters arranged around the nuclear membrane and often dispersed throughout the nucleus. Poorly differentiated tumours (grade III) had evidence of intense membranous staining (70%) than well diffenentiated (grade 1 -54.5%) or moderately differentiated tumours (grade II -44.4%). When subjected to AgNOR counting, these showed a range between 10.23 (grade I), through 11.70 (grade II) to 12.45 (grade III) mean dots/ nucleus. Statistically the histological grade and c-erb B-2 oncoprotein over expression were significantly correlated but not the histological grade and AgNOR count. Of the 27 c-erb B-2 positive neoplasms, 21 were negative for ER, 18 for PR, and 17 of both ER and PR of which 7 were grade III tumours. c-erb B-2 oncoprotein over expression was found to be significantly correlated to ER and PR negtivity. Staining for vimentin showed positivity in 21 of 50(42%) invasive ductal carcinomas. A total of 16 tumours showed positivity for both vimentin and c-erb B-2 of which 7 tumours were negative for ER and PR (4 were of grade III). Vimentin was expressed in 90 percent of grade III tumours compared to 33.3 percent of grade I tumours. The tumours showing ER and PR positivity did not express vimentin. Nine tumours were negative for ER and PR but showed vimentin expression in cytoplasm. The expression of vimentin in ER and PR negative tumours was statistically significant. It is thus concluded that c-erb B-2 marker can be used to predict disease progression in ER and PR negative tumours. Vimentin and c-erb B-2 oncoprotein help in detection of tumours which do not respond to routine management. However, AgNOR counts, c-erb B-2 oncoprotein and histologic grade did not show mich correlation. Thus AgNOR alone cannot be taken as a marker of tumour proliferation.



CLINICAL APPLICATION OF TUMOURGROWTH FRACTION ASSOCIATED MARKERS IN THE RADIOTHERAPY OFCANCER OF THE UTERINE CERVIX  

Clinical application of tumour growth fraction associated markers in the radiotherapy of cancer of the uterine cervix. The study was undertaken on 312 cervical cancer patients (stage IIb or III) to define the role of tumour growth fraction and its relationship to the clinical course of the disease, as also to know whether cell knetics had an association with response of the tumour to radiotherapy. Tissue biopsies and smears of exfoliated cells were taken from all patients before treatment. All pre-treatment biopsies were analysed for labelling index using bromo deoxy uridine (BrdUrd) labelling of DNA and Ki 67 antigen expression. In addition, immunocytochemical localization was carried out for argyrophillic nucleolar organiser region (AgNOR), expression of epoidermal growth factor (EGF) and its receptor (EGF-R), transforming growth factors alpha and beta (TGF alpha and TGF beta), HPV transforming protein E6, tumour suppressor protein p53 and the anti-apoptotic product bcl-2. Of the 312 patients, 152(106 disease free- Group 1 and 46 with residual/ recurrent disease - Group 2, after treatment) were evaluated through their course of radiotherapy and upto a follow up of 12 months and correlation was made between pre treatment status of tumour growth fraction associated markers and clinical outcome after radiotherapy. To study the transition of parameters analyzed during radiotherapy, cervical smears were obtained before the therapy, and after 1000, 2000, 3000 and 4000 cGY. Pre treatment analysis of AgNORs significantly correlated to disease status treatment. Lower AgNOR counts were significantly associated with Group 2 patients suggesting that increased proliferative acitvity may be a positive prognostic indicator. Observations made during various phases of radiotherapy revealed consistent higher AgNOR counts in Group 1 patients. However, the other proliferative associated markers did not show such an assiciation. EGF and EGF-R showed significant pre treatment ciorrelatoins with the final disease outcome. Both these markers were expressed more by patients belonging to Group 2, whereas the TGF alpha was expressed more by patients belonging to Group 1. Group1 patients mostly showed mild to moderate expression for TGF beta while most Group 2 patients were negative for this growth factor. This conspicuous difference was also evident during the various of radiotherapy. Pre treatment expression of bcl-2 was different in the two groups of patients. Most patients in Group 1 showed mild expression while in Group 2, Teh majority showed a moderate to intence expression. Similarly, during the various phases of radiotherapy, Group 2 patients showed consistently higher levels of bcl-2.



TUMOUR CELL KINETICS IN GESTATIONAL TROPHOBLASTIC DISEASE - AS A PREDICTIVE INDEX OF TUMOUR AGGRESSIVENESS 

The expression of markers of cell proliferation in estational trophoblastic disease, was evaluated immunohistochemically. Efforts were also made to correlate the status of markers of cell proliferation with the clinical profile, particularly the regression pattern of the tumours, the level of Beta hCG and histologic nature of the issue. The markers included proliferating cell nuclear antigen (PCNA), nuclear organizer region associated proteins (AgNORs), cell kenetic maker (Ki67) and epidermal growth factor receptor (EGFR). The study material comprised histologically ggraded trophoblastic tumours obtained from 122 patients of gestational trophoblastic disease and 50 normal placetae of comparable gestational age collected after medical termination of pregenancy and term delivery. Both norma normal and pathological placentae belonged to gestational ages between 6-36 weeks, with the mean ggestational age of normal placentae being 17.75+_ 11.21 weeks and that of the pathological placentae being 15.96+_6.11 weeks. The PCNA, Ki67 and EGFR expressed more strongly by the molar placentae in comparison to normal placentae of similar gestational age. EGFR and PCNA were expressed strongly by the molar placentae of all gestational ages unlike normal placentae where the expression was restricted to the first and second trimesters. The PCNA staining pattern could differentiate spontaneously regressing tumours from slowly regressing tumours and Ki67 staining pattern could identfy those tumours which require chemotherapy for regression. It is thus clear that the staining indices of PCNA in combination with Ki67 would serve as a better marker in the assessment of the prognosis of trophoblastic tumours



ROLE OF SOME OF THE NONSTEROIDAL ANTI-INFLAMMATORYDRUGS IN CARCINOGENESIS  

Role of some non-steroidal anti-inflammatory drugs in carcinogenesis. The study was carried out in male weanling inbered Swiss mice to evaluate the role of non-steroidal anti-inflammatory drugs (NSAIDs) viz, piroxican, diclofenac and naproxen in carcinogenesis. The animals were fed the NSAIDs through diet. For mechanistic studies the doses of diclofenac, naproxan and piroxican were 25, 75 and 375 ppm; 150, 500 and 1500 ppm; and 3, 10 and 30 ppm respectively for 4 weeks. For tumour studies the highest doses of the NSAIDs were used with carcinogen Nitrosodiethylamine (NDEA) in the dose of 80mg/kg body wt. at 4 weekly intervals thrice. Diclofenac in a dose of 375 ppm in the diet inhibited NDEA induced lung tumourigenesis by acting at the initiation and post-initiation phases of of carcinogenesis, whereas naproxen in a dose of 1500 ppm stimulated the promotional phase of carcinogenesis. Ornithine decarboxylase (ODC), which is the rate limiting enzyme in mammalian polyamine biosynthesis required for tumour promotion and cell proliferation in various organs, was inhibited by diclofenac and piroxcan, byt was stimulated by naproxen indicating that this enzyme is a good marker for tumourigenesis and for agents with anticarcinogenic properties. Prostaglandin E2 release, which is involved in inflammation and tumour promotion. was decreased to a variable degree in plasma, liver and lung by all three NSAIDSs tested; diclofenac biing the storngest, and naproxen the weakest inhibitor. Diclofenac and piroxican slowed down the NADPH and ascorbate dependent microsomal lipid peroxidation in vivo and in vitro, whereas naproxen at a concentration of 10 mM stimulated the process. It seems that besides being a scavenger of free rdicals, enzymatic metabolism of naproxen at higher concentration formed reactive intermediates/ free radicals which exceeded is scavenging capacity. The feeding of NSAIDs to mice inhibited the process of lipid peroxidation and stimulated the tissue antioxidant defence system comprising superoxide dismutase, catalase, glutathione peroxidase, glutathione recductase and reduced glutathione. It also resulted in the decrease in cytochrome P-450 levels. Increased activities of glutathione-s-transferase, glutathione reductase and reduced glutathione by NSAIDs may detoxify the xenobiotics more efficiently. The enhanced levels of reduced glutathione, in addition may provide protection to tissues against the damaging action of free radicals. The enhanced tumourigenesis and ODC levels produced by naproxen requires further evaluation for its safety.



ROLE OF 'S' TRANSACTIVATOR IN HEPATOCARCINOGENESIS : INVITROEXPRESSION AND PURIFICATION OFTRANSACTIVATING PROTEIN  

Role of 'S' transactivator in hepatocarcinogenesis: In vitro expression and purification of transactivating proteins. The study was carried out to elucidate the effect of 'S' (463-851) transactivator on various promotor enhancer systems; raise monoclonal/ plyclonal antibodies against this transactivator protein prepared either by recombinant DNA method or by using synthetic peptides; and demonstrate and localize the transactivator protein in hepatocarcinoma cells. This association of hepatitis B virus (HVB) in hepatocellular carcinoma (HCC) is well established. Insertional mutagenesis, transactivation by truncted X or pre S2/S regions and activation of growth regulated genes or oncogenes have been suggested as possible mechanisms for carcinogenesis. The transactivating properties of a frequently detected fragment, which form part of the 'S' reading frame, extending from 426 nt, to 855nt., has been reported The clones were sequenced and subcloned into pRSET vectors in all the three reading frames and expressed using IPTG induction. A Western blot analysis was done using polyclonal anti HBsAG and the presence of 19.24 kDa functinal protein in the correct reading frame was obtained. The expressed protein was checked for by commercial ELISA. However, the antigen could not be detected by ELISA in any of the three reading frames. The 'S' proteins was purified by SDA-PaGE and was used to raise polyclonal antibodies in rabbits. These polyclonal antibodies were then used to screen tissues. The 'S' region being a very small part of the surface region of the virus (426-824 ntds), could not be detected by immunofluorescence. The region, extending from 426-824 ntds was proved to be a generalized transactivator of the viral promoters and enhancers. Using chloramphenicol acyl transferase as a reporter gene, in transfection assays it was found to be capable of activating HIV promoter, HTLVI promoter, RSV promoter and enhancer, c-Fos and c-Jun promoter to a high degree.



C-MYC LOCALIZATION IN PREMALIGNANT AND MALIGNANT LESIONS OF HUMAN COLON : SEMI AUTOMATIC IMAGE ANALYSIS AND IMMUNOHISTOCHEMICAL STUDY  

The correlation of the expression of c-myc oncogene with the degree of differentiation of premalignant and malignant lesions of the colon was examined immunohistochemically using monoclonal antibodies against c-myc protein and by morphometry. The colonic lesions studied included polyps with dysplasia, well differentiated, moderately differentiated and poorly differentiated carcinomas, signet ring carcinoma, mucinous carcinoma and adenosquamous carcinoma. Paraffinsections from normal colonic mucosa served as controls. Sections representing the normal colonic mucosa showed the highest nuclear c-myc positivity (84%). The percentage of nuclear positivity declined with the increase in histologic grades - the highest (59%) and lowest (2%) nuclear c-myc positivity being recorded in cases of well differentiated carcinoma and adenosquamous carcinoma respectively. There was a significant increase in the cytoplasmic positivity of the c-myc oncoprotein in various lesions (except for signet ring carcinoma which showed zero cytoplasmic positi- vity) as compared to controls. The nuclear diameter was found to be a reliable cytological variable in distinguising severe dysplasias with well differentiated carcinomas. Moderately and poorly differentiated carcinomas and adenosquamous carcinoma showed signifi-cantly higher values for nuclear diameter compared to controls and well differ- entiated carcinoma. There was a significant decline in nuclear volume with an incrase in the histological grade. With an increase in nuclear volume the nuclear expression of c-myc oncorprotein declined. Well differentia-ted carcinomas showed nearly equal distribution of c-myc positivity in the nucleus and cytoplasm. Moderately differentiated carcinomas showed predominan-tly cytoplasmic c-myc positivity. Poorly differntiated carcinomas showed very weak nuclear but intense cytoplasmic positivity. It may be concluded that cytoplasmic c-myc over expresssion is related to the aggressive behaviour of the tumour as it is a growth regulating protein. Hence the assessment of c-myc over expression by immunocytochemistry is an additional prognostic indicator. Combining immunocytochemistry of c-myc oncogene with morphometric analysis will give new dimensions in predictingthe prognosis of colonic neoplasms. However, a large number of cases need to bestudied for a more reliable assessment of c-myc expression.



OVEREXPRESSION OF P53 AND CELLPROLIFERATION INDICES IN NON- HODGKIN'S LYMPHOMA - A CORRELATIVE STUDY  

The study was carried out to assess the morphological classification of non Hodgkins lymphoma NHL) on H&E stained sections and the oer expressin of p53, argyrophilic nucleolar organiser regions (AgNORs) and proliferating cell nuclear antigen (PCNA) in immunohistochemistry of formalin fixed, paraffin embedded sections. According to the NCI Working Formulatins for Clinical Usage, 1982 the 50 NHLs were classified as small lymphyocytic lymphoma-17; follicular, small cleaved lymphoma-6; follicular, mixed small and large lymphoma-2; diffuse small cleaved lymphoma-11; diffuse, mixed small and large lymphoma-7; diffuse, small non cleaved lymphoma-4; immunoblastic lymphoma-2; and lymphoblastic lymphoma-1. Out of 50, 25 were low grade. Six(12%) NHLs (1/25 low grade, 2/18 intermediate and 3/7 high grade. Six(12%) NHLs (1/25 low grade, 2/18 intermediate and 3/7 high grade lymphomas) were positive for p53 (>10% of tumour cells showing positivity). PCNA labelling index was >1% in 4(16%) of the low grade lymphomas, 6(33%) of the intermediate and 5 (71.5%) of the high grade lymphomas.It was found that all the p53 positive lymphomas had a PCNA labelling index of >30%. The range of AGNORs was 2.0 to 7.0 in all but one low grade lymphomas, whereas the intermediate grade lymphomas showed a range of 7.0 to 9.2 and high grade lymphomas a range of 8.0 to 14.4 It was found to PCNA showed a higher labelling index in higher grade cases, indicating the presence of higher proportion of proliferating cells. AGNOR scores showed a direct relationship PCNA lebelling index in that both were increased in high grade lymphomas. p53 and PCNA immunoreactivity showed a relationship in that PCNA labelling index was high in tumours with p53 positivity. Findings of this study indicate that p53 expression in malignant lymphomas is related to the grade lymphomas and that p53 positivity may also be a marker for cell proliferation.



EVOLVING STRATEGIES FOR THE PREVENTION OF ORAL CANCER IN INDIA  

The study was carried out on 79 oral cancer patients (45 male and 34 female in the age of 24-82 years) to determine the factors causing delay in seeking medical advice, Majority patients (57/79; 72%) were illiterate and 22/79(28%) had education upto higher secondry level. Eighty percent patients were from lower and 20% from higher socioeconomic group. The delay in months was used as a unit to measure delay. The primary delay was defined as the time taken by the patients to seek first medical opinion and secondary delay as the time taken to consult a specialist where treatment was possible. The period of primary delay ranged from 3 days upto nearly 3 years and of secondary delay from day zero to 4 years. Only 5/79 (6.3%) patients visited a qualified doctor within the first week after they perceived a problem in their month while 6/79 (7.6%) and 2/79(2.5%) patients delayed untill the 2nd and 3rd year, respectively. A relationship between primary and secondary delay was investigated by linear regression analysis. It was found that for patients with primary delay of less than 6 months secondary delay was longer in majority. However for patients with primary delay of more than 6 months the secondary delay seemed shorter than primary delay. This refelects that patients who present themselives for medical opinion are simply not reffered to a hospital with treatment facilities as long as the problem is not too serious. Age,sex, socio-economic status and educational level of patients had no significant relationship with the proportion of early and advanced clinical stage of cancer. Stage of cancer presentation to a specialist was found tto be related with primary delay. Longer the duration of the primary delay more was the probability having advanced cancer. The stage of cancer did not relate to secondary delay. Important psycho-social factors responsible for the primary delay (on patients account) emerged out to be ill fated to have cancer, cancer a curse., non availability of transport., prolonged treatment renders family stressful,etc. It is thus clear that there is a need for a health education campaign for the masses in India. To be effective this will have to be in vernaular and scientifically elaborated and directed towords population at risk. It should have components of education towards early self detection of oral cancer by recognising the need of developing a plan of motivation exercise. The secondary delay detected during the study equally emphasises the need to educate the primary care physicians for early recognition of oral lesions and referral of such partients.



EXPRESSION OF ANTIMETASTATIC MARKER-NM23 AND PROLACTIN IN HUMAN BREAST CANCER: ITS RELATION TO RELAPSE SURVIVAL AND OTHER PROGNOSTIC FACTORS  

The study was carried out in 100 patients (25 with benign breast disease and 75 with breast cancer) to investigate the combined utility of an antimetastatic marker nm23 and prolactin a marker of tumour aggressiveness, in the management of patients with benign breast disease early and advanced breast cancer. The parameters studied included prolactin (PRL) estimation pretherapeutic and serial monitoring as an indicator of disease status, immunohistological localization of (PRL), expression of PRL mRNA and nm23 mRNA by reverse transcriptasepolymerase chain reaction (RT-PCT). Hyperprolactinaemia was observed in 52% (13/25) of patients with benign breast disease and 55%(41/57) of breast cancer patients. Hyperprolactinaemaia was observed more in patients with advanced stage cancer (41%). The levels of PRL increased with appearance of recurrence/ metastatic disease and decreased in patients who responded to therapy. It was found that rise in PRL level preceded disease progression. The PRL level remained elevated througout the course of the disease in patients who did not respond to adjuvant therapy. In 72% (18/25) of patients with benign breast disease and 79%(53/67) of breast cancer patients the tumours showed immunoreactivity with PRL anibody. A total of 56%(40/71) breast cancer tumours and 40%(4/10) of the benign tumours expressed PRL-mRNA. Immunoreactivity of nm23 was found to be 84,69 and 80% in benign, stage II and advenced stage cancer patients, respectively. The nm23 mRNA expression was observed in 70% of benign and 100% breast cancer patients. Eighty six percent(44/51) of the nm23 posittive and 56% (9/16) of the nm23 negative tumours showed immunoreactivity with PRL antibody. The results showed that PRL is a superior marker for monitoring the disease course whearas nm23 cancer be used as an antimetastic marker but is a marker of tumour aggressiveness.



FRAGILE SITES AND MUTAGEN SENSITIVITY STUDIES IN COLON CANCER FAMILIES  

Fragile sites and mutagen sensitivity in colon cancer families. The study was carried out to determine the presence if any, of constitutional chromosomal markers and chromosomal fragile sites in familial and non familial colon cancer patients and their close relatives: and to see whether any family member showed mutagen sensitivity or resistance with regard to chromosomal braakege. The subjects studied comparised 44 familial colon cancer patients from 34 families (23 males and 21 females), their first/ second degree relatives(189) and 44 sporadic colon cancer patients (no family history of colon cancer). Cytogenetic analysis of peripheral blood lymphocytes of colon cancer patients and their relatives showed a normal chromosomal pattern without any constitutional or sturctural anomalies. No consistent fragile sites were detected in colon cancer patients and their relatives.All familial colon cancer patients showed bleomycin induced chromosome hypersensitivity. Of the 189 relatives of familial colon cancer patients, 13(6.9%) showed hypersensitivity, 33(17.4%) normal sensitivity and 143(75.7%) hyposensitive. Among the 44 sporadic patients, 7(15.9%) were hypersensitive, 31(70.5%) sensitive and 6(13.6%) hyposensitive. Of the 13 hypersensitive relatives 2(15.3%) developed colon cancer. Subsequently Pedigree analysis showed that familial colon cancer was inherited in an autosomal dominant pattern. The observations indicate that the genetic suseeptibility risk in familial colon cancer is transmitted from generation to generation. There was a good correlation between blemycin induced chromosome hypersensitivity and cancer susceptibility. Results of this study indicate that the blemycin induced chromosome sensitivity analysis can be utilized as a biomarker for identification of high risk members in cancer families.




OPHTHALMOLOGY
CELLULAR MANIFESTATIONS OF P53AND bcl-2 GENES IN OVARIAN CARCINOMA  

The study was carried out to determine the extent of apoptosis in various ovarian tumours and correlate it to the expression of apoptosis regulatory genes p53 and bcl-2 as well as the total proliferative compartment of the tumour defined by the expression of the proliferating cell nuclear antigen (PCNA). Patients (138) with various histopathological types, stages and grades of ovarian carcinoma were included in the study. Of these, 24 patients had benign cystadenomas and were grouped as controls. The malignant tumours were further sub-divided to include 51 patients with serous, 26 with mucinous, 18 with endometroid, 6 with clear cell and 13 with undifferentiated carcinomas. Expression of p53 and bcl-2 oncogenes were analysed by immunocytochemistry and Westren blot assay. The levels of mutant p53 was estimated using a mutant specific p53 ELISA. Apoptosis was evaluated by analysing the presence of nucleosomes by ELISA and further confirmed by the TUNEL (Tdt-mediated dUTP biotin nick end labelling) assay. The bcl-2 gene was found to be expressed predominantly in benign cystadenomas. It was also expressed by endometroid carcinomas. However, it was mostly absent in serous tumours, clear cell carcinomas and undifferentiated carcinomas. None of the benign lesions expressed p53 while most of the undifferentiated (77%) and serous carcinomas (59%) expressed the protein. ELISA results for mutant p53 closely correlated with the immunocytochemistry data. The tumour proliferative compartment analysed by PCNA expression was also maximum in undifferentiated carcinomas and serous tumours. The presence of p53 protein and expression of PCNA were segnificantly correlated, supporting a major role of p53 in the regulation of cell proliferation in ovarian tissue. The lack of correlation between bcl-2 and PCNA suggested that bcl-2 is not a major regulator of cell proliferation. Higher levels of apoptosis were seen in mucinous carcinoma compared to the other tumour subtypes. Mucinous carcinoma also had lower levels of bcl-2 expression. There was an increased apoptosis and bcl-2 expression in benign cystadenomas.



STUDIES ON THE EVALUATION OF POTENTIAL DRUGS FOR THE MEDICAL THERAPY OF CATARACTS, AND THEIR MECHANISM OF ACTION  

A group of non-steroidal anti-flammatory drugs (NSAIDs) viz naproxen aspirin ibuprofen piroxicam sulindac indomethacin and flurbiprofen were screened for their anticataract potential (both prophylactic and curative) mechanism of action and toxicity in both topical and systemic application using Wistar strain galactosemic rats. Attempts were also made of find out the relationship between aldose reductase inhibiting capicity and anti- cataract potential of these drugs. Flubiprofen eye drops (0.3 and 1%) exhibited significant anticataract activity. Prophylactic effect of flurbiprofen (50mg/kg diet) also exhibited anticataract activity. treatment of galactosemic rats with aspirin (DL Lysine) in in the dose of 50 and 100 mg/kg diet and 0.3 and 1 per cent eye drops showed significant anticataract activity. Piroxicam did not show any anticataract activity prophylactically. Indomethacin was not found effective in slowing down or regression of the cataractous process. It was found to be toxic above 50mg/kg diet dose. Naproxen and sulindac were found to be the most potent anticataract drugs. All the NSAIDs studied had aldose reductase inhibiting capacity out of different magnitude. It was observed that the anticataract potential of the most potent inhibitor of aldose reductase, ie sulindac and mild aldose reductase inhibitor ie naproxen were at par and no direct relationship cou be established between the two properties. Naproxen also offered protection against selenite induced cataract probably by preventing the lens from oxidative stress. a constant ratio in water soluble and insoluble proteins till 48 days of glactose feeding was observed in naproxen treated rats. No significant change in polyol contents in lens was observed. The blood glucose and galactose levels were not elevated significantly. The dosage of naproxen tested orally or topically did not exhibit any ocular or systemic toxicity. However sulindac in the dose of more than 480mg/kg diet was found to be toxic.



PRIMARY GLAUCOMA- A STUDY ON ITS GENETIC, BIOCHEMICAL AND IMMUNOLOGICAL ASPECTS  

The present study was carried out to isolate and characterise the exometabolites(EXOM) of Leishmania donovani promastigotes from defined synthetic culture medium. and to evaluate their potential in serodiagnosis and prophylaxis of kala azar. Biological significance of EXOM in host-parasite interactions was also studied. A chemically defined medium using commercially available Alpha MEM supplemented with hemin, HEPES, L-glutamine, D-glucose, folic acid, D-biotin and adenine (S-alpha MEM) was developed which supported luxuriant growth and propagation of L. donovani promastigotes. The medium was found suitable for transformation of isolated amastigotes form infected hamster spleen. Promastigotes could be detected by culturing in this medium, the bone-marrow aspirate or spleen puncture material from kala azar patients. The medium supported cultivation of primary isolates. Macroscopic colonies appeared on agar plates prepared with the medium within 16-20 days after incubation. The cloning efficiency was increased about five- fold by glycerol supplementation. EXOM of L. donovani promastigotes consisted of proteins, glycoproteins and lipophosphopolysaccharide (MW 28-40 kDa). Lipophoshophopolysaccharide (LPPS) could be metabolically labelled with 32 P. The protein media, but the major LPPS could be isolated detected by TLC using saturated phenol- H2O solvent system and stained with H2SO4 +K2CR2O7 spray reagent. LPPS could be isolated from EXOM by phenol-extraction or by DE-52 column chromatography of EXOM with 200 mM NaCl (D2 fraction). Antibody against LPPS of EXOM of L. donovani (URG stain) promastigotes could be detected in kala azar patients sera. The EXOM contained at least 4 distinct antigens of which two werre slow moving and 2 fast moving on immunolectrophoresis (IEP). ELISA using EXOM or its fractions D3 and D4 (the fractions eluted from DE-52 column chromatography of EXOM with 300 mM and 400mM NaCl respectively or PE-O (the organic phase of phenol-extractin of EXOM) as antigen source, for serodiagnosis of visceral leishmaniasis was highly significant in parasite positive patients as compared to control group consisting of normal sera (10) and sera from tuberculosis, lepromatous leprosy and malaria patients (10 each). It was found that the attachment of promastigotes to macrophages was not significantly inhibited by EXOM indicating that there may be some other major ligands responsible for the attachment of promastigotes prior to endocytosis of the parasites.



INTRA-LENTICULAR DISTRIBUTION OF CU,ZN,CD & SE IN NORMAL,HU-MAN SENILE CATARACTOUS LENSES AND EXPERIMENTAL RABBIT MODEL AND ROLE IN CATARACTOGENESIS*  

Intralenticular bdistribution of various trace elements viz. zinc, copper cadmium and selenium was studied in different regions (equator, cortex and nucleus) of human normal and senile catractous lenses. Normal lenses were obtained from eye bank whereas cataractous lenses were obtained by intra- capsular extraction from patients. Concentration of all these elements were higher in various categories (immature, cortical and nuclear) of cataractous lenses compared to normal lenses. In cataractous lenses maximum concentration of these elements was found in the cortical and equitorial zones. Nucleus has least amount of these elements. Antioxidant status of lenses with and wihtout senile cataract was studied Levels of glutathione-reductase, glutathione peroxidase and superoxide dismutase showed a significant decrease in cataractous lenses compare to normal lenses. Levels of ascorbic acid in aqueous humour and leses of cataractous patients decreased significantly compared to normal eyes. Metal binding proteins different regions of the lens viz. cortex and nucleus were isolated and characterised. A 10 per cent lens homogenate labelled with Cd and subjected to G-75 chromatography showed two peaks corresponding to a small high molecular weight protein and a low molecular weight metal binding protein. Cortical portion of lens bound more Cd than the nucleus. Levels of Zn and Cu which co-eluted with Cd were also higher in peaks from cortical regions. Molecular weight of the metal binding protein was found to be 10,000. Ultraviolet spectra showed more absorbance at 254nm which is characteristic of low molecular weight metal binding protein. Antibodies against purified lens protein from cortical and nuclear regins, cross-reacted with lens protein only and not with the low molecular weight metal binding protein from liver or kidney showing that the lens protein is an organ specific protein. Increased concentration of trace elements in the equator and cortex which are metabolically more active than the nucleus and decreased levels of metal binding proteins in this region are probably factors in the pathophysiology of cataract formation.



FUNCTIONAL EVALUATION OF CORNEA IN DIABETES MELLITUS  

Studies involving functional evaluation of cornea in patients of diabetes mellitus were designed to observe measurable effects on corneal senstivity and its correlation to status of diabetic retinopathy and autonomic dysfunction. Functional capacity and reserve of corneal endothelium pump activity was quantified by measuring levels of corneal hydration and regression rates of contact lens induced stress leading to corneal swelling. In addition, the effects of cataract surgery on corneal endothelium have been quantified in eyes of diabetics. In 100 eyes of diabetics with retinopathy corneal sensation (touch threshold) was 1.72+1.16 g/mm2 which was significantly higher than in the diabetics without retinopathy (1.11+0.26 g/mm2) and normal controls (1.03+0.64 g/mm2). Autonomic dysfunction was objectively evaluated by calculating the Valsalva ratio. A significant correlation was present between the corneal senstivity and Valsalva ratio. From data obtained on pachymetry it was possibleto demonstrate that the cornea in patients of diabetes mellitus was significant-ly thicker by five per cent than in non-diabetics. Studies on regression rates of contact lens induced corneal hydration demonstrated that Percentage Recovery Per Hour (PRPH) in the diabetic group (44.79=9.81%) was significantly lower thanin normal controls (65.27+12.01%). Eyes with manifest diabetic retinopathy demonstrated the worst PRPH (37.28+6.01%). Following cataract surgery in 10 eyes of diabetics, corneal thickness at 24 weeks post surgery was 0.58+0.023 mm which was comparable to that in non- diabetics (0.57+0.028mm). Observations from this study document significant loss of corneal sensation in diabetics indicating existence of corneal neuropathy. Strong correlation ofdiminished corneal sensations to Valsalva ratio suggests the possibility of identifying subjects at risk of diabetic autonomic neuropathy by the assessment of corneal sensation. More importantly the observations documentin vivo diminished corneal endothelial pump activity, capacity and reserve in patients of diabetesmellitus as indicated by lowered PRPH.



ROLE OF OXIDATIVE STRESS ENZYMES IN SUGAR CATARACT AND PROTECTION BY ANTI-OXIDANTS (VITAMINS C & E)  

The study was undertaken in Wistar strain albino rats to find out the effect of galactose feeding on activities of aldose reductase (AR), glutathione reductase (GR), glutathione peroxidase (GP), catalase and super- oxide dismutase (SOD) at different stages of galactose cataract. The vivo effect of vitamin C and E (antioxidants) fed individually and in combination on the enzymes and lens opacification was also studied. There was a signifi- cant alteration in the acitvity of all these enzymes in the lenses of galactose fed rats. The observed decrease in GP and GR could be prevented by supplementing the galactose diet with vitamin C or vitamin E. The AR activity gradually decresed from day 2 to day 30 of feeding galactose plus vitamin C and E. The GP activity incresed 3 times on day 23 as compared to day 2, whereas the GR activity increased one and a half times upto day 14 and then decresed upto day 28. Similarly there was a gradual increase in catalase and SOD activities upto day 23 on administration of vitamin C and E together with galactose. The study revealed that the activity of defence system enzymes against oxidative stress was affected in the lenses of galactose fed rats. This effect was reversed to some extent by administering either vitamin C or vitamin E alone in the galactose diet. However, the effect of galactose feeding on lens enzyme activity was reversed more significantly by adminstering both vitamin C and E together with the galactose diet.



CRITICAL EVALUATION OF LASER THERAPY IN THE MANAGEMENT OF DIABETIC RETINOPATHY  

The study was undertaken to assess the relative efficacy of argon green (514 nm) versus krypton red (647 nm) laser photocoagulation in the management ofdiabetic retinopathy and maculopathy respectively and to evaluate the efficacy of peripheral scatter laser photocoagulation in preproliferative diabetic retinopathy (PPDR), The patients were categorised into different groups. Group 1 included 75 eyes of 68 diabetic patients with proliferative diabetic retinopathy (PDR) and high risk characteristics (HRC) while group 2 included 60 eyes of 47 patients with PDR but without HRC. The high risk characteristics included new vessels onor within 1 disc diameter of the optic disc with and without vitreous or preretinal haemorrhage; new vessels on the optic disc (NVD) of approximately 1/4 to 1/3 disc area associated with vitreous and/or preretinalhaemorrhage; and new vessels elsewhere more than 1/2 disc area associated with vitreous and/or preretinal haemorrhage. All these eyes were prospectively ran- domised to ttreatment with argon green (38 and 30 eyes respectively in groups 1 and 2) or krypton red (37 and 30 eyes respectively) laser photocoagulation. At the end of one year follow up, there was no significant difference in the two groups treated with different wavelengths in causing regression of neovasculari-sation and improvement/stabilization of visual acuity. Group 3 included 121 eyes of 88 diabetic patients with clinically significant macular oedema (CSME). The eyes were randomised to treatment with argon green (60) or krypton red (61) laser photocoagulation. At the end of one year follow up the eyes treated with both argan green or krypton red laser behaved similarly as regards the resolution of CSME and preserving/ improving visual acuity from baseline levels. Studies carried out on another group of 60 eyes (26 treated and 34 untrea- ted of 42 patients) to evaluate the efficacy of peripheral scatter laser photo- coagulation in PPDR demonstrated, that the number of eyes showing progression was much less in the treated group (34.6%) compared to control (55.9%). However, no conclusion could be drawn from these findings as the duration of follow up was too short (3 months) to comment upon the beneficial role of this treatment. The results of the study suggest that the quality of care is not compro- mised by the availability of specific laser wavelength for fundus photocoagu- lation. However, a longer wavelength like krypton red offers the additional advantage of better penetration through hazy media and cataract and thus can be used with equal efficacy as the standard argon green laser in eyes with cataractand media opacities.



ETIOPATHOGENESIS AND MANAGEMENT PROBLEMS IN PSEUDOEXFOLIATION SYNDROME  

Patients with cataract (2792; 1695 males and 1097 females) and open angle glaucoma (147) were screened for pseudoexfoliation (PXF) and to find out how such eyes react to surgery foor cataract and glaucoma and their management. One hundred and sixty two of the 2792 (5.8%) patients with cataract had PXF(usually bilateral) with, the highest incidence being found in the age group of 60-70 years. Incidence of PXF was more in males (61%) as compared to females (39%). There was an association of PXF with nuclear cataract, advanced stage ofsenile cataract and sublerxation of the cattaractus lens. In patients with PXF extracapsular cataract extraction reduced the risk of vitreous loss and accidental capsule rupture, but poor mydriasis, zonular weakness and pigment dispersal may pose difficulties. Surgery in PXF syndrome was often complicated by an intense postoperative iridocyclitis and sometimes by hyphaema and vitreous haemorrhage. It is felt that adequate surgical planning and gentle handling of tissues can prevent or reduce the complications. Nearly one-fourth of all the patients with PXF had glaucoma. There was a strong correlation between the presence of PXF material in the eye and glaucoma-tous damage. In addition to pigment deposition in the trabecular meshwork, the accumulation of PAS-positive mucopolysaccharide-like PXF material in the outflowchannels may be a causative factor in pseudoexfoliation glaucoma. Trabeculectomy yielded better results than medical therapy in PXF glaucoma though the results were worse than in primary open angle glaucoma (POAG). Hyphaema and uveitis were more commonly seen following trabeculectomy in PXF glaucoma than in POAG. Intraocular haemorrhage and uveitis might be due to the deposition of PXF material in the iris vessels and the resultant neovas- cularization seen as profuse dye leakage at the pupillary margin during iris fluorescein angiography. This test can be used as a sensitive and specific indicator of PXF syndrome in doubtful cases where even after full mydriasis, slit lamp biomicroscopy does not reveal the presence of PXF material. Findings of the study thus suggest that an awareness of the PXF status will help in proper surgical planning, reduction of operative complications and identification of patients suspected to have glaucoma for long-term follow-up.



ROLE OF PEPTIDE M, UVEITOPATHOGENIC FRAGMENT OF 45 KDA RETINAL S-ANTIGEN IN HUMAN UVEITIS  

Role of peptide M, uveitopathogenic fragment of 45 kDa retinal S-antigen in human uveitis. The study was carried out in patients of uveitis (38), diabetic retinopathy (27) and systemic connective tissue disease (CTD, 30) and 30 healthy volunteers to investigate the role of retinaal S-antigen and its uveitopathogenic peptides (M and G) and uveitopathogenic fragment of interphotoreceptor retinoid binding protein (IRBP) in idiopathic human uveitis. Lymphoproliferative responses were tested in vitro against native S-antigen, peptides M and G, yeast histone H3 peptide and IRBP:R 16 to establish their role in pathogenesis of human uveitis. Seven of 38 (18.4%) patients with uveitis, and none among the diabetic retinopathy and CTD patients and healthy volunters responded (stimulation index >3) to at least one antigen used. One uveitis patient showed response to native S-antigen and peptide M and yeast histone H3. One responded to both S-antigen and peptide M another to responded to both S-antigen and peptide M and another to both peptide G and R-16 peptide. Two responded to S-antigen only, one to peptide M and one to peptide G. In addition, one uveitis patient and two patients of diabetic retinopathy responded to histone H3 only. These findings thus suggest that retinal antigens may play a role in the etiopathogenesis of a subset of idiopathic human uvveitis.



EVALUATION OF INTRA-OCULAR LENS IMPLANTATION IN CHILDREN  

Evaluation of intra-ocular lens implatation in children. Intra-ocular lens(IQL) implantation was evaluated in 75 eyes of 45 patients aged 14 months to 12 yr, with congential, traumatic and developmental cataracts. Of the 75 eyes, 30 eyes were from 15 patients with congenital cataract, 15 from 15 patients with traumatic cataract and 30 from 15 patients with developmental cataract. For comparison 15 eyes(4 with congenital, 6 with tramatic and 5 with developmental cataracts) had surgery without IOL implantation (aphakic eyes). Eyes with associated systemic or ocular pathologies (other than amblyopia and strabismus) likely to affect visual prognosis later were excluded from the study. Posterior chamber intra-ocular lenses in the range of +20 to +24 D were implanted after computerized power calculation keeping the ambyopia factor and prospective adulthood refraction in mind, and managing the school age children with low powered biofocals. IOL implantation was found to be difficult in children compared to adults.Pseudophakes were found to have distinct advantages of better quality good binocular vision compared to aphakes who suffered from problems of restrictions, anisometeropia and diplopia caused by aphakic correction. Porblems of poor compliance, corneal complications and costly replacements seen with contact lens use were also eliminated with IOL implantation. Placement of the IOL in the bag is close to the physiological and anatomical positions of the natural lens and hence comparatiely safe. Intra-operative problem of scleral collapse in children was managed by application of Fleringa's ring under general anesthesia. Post-operative severe uveitis responded to frequent administration of steroid drops for 8 to 12 weeks as well as oral steroids in the first week. Pupillary capture in 80% cases of cataract was prevented by keeping the pupil mid dilated. Over 86% of patients showed early after cataract (post capsular thickening) which responded to early YAG-laser capsulotomy in children aged above 5 yr, while this complication would be handled by secondary discission within 6 months of surgery in the age group below 5yr. Best results were obtained in 14% of patients where primary post capsulotomy/ rhexis with anterior vitrectomy was donr followed by IOL implantation. It can thus be concluded that IOL implantation is a safe and effective aphakic rehabilitation method in the management of developmental and congenital cataracts as also in the case of traumatic cataract depends upon the amount of damage that had occurred to various ocular structures due to the injury. Visual recovery may be further improved and rate of complications reduced by lowering of intra-ocular pressure preperatively, use of scleral support to prevent scleral collaspe, control of iritis and maintaining pupillary mobility, promt management of posterior capsular opacification along with vigrous amblyopia therapy.




ORAL HEALTH
ROLE OF FREE RADICALS & ANTIOXIDANT STATUS IN RBCS & THERAPEUTIC RESPONSE OF ZN & VITAMIN E IN AGE RELATED MACULAR DEGENERATION *  

A prospective study was carried out in 51 patients (age > 55 years) with reduction in central visual acuity due to age related macular degeneration (AMD) and presence of characteritersic lesions in the fundus, and in 20 healthy individuaals matched for age and sex to assess the status of antioxidant enzymes (superoxide dismutase, catalase and glutathione peroxidase) as well as oher antioxidant factors like ascorbic acid, glutathione and lipid peroxidation. Efforts were also made to evaluate the effect of oral zinc supplementation on the activity of antioxidant enzymes and the clinical course of the AMD. Patients with media opacities due to any cause precluding proper fundus examination, presence of any other ocular disease causing poor vision or distorting the anatomy of the macula and patients with malignancies, diabtes mellitus, renal and chronic liver diseases were excluded from the study. The levels of catalase, glutathione peroxidase superoxide dismutase and glutathione and lipid peroxidation were significantly lower in the AMD patients at base-line with no difference in the levels of ascorbic acid, haemoglobin and zinc. The AMD patients were divided into 2 groups: group A includced 29 patients who received zinc supplement (89.2 mg of elemental zinc given orally) and group B included 22 patients who did not receive any zinc therapy. Patients were followed up at 3 monthly intervals. At base line, serum Zn levels were 96.73+-18.62 and 99.97+-13.3 mg% in groups A andB respectively. These was a significant rise in the Zn levels rising to 258+-12.60 in group A vs 103.60+-14.72 in group B. There was a rise in the level of glutathione peroxidase at 3 months to 140.17+-42.38 units/g Hb in group A compared to 92.36+=37.22 units/g Hb in group B and this rise was maintained till 24 months. The levels of superoxide dismutase did not show any rise till 9 months after which rose significantly. The levels of catalase showed a significant rise after 3 months of Zn administration but there after there was a gradual decrease. There was no change in the levels of glutathione, Hb and ascorbic acid in both the groups. There was no significant improvement in ocular status or visual improvement in the treatment vs non treated group.




ORTHOPAEDICS
CLINICAL AND BACTERILOGICAL STUDY OF DENTAL CARIES IN CHILDREN  

The study was carried out to determine the prevalence of dental caries in school children aged 10-15 yr from different socio-economic stra- ta. The bacterial isolates, the serum and salivary immunoglobulins and CMI responses to Streptococcus mutans, were also evaluated in these children. Among the 1092 children studied, the prevalence of dental caried was found to be 39.19 per cent with an average mean DMFT (delayed, missing and filled teeth) per child being 1.02. A significant relationship was found between dental caries and the source of drinking water, oral hygiene measures used, type of cleaning agents used, frequency of cleaning and intake of sweets. However, no significant relationship was found between dental caries and the type of diet or the type of sweets and the type and frequency of drinks. The prevalence of caries was maximum at the age of 10 years. A total of 285 specimens comprising 116 specimens each of dental plaque material and carious material from children with dental caries and dental plaque material from 53 matched controls, were studied for the presence of microorganisms. The incidence of Bacteroids melaninogenicus assacharolyticus group and aerobic Gram negative bacilli was higher in controls as compared to children with caries. There was no significant difference in the isolation rate of Streptococcus mutans in patients and controls. Immunoglobulin levels were estimated in 379 children (169 with low caries, 57 with high caries and 153 controls). Serum IgG levels were higher but salivary levels were lower in carious children as compared to controls. The serum IgA and IgM levels of patients and controls were not significantly different. The salivary IgA levels were lower in children with caries, whereas salivary IgM levels were below detectable levels in both children with caries and controls. Significantly high levels of S.mutans specific serum IgG were found only in children with high and low caries whereas specific salivary IgG levels were more or less in the similar range in children with low and high caries as well as controls. S. mutans specific serum IgA levels were high in children with caries compared to controls. An interesting finding was that children with high caries showed low levels of S.mutans specific salivary IgA levels whereas those with low caries showed high levels of specific salivary IgA. This clearly indicated that S.mutans specific salivary IgA plays an important role in protection against dental caries. Cell mediated immunity was studied in 360 children (160 with low caries, 45 with high caries and 155 controls). Low stimulation index was observed in case of high caries while children with low caries showed high stimulation index indicating that in case of recent infection CMI plays an imporant role. The control children revealed stimulation index levels between those of children with low and high caries. It is thus concluded that humoral and CMI immune responses to S.mutans in dental caries play an important role.



EPIDEMIOLOGICAL AND CLINICAL STUDY OF LEGG-CALVE-PERTHES DISEASE  

Retrospective and prospective studies were carried out on 781 children (age group 4-16 yr) with Legg-Calve Perthes disease to identify epidemiolo- gical factors of etiological significance and to explore prophylactic measures of socio-economical significance. A clinical study was also carried out to identify the pattern of the disease and to analyse the effect of the various treatments employed. Incidence of Perthes disease in relation to the total number of orthopaedic outpatient cases in Kottayam Medical College was found to be 1 in 750. The mean age at onset of the disease was 8.83 yr., 8.84 yr for boys and 8.79 yr for girls. The male: female ratio was 2.49: 1. Most patients(71%) were from low income group families. Significant antecedent trauma was noticed in 25 percent patients. Associated developmental anomalies like CTEV, inguinal hernia, hypospadias, undescended testis etc. were seen in 24 children. The disease occurred more in children who were born late in the family and to older parents. Of the 781 patients, 734 had unilateral involvement while only 47 (6%) had bilateral involvement. In boys the right hip was affected more, whereas in girls it was the reverse. Average duration of symptoms at diagnosis was 19 weeks (ranging from 1 week to 18 months); pain and limping being the most common (66%) presenting symptoms; followed by hip pain alone (16%) limping alone (13%) knee pain (4%) and stiffness of hip (1%). Signs of head (femoral) at risk were noted in 70 per cent patients . Eighty per cent patients had extensive involvement of the femoral head at presentation. Increase in inferomedial joint space was found to be constant and the earliest radiological evidence of the disease which was influenced by the age of the patient and duration of symptoms. Reduction in the vertical height of capital femoral epiphysis was also one of the earliest radiological features of the disease. Acetabular osteoporosis was observed in 90 per cent patients. Age of onset of the disease sex of patients and the extent of involve- ment of epiphysis and definite influence of the final outcome. Ambulation with containment of femoral head was the main guiding principle of treatment of all new cases. Abduction orthosis and varus derotation osteotomy were the two methods of treatment employed for containment. Other factors being identical these two methods of treatment were found to be superior to all other treatments employed. It was concluded that Legg-Calve-Perthes disease is relatively common in Kerla and ambulation in containment of the femoral head is the best method of treatment of this disease.



IMMUNOPATHOGENETIC ROLE OF FOOD ANTIGENS IN RHEUMATOID ARTHRITIS (RA)  

Fifty patients with rheumatoid arthritis (RA) were studied to determine whether food related antigens caused exacerbation and their removal caused remission in the inflammatory activity in RA. This was done in order to see if this dietary elimination technique could become one of the adjuctive techniques and whether it could be predicted as to which patient would respond to this therapy and to which of the food item(s). Patients receiving any disease modifying drugs or having taken corticosteroids were excluded from the study. The patients were subjected to dietary modification therapy. This comprised a protein free diet of salads, fruits, vegetables, sugar and oils for 2 weeks. Patients who responded to this dietary elimination were then allowed step-wise dietary addition. Seventeen of the 50 patients (34%) responded to diet elimination and showed decreased synovitis. Only 10 of these 17 patients could be followed up in the dietary addition phase. Six of them were intolerant to one of three dietary items (one to milk, two to rice and three to wheat), two were intolerant to more than one dietary items (both milk and wheat caused flare in these two) and two patients were intolerant to some food items which could not be identified. Wheat, milk, and rice were found to be the common offending dietary items causing flare up of the synovitis. Analysis of relative risk for the various HLA antigens revealed that RA patients carrying HLA DRI phenotype had over 26 times more chance of responding to diet elimination compared to those without DRI. Similarly RA patients carrying DR5 had 2.75 times higher chances of responding to diet elimination than those without this antigen. Nonspecific inflammatory immunoglobulin levels, immune complex levels, lymphoproliferative response and anti-food item antibodies did not show any correlation with response to dietary elimination. Patients with active rheumatoid arthritis showed decreased lymphopro- liferative response both to specific antigens as well as nonspecific mitogens. They also showed perturbed ratio between 'native' and 'memory' CD4+ subset of helper lymphocytes. Those who responded to dietary elimination showed a trend of this ratio to revert towards normal levels. High levels of anti-food item antibodies seen in RA patients could be related to the increased gut permeability in patients with rheumatoid arthritis. Those who showed reponse to dietary elimination also showed a fall in the IgA class of anti-food antibodies indicating that, somehow their gut permeability could have improved.



STUDY OF NORMAL RANGE OF MOTION AT THE NECK  

The study was carried out to estimate the normal range of various movements of the neck in individuals of various age groups using an objectively reliable, simple instrument - the cervical goniometer, which is based on the principle of gravity and pendulum. Four basic movements at the neck were evaluated on 250 normal healthy subjects (125 male and 125 female) in the age group of 20 to 70 years. The mean normal values for the individual movements of flexion, extension, lateral-flexion to right and left and rotation to right and left were 53.3, 70.9, 46.6, 48.6, 81.9 and 82.3 degrees respectively. The mean normal values for the range of motion from flexion to extension, total lateral-flexion and total rotation were 124.5, 95.6 and 164.3 degrees respectively. A significant decrease in each movement was observed with increase in age in both sexes. Although females showed slightly better mobility, differences in the mean values amongst the males and females was not significant. In 25 patients of ankylosing spondylitis in the age group of 20-30 years, restriction of all the movements in general and lateral-flexion in particular was observed. Cervical goniometry thus appears to be useful for (i)estimating the normative values of the neck movements (ii) assisting in the detection of ankylosing spondylitis at an early age; (iii) systemati- cally planning and evaluating outcome of therapeutic procedures; and (iv) evaluating the degree of disability.




PATHOLOGY
ANALYSIS OF THE NATURAL HISTORY OF PERTHES' DISEASE IN INDIA  

A retrospective study involving review and analysis of radiographs and records of 518 patients of Perthes disease was undertaken to determine the natural history of the disease and to know whether the natural history of the disease is altered by the current treatment protocol as alos to decide whether current treatment protocol needs to be modified. The study revealed that the onset of Perthes disease was around 8.5 Yr of age; the male : female ratio of the affected children being 70:30. Bilateral involvement was noted in 5% of the patients and extensive femoral head involvement was very common. It was found that the disease runs a very slow course and the average total duration of the disease is around 4 years. In children under 12 years the disease could be divided into clearly identiable stages (IA, IB, IIA, IIB, IIIA, IIIB, and IV) based on plain radiographic appearances. The duration of the different stages increased in ascending order (IA



METABOLISM OF LIPIDS AND GLYCOCONJUGATES IN ALCOHOL TREATED ANIMALS  

The study was carried out on Sprague Dawley strain male albino rats to ascertain the effect of different concentrations of ethanol on the metabolism of lipids and glycoconjugates, effect of paracetamol on normal and alcohol treated rats as also the protective effect of testosteron and N-acetyl-D-cysteine on the alcohol and paracetamol treated rats. Comparative studies of the hepatotoxic effects of alcohol and paracetamol as also histopathological study of the changes in the liver tissue of the alcohol and paracetamol treated rats were also undertaken. Serum glutamate oxalacetate transaminase (SGOT), serum glutamate pyruvate transaminase (SGPT) and serum acid and alkaline phosphatases activities incresed after alcohol and paracetamol treatment. SGOT, SGPT, alkaline phosphatase and acid acid phosphatase activities were further increased when paracetamol was administered to the alcohol treated rats. Liver and kidney alcohol dehydrogenase (ADH) acitivities decreased in alcohol and paracetamol treated rats. This was further decreased after paracetamol administration in alcohol fed rats. Changes were also observed in the levels of liver GOT and GPT as also in the metabolism of lipids, lipid peroxidase and glycoconjugates in the liver and brain. As a result the level of malondialdehyde increased in the liver and brain of both alcohol and paracetamol treated rats. Alcohol administration also increased the deposition of glycolipids in the brain. Alterations in the metabolism of lipids, lipid peroxides and glycoconjugates were also observed in extraneural and extrahepatic tissues (eg. heart kidney and aorta). Testosterone and N-acetyl-D-cysteine offered some protection against the toxic effects of alcohol and pracetamol singly or together. This protective effect was evident from the decrease in SGOT, SGPT and serum acid and alkaline phosphatase activities as the reduction in the levels of triglycerides, peroxidation products, serum lipid and glycolipids. Hepatic histopathology of alcohol and paracetamol treated rats showed changes in the form the high degree of nuclear disintegration, chromatolysis, cytoplasmic and hydropic vacuolation as also necrosis necrobiosis and central venous dilation. Liver cells of rats treated with alcohol alone showed appearance of binucleate cells, hyaline bodies, necrobiosis, portal inflammation and kupffer cell hyperplasia Administration with only paracetamol produced nuclear disintegration, chromatolysis and necrosis. These changes confirmed the hepatotoxic effect of alcohol and paracetamol. Paracetamol produced synergistic effect when administered to alcohol fed rats.



FLOW CYTOMETRY IN ARCHIVAL MATERIAL  

DNA contents and cell cycle distribution were measured in 27 routinely processed paraffin embedded biopsy specimens of prostatic adenocarcinoma and 18 of breast carcinoma using DNA flow cytometry. The analysis of proliferative activity, which is defined as flow cytometric S and G2-M fractions was also carried out to determine whether these have any independent prognostic value or not. Twelve of the 27 (44.4%) prostatic carcinoma and 6 of the 18(33.3%) breast carcinoma specimens yielded >10 cell/mm which were processed further for analysis of S phase fraction (SPF) and G2-M phase fraction. SPF was found to be between 19 and 36 percent in prostatic carcinoma specimens (the mean being 28.9%). Near diploid tumours had significantly higher SPF compared to hyper and hpo diploid tumours. Hupodiploid tumours had higher G2-M fractions compared to near diploid and hyper diploid tumours. Near diploid tumours had a higher mean S+G2-M fractions compared to hypo and hyper liploid tumours. There was a significant difference between the mean values obtained for SPF when grade III and IV prostatic tumours were compared. All the 6 breast carcinomas were grade III tumours and near diploid breast carcinoma tumours had a significantly higher mean S+G2-M fraction compared to hypo diploid tumours. The ploidy status, SPF and S+G2-M were found to be associated with reevaluated histological grade of tumours. The ploidy, proliferating fractions and histological grade and individual prognostic significance. it is concluded that there was a direct correlation between the incidence of aneuploidy (hyperploidy) in prostatic adenocarcinomas and Gleasons grade. The high proliferative activity was an independent predictor of high histological grading in prostatic carcinomas. There was no correlation between the ploidy status and histological grade of breast carcinoma.



STRUCTURAL AND FUNCTIONAL ALTERATIONS OF LIVER FIBROSIS  

Structrual and functional alterations in liver during fibrosis. The study was carried out on adult male albino rats to find out the alterations in liver functins during dimethylnitorsamine (DMN) induced hepatic fibrosis. The rate of biosynthesis and merabolic degradation and accumulation of different types of collagen in hepatic fibrosis as also the role of trace elements during the progression of hepatic fibrosis were also studied. Hepatic fibrosis was induced by intraperitoneal injections of DMN (1 mue l/100g body wt.) on 3 consecutive day of ech week for 21 days. Histopathological examination of the fibrotic liver tissue showed intense neutrophilic infiltration, bile duct hyperplasia, Mallory's hyaline within cytoplasm, apoptosis of hepatocytes oysplasia, bridging recrosis and extreme centrilobular necrosis and fibrosis. A four fold ncrease of collagen content was found in the liver tissue. A significant increase of serum aspartate transaminase (AST), alanine transaminase (ALT), gama glutamyl transpeptidase ( gama-GT), alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) was a significant in crease in serum globulin levels. While an induction in the biosynthesis of gama-GT and ALP in the liver was found, the AST and ALT activities were significantly reduced in the hepatic tissue. The deterioration of liver functions and modulating enzyme activities may be contributing to an extent towards the development of hepatic fibrosis. Study of the rate of biosynthesis and metabolic degradation of collagen in DMN induced fibronic liver revealed enchanced anabolism and catabolism of hepatic collagen. It was also found that the balance synthesis and degradation was almost maintained in the early stages of DMN treatment as a self defence mechanism, but it was totally impaired in the later stages with a net result of accumulation of collagen in the liver. Studies on the molecular characterristics of fibrotic liver collagen demonstrated a significant increase of beta- chains with a notable decrease of alpha/beta ratio after DMN administration. Reduction with beta-mercaptoethanol indicated the presence of type III collagen in the electrophoretic field with a prominent increase in its level on day 21. An increase in the aldehyde content and enhanced rate of fibril formation in DMN induced liver collagen indicated a higher degree of cross linking. Studies on minerals and trace elements during the progression of hepatic fibrosis revealed significant decrease of Ca, Mg, K, Na, Se, and Zn in serum. The results indicate that exacerbation of hepatic fibrosis with ascites plays a major role in the alteration of essential elements which can further aggravate the disease.




PHARMACOLOGY
IN SITU HYBRIDISATION STUDIES OF EPSTEIN-BARR VIRAL DNA IN HODKIN'S DISEASE  

In situ hybridization studies of Epstein-Barr virus in Hodgkin's disease. The study was carried out on 40 patients of Hodgkin's disease to demonstrate the presence of Epstein-Barr viral (EBV)RNA in the malignant cells by a sensitive non-isotipic in situ hybridization and to localize the EBV encoded latent membrane protein-1 (LMP-1) in the malignant cells. Efforts were also made to correlate the results of in situ hybridization with immunohistochemistry for EBV encoded LMP-1 and analyse the percentage of positivety in relation to various subtypes of Hodgkin's disease. Of the 40 patients studied, 30 were adults and 10 children; male: female ratio being 37:3. The most common histological subtypes was nodular sclerosis (24/40; 60%), followed by mixed cellularity (12), lymphocyte predominance ( (3) and one case of lymphocyte depletion. EBER-1 transcripts were identified in 28 of the 40 (70%) patients (18 adults and 10 children). In the positive cases, EBER-1 positivity was confined to the Reed Sternberg and mononuclear Hodgkin's cells. A higher rate of EBER-1 positivity was observed in mixed cellularity (83.33%) and lymphocyte deplection (100%) followed by nodular sclerosis (70.83%). No positivity was observed in lymphocyte predominance. Twenty six of the 28 patients (except one with mixed cellularity and the only case with lymphocyte depletion.) showing positivity with in situ hybridization, showed reactivity for EBV encoded LMP-1. The 100 percent EBER-1 positivity in peadiatric patients strongly support the role of EBV in the etiopathogenesis of Hodgkin's disease. The demostration of LMP-1 along with the detection of EBER-1 sequences has important implications since it is one of the latent gene products which produces B lymphocyte transformation. The data thus strongly support the hypothisis, that when present, EBV is not merely a silent passenger in Hodgkin's disease but contributes directly to the pathogenesis. The results also indicate that the procedures used in this study can be applied on archival material allowing retrospective analysis of many neoplasms suspected to be EBV related.



EFFECT OF A CALCIUM CHANNEL BLOCKER, NIFEDIPINE ON EXPERI-MENTAL DIABETIC CARDIOMYOPATHYIN RATS : A BIOCHEMICAL AND HISTOPATHOLOGICAL PROFILE  

Study was conducted with the aim to investigate the beneficial effect of calcium channel blocker, nifedipine in streptozotocin induced diabetic cardiomyopathy, to study the cardiac metabolism and alteration produced by nifedipine in diabetic cardiomyopathy and to assess the mitochondrial function in relation to cardiac energy balance and the effect of nifedipine in diabetic cardiomyopathy. The primary defect in carbohydrate metabolism in the heart during diabetes mellitus is an impairment in the glucose transport across the sarcolemmal membrane into the myocytes. The present study was designed to investigate the effects of a calcium channel blocker, nifedipine (NIF) on diabetic cardiomyopathy and non diabetic rat heart. To understand the basis of these effects diabetes was produced in rats by streptozotocin (STZ) (65 mg/kg i.v. single dose) and the animals killed after four weeks. Isoproterenol (ISO) (85 mg/kg sc for two days) administration in normal rats resulted in a reduction of cardiac glycogen, ATP and creatine phosphate (CP) levels to a great extent, accompanied by the manifestation of myocardial necrosis and marked increase in serum lactate hydrogenase (LDH). Pretreatment with NIF afforded significant protection to the isopropendol (ISO) -induced injury of myocardium. Further NIF administration signi- ficantly increased the myocardial glycogen content and ATP & CP levels, lowered the myocardial lactate content and the serum LDH levels. In the present study a lack of the cardiotoxic effect of ISO in STZ-induced diabetic rat heart has been observed. To elucidate this observation, the effect of ISO on the levels of cardiac glycogen, ATP and CP have been determined in non-diabetic and diabetic rats. Hearts from diabetic rats did not show any significant high energy phosphate exhaustion nor an enhanced level of myocardial lactate and serum LDH, although the glycogen levels were significantly lowered. This finidings may be due to the altered ion permeability of sarcolemma due to hyperglycemia seen in diabetes alongwith its metabolic consequences. The study indicates that NIF protects the myocardium from ISO-induced injury to a significant extent, NIF improves the energy status of the normal heart and NIF treatment in diabetes appears to favourably affect the cardiac metabolism. However, the exact potential of NIF in the treatment of diabetic cardiomyopathy needs to be further investigated.



BEHAVIORAL NEUROBIOLOGY OF SEROTONIN NEURONS, BENZO- DIAZEPINE RECEPTORS AND LOCUS COERULEUS IN ANXIETY STATES IN RATS  

This project was aimed at examining the neutral subtrates that mediate anxiety and to standardise certain paradigms of animal behaviour, with a view to providing an ethoexperimental framework that can be utilized for studying the pharmacology of novel anxiolytics. The project focussed on the three major neural substrates: (1) Septo- hippocampal system, (2) Limbic system, (3) Locus coeruleus (LC). The major objectives of the present project were to understand the neural basis of anxiety; and to develop tests of anxiety in which behaviour of laboratory animals were studied after exploiting the environmental condi- tions or; the internal states of the animals. The models employed in these studies mimic like behaviour and alterations that may be observable in humans. The paradigms designed were: (1) Two way active avoidance paradigm (shttle box); (2) Social interaction test; (3) test for exploratory behaviour; (4) test for neurological status; (5) whole board test; (6) Elevated plus maze; (7) Bright dark arena; (8) Novel maze; (9) Fear potentiated post startle paradigm. Techniques developed and standardised were: (1) Stereotaxic procedure-To administer pharmacological probes into target areas in brain; lesions and electrical stimulation; histological verifications; (2) HPLC assay of biogenic amines and metabo- lites; (3) EMG recording; (4) Ethological studies in rodents; (5) Straining methods (Nissle staining, cresyl violet staining etc.; (6) Cytogenetic methods. The present study adopted an ethoexperimental approach to examine the development subsequent to alterations in serotonin (5-HT) neurotransmission following treatment with site specific neuropharmacological probes. The impact of perturbation in 5-HT neurotransmission on baseline behaviour was analysed employing three animal models of anxiety, i.e., hole board, elevated plus maze and bright/dark arena. Selectively rared rats (Wistar strain, weighing between 150-200 gm) were used in this study. The vivarium and the behavioural and laboratory were specially designed to permit opera- tion or reversed light-dark cycle and all experiments were performed during the dark period. Pharmacological tools selected to influence 5-HT levels include: (1) a combination of tranylcypromine and tryptophan (TCP + TRYPT) (0.75 mg/kg + 40 mg/kg) which augment 5-HT biosynthesis, (2) p-chlorophenyl- alanine (PCPA : 200 mg/kg) an inhibitor of 5-HT biosynthesis and (3) 5-HT re-uptake blockers namely zimelidine (ZIM) (40 mg/kg) and fluoxetine (FLU) (10 mg/kg). Rats under the influence of PCPA exhibited anxiolytic response, whereas treatment chosen to raise 5-HT levels, viz., TCP + TRYPT, ZIM and FLU, displayed anxiogenic-like reaction. Several other agents known to specially interact with 5-HT receptor subtypes were also tested. 5-HT receptor stimulants, such as quipazine (5 mg/kg) and MK 212 (0.5 mg/kg), were found to be anxiogenic. Buspirone ( 2 mg/kg), a 5HT-1 agonist, surmounted normal behavioural inhibition. However, another 5HT-1 stimulant, 8-OH-DPAT (0.025 mg/kg), displayed anxiogenic action. Pre-treat- ment with 5-HT3 antagonists zacopride (2mg/kg) and GR 38032F (0.1 mg/kg) and putative 5-HT1 antagonist [propranolol (10 mg/kg)], resulted in border line disinhibition of normal behavioural inhibition to novel environments. In contrast, cyprohepatadine (0.5 mg/kg), a 5-HT2 antagonist, prevoked anxiogenic-like behaviour. Altogether uniform results were obtained for each probe in all the three models, suggesting that the battery of anxiety tests chosen in this study are reliable and sensitive to detect unknown pharmacological responses. The results support the hypothesis that stimulation of serotonergic neurotransmission hightens normal anxiety, whereas its blockade releases normal behavioural inhibition. Furthermore, this work establishes the validity of using the three paradigms in evalu- ating the involvement of multiple neurotransmitter receptors in the control of behaviour of rodents under natural circumstances and also detect any aberration following exposure to novelty and stress. An in vivo study was undertaken to examine the cytogenetic changes following single dose treatment with buspirone, a novel anti-anxiety agent employing the bone marrow chromosomal aberration and mitotic index was studied at 6, 24, 48 and 72 hr. by administering 0.5, 1, 2, 4 and 8 mg/kg of buspirone intraperitoneally. Results obtained from chromosomal aberration data such as (a) breaks and gaps in chromatid continuity and (b) exchanges between chromosomes were evaluated to determine the clastogenic effect of the drug. The drug proved to be mitodepressive and induced chromosomal aberrations mainly in the form of chromatid gaps. From the present study, it is surmised that buspirone can be ranked as a moderately weak clastogen. The criteria for each catagory of responses on the rating scale was clearly defined and depended on the state of the nervous system. The neural substrates that modulate post-startle behaviours was studied by modifying neural transmission (localised drug infusion and electrical stimulation). The rating scale designed in this study helped in evaluating the influence of the mediating systems (along the startle pathway in different brain centres) on post startle response. Consistent and charac- teristic behaviour was observed with the anxiogenic agent yohimbine. It would be pertinent at this stage to note that any characteristic posture, or behaviour, observed during the post-startle period would be significant indicator of the level of anxiety or fear. Measuring post-startle behaviour will definitely provide precise information about startle relevant viz., crouching and freezing, that are common indexes of fear. The "TEN POINT RATING SCALE" was validated for variability and reliability.



PRODRUGS AS DRUG DELIVERY SYSTEMS  

One of the promising approach for optimisation for drug delivery and hence increasing drug efficacy is the prodrug concept which is defined as the chemical modification of biologically active compound to form a new compound, which upon in vivo enzymatic attack will liberate the parent compound. The prodrug approach has been successfully applied to a wide variety of drugs to overcome various pharmacokinetic and pharmaceautical barriers. The present work is aimed at designing prodrugs of norfloxacin. four derivatives of norfloxacin have been prepared. The derivatives synthesised should be bioreversible. To confirm the bioreversibllity of these derivatives in vivo methods have been applied. The derivatives synthesised are 'N' alkylated derivatives and their major metabolic site is the cytochrome P 450 enzyme system in the liver. For in vivostudies rat was selected as model animal because its enzyme systems possess the closest biochemical and physiological resemblance to those of man. the dose of 200 mg/kg was arrived at after studying the various doses used clinically and the blood levels achieved by these doses in vivo. Analysis for release of norfloxacin from derivatives N1 in plasma samples was carried out by HPLC. Results of plasma analysis clearly indicated nonreversibility of N1 derivative to give norfloxacin. These derivatives also failed to exhibit invitro antibacterial activity against E. coli, S. aureus. therefore, these derivatives cannot be considered as prodrugs.



CENTRAL THERMOREGULATORY EFFECTS OF BENZODIAZEPINES  

Four benzodiazepines - diazepam, lorazepam, nitrazepam and oxazepam and a reference drug, chlorpromazine were studied for their action on thermoregulation. They were injected into a lateral cerebral ventricle (icv) through a permanently implanted cannula or intravenously (iv)/intraperitoneally (ip) to distinguish between their central and peripheral actions. The benzodiazepines resembled in their thermoregulatory action to the reference drug, chlorpromazine: small doses evoked hyperthermia and large doses evoked dose-dependent hypothermia. The hypothermic effect was not entirely mediated through thermoregulatory neurons as the ratio between the effective doses by the two routes was marginal. Diazepam reduced the hyperthermic effect of heat stress and aggravated the hypothermic effect of cold stress. Similarly, nitrazepam evoked greater hypothermia at low than at high ambient temperature, prevailing over the year. Indeed, it caused hyperthermia at high ambient temperature. Benzodiazepines facilitate GABAergic transmission in brain. Failure of icv injected GABA to modify body temperature may, perhaps, be due to its failure to reach the target neurons or absence of its effect on them. The initial hyperthermic effect of chlorpromazine is centrally mediated resulting from stabilization of neuronal membrane similar to that of procaine. It is likely that similar effect of benzodiazepines is responsible for their hypothermic action. The presence of benzodiazepine receptors at a variety of neuronal sites (adrenergic, cholinergic, dopaminergic, serotonergic, GABAergic and calcium uptake mechanism) raises the possibility that alteration in the function of one or more of these systems may be involved in the action of benzodiazepines.



BIOCHEMICAL STUDIES ON THE MODE OF ACTION OF ANTIFUNGAL DRUGS ON DERMATOPHYTES  

Investigation was designed to study biochemical mode of action of tolnaftate and griseofulvin in Microsporum gypseum. In order to investigate the possible target sites for the antifungal drugs, macromolecular composition of cultures grown in absence od presence (IC50) of the drug and macromolecular biosynthesis from radiolabelled precursors in drug treated (X10MIC) mycelia were studied. Effect of drugs on certain membrane functions was assessed by measuring the leakage of labelled intracellular components (32p-leakage), K+-efflux and activity of membrane bound enzyme such as phosphodiesterase (PDE), ATPase and 5'- nucleotidase. Studies on macromolecular composition and macromolecular biosynthesis revealed that tolnaftate exerts an inhibtory effect on phospholipids and sterols without significantly altering the level of proteins and DNA. Tolnaftate caused an increased in 32p-leakage without affecting the level of K+ efflux and activity of PDE, 5'-nucleotidase and ATPase. On comparision of control and tolnaftate resistante mycelia, both the strains were found to have similar macromolecular composition except for significantly higher level of total phospholipids in tolnaftate resistant mycelia. Activity of PDE was decreased and 5'-nucleotidase and ATPase activity was increased in the tolnaftate resistant mycelia. Tolnaftate did not affect the total phospholipid content in the tolneftate resistant mycelia and the level of 32p-leakage induced by tolnaftate in drug resistant mycelia was found to be less than the corresponding leakage in the drug treated control mycelia. Inhibitory effect of tolnaftate on phospholipids appeared to be critical to its growth inhibitory activity in M. gypseum. studies on griseofulvin action showed an inhibitory effect of the drug on macromolecules including phospholipids sterols proteins and DNA and treatment with griseofulvin also resulted in impairment of some membrane functions in control mycelia as indicated by a stimulation in 32p-leakage and K+ efflux. Comparision of griseofulvin resistant and control mycelia revealed a simalar macromolecular composition except that griseofulvin resistant mycelia possessed a higher protein content. Grisoefulvin failed to cause any significant inhibtion in synthesis of phospholipids and sterols and it decreased the leakage of intracellular components in griseofulvin resistant mycelia. Inhibtion of macromolecular synthesis by griseofulvin appears to cause a delay in S-phase and thereby interfere with the normal growth of dermatophyte mycelia.



STRATEGY FOR BROAD SPECTRUM ANTHELMINTICS  

Synthesis and evaluation of certain novel analogs of diethyl carbamazine and amoscanate, broad spectrum filaricidal agents was undertaken. Molecular modifications of diethyl carbamazine (DEC) led to centperazine, an analog of DEC with rigid conformation (with greatly reduced conformational mobility of diethyl carbamoyl side chain). Centperazine had microfilaricidal activit greater than DEC. The analogs of DEC had restricted geometry around sp2 hybridised aromatic ring. A nitro group was introduced in the aromatic ring to further enhance biological activity. Compounds (1-25) were synthesised from which the representative compounds were screened for antifilarial activity. Compounds 12,44,60,64,84,87 and 123 showed good response for antifilarial activity in comparison to DEC. Compound 12 (with R' =piperidine ) could show sustained microfilariaemia till day 42, but again it failed to show any adulticidal activity. Compound 44 with R=dimethyl amine exhibited lethal effect on adult worms. In case of 12 and 44 R' =piperidine was common and it appeared that a piperidine moiety in R' position evoked a better antifilarial profile than the other heterocyclic amines. Compounds 60 and 64 showed effective adulticidal action. The presence of dimethyl amine moiety in compound 64 and 44 had favourable effects. Besides compounds 69 and 64 also had an aniline ring. Compounds 84 and 87 and 123 showed 81.5, 93.8 and 75.0 % adulticidal action against L. carinii at 30 mg/kg injected intraperitoneally. Presence of a substituted benzolinone/benzthiazolinthione/amino methylated heterocyclic moiety alongwith N-Me-piperazine appeared to evoke excellent antifilarial response.



THE TOXIC EFFECT OF AN ORGANOCHLORINE INSECTICIDE DIELDRIN ON BEHAVIOUR AND NEUROCHEMISTRY OF RATS : ITS INTERACTION WITH CENTRALLY ACTING DRUGS  

Chronic (90 days) treatment (orally) with cyclodiene insecticides, endosulfan (2 mg/kg) and aldrin (1 mg/kg) inhibited food intake and weight gain in rats. These effects were more marked in female rats than in males. A deterioration of muscular activity was found in aldrin treated animals. A greater muscle weakness occurred in male rats. Chronic treatment with these compounds produced an impairment in learning and memory processes too. This behavioural defect was accounted to an activation by them of 5-HT mechanism in the brain, since increased 5-HT concentrations were recorded in the cerebrum and midbrain regions of endosulfan treated animals. Endosulfan increased spontaneous motor activity (SMA) and it was inhibited by aldrin in both the sexes. Aldrin and endosulfan are known to activate hepatic microsomal enzymes, and as an evidence a shortening of pentobarbitone sleeping time was found in animals treated chronically with them. Their interaction with diazepam which is known to be metabolized by this enzyme system to a long acting metabolite, oxazepam, is therefore, likely to result in a prolongation of the effects of the latter. Consistently, in the present study the hypnotic, muscle relaxant and sedative actions of diazepam were prolonged in rats exposed chronically to these insecticides. The tranquili- zing actions of CPZ (inhibition of SMA and CAR) were, on the other hand, initially enhanced and then suppressed in these animals. CPZ failed to prolong pentobarbitone sleeping time in test animals. A fast degradation of CPZ in these animals to an active metabolite and then to inactive compound seem to explain these findings. The doses of aldrin and endosulfan employed seem to have convulsant potentiality after chronic administration, since the convulsant latency of picrotoxin was shortened in these animals. Ethanol sleep was prolonged in female and not in male animals.



METABOLIC FATE OF MEMBRANE PROTEINS IN RAT BRAIN DURING CHRONIC ETHANOL CHALLENGE  

The main aim of this study was to identify the changes in morphological, macromolecular and membrane architectural properties of neurons and astrocytes and to ascertain specific cell type vulnerability in brain following chronic ethanol in vivo treatment. 5% (v/v ethanol in drinking water, yielding comparable blood alcohol concentration without adversely affecting total calorie, food and fluid intakes of alcoholic rats, was chosen in the present study. Assessment for changes in body weight, food and fluid intake revealed slight decrease in the body weight (9%) of ethanol treated rats. Scanning electron microscopy indicated that chronic ethanol treatment in vivo might alter the morphology of astrocytes. Astrocytes derived from chronic ethanol treated rat showed a characteristic smooth surface. Neurons were not affected morphologically in this study. RNA and protein appeared to be more vulnerable to ethanol influence than the DNA. The data, in the present study demonstrated that ethanol affects transcriptional and translational ability in both neurons and astrocytes indicating that the overall cell growth is affected. The data, in the present study suggest, in overall, that cell growth might be affected in both astrocytes and neurons but to a greater extent in astroglial cells than in neurons. Further, reduced 3H - leucine incorporation into proteins of neurons and astrocytes supports such a concept. The enolase activity was not affected in neurons, while it was reduced by 51% in astrocytes, due to ethanol treatment. Further, reduction in the glutamine synthetase activity by ethanol was noticed in the present study. In the present study, the data shows variation in the composition of membrane proteins from neurons and astrocytes and ethanol seems to be inducing a loss in several proteins, while it also causes the appearance of a set of new proteins. Further in the present study, iodinated WGA binding studies showed prominent appearance in N-linked glycoprotein (97, 116 and 160 kDa) in astrocyte membrane fraction on ethanol treatment which suggests that cell surface modification is possible during ethanol treatment. Thus it was found that astroglial cell morphology is modified, growth is impaired in both neurons and astrocytes in terms of macromolecular composition, astrocytes appear to be more vulnerable than neurons basing on cell type specific marker enzyme activities. The membrane protein composition is altered, which might be responsible for membrane disorders as many number of membrane proteins disappear (compared to newly appearing proteins) and N-terminal glycoproteins are increased in astrocyte cell membranes which might involve cell surface modifications during chronic ethanol in vivo treatment in rats.



TREATMENT FOR CLEISTANTHUS COLLINUS POISONING  

Acute oral toxicity-induced clinical symptoms, biochemical alterations and histological lesion due to Cleistanthus collinus poisoning in laboratory animals (rats, gunea pigs, rabbits) were studied to provide a better understanding of the mode of action of C. collinus toxin in turn, could pave the way for the development of antidotal therapy. Correlation of clinical symptoms with biochemical alterations and histological lesions suggested the involvement of autonomous nervous system in C. collinus poisoning. Study on the biochemical alterations at cellular level like monitoring the activity of certain enzymes such as cholinesterase, lactate dehydrogenase and transferases indicated the interaction of C. collinus toxin with sulfhydryl of thiol (-SH) dependent enzymes. This observation prompted the investigation to study antagonistic effect of 'thiol' rich compounds like L-cysteine, N-acetylcysteine, L-methionine and glutathione, in C. collinus poisoning. the reduced mortality rate, quick reversal of biochemical alterations and prevention of histological lesions in L-cysteine treated animals, following other compounds tried. It is concluded that L-cysteine is the most promising antidote in the treatment of C. collinus poisoning.



MONITORING OF EPIDEMIOLOGICAL PROFILE & FACTORS RESPONSIBLE FOR ADVERSE DRUG REACTION IN INDIA  

The freequency and epidemiology of adverse drug reactions (ADR) was studied through comprehensive drug surveillance. ADRs were recorded by anec- dotal reporting in which participating physicians reported the patients if they suffered from any ADR after drug use. Out of 100 ADRs spotted in 1991- 92, 25% were of A type (augmented type) while 75% were of B type (allergic type). Many of the chemotherapeutic agents were found to have potential for causing adverse drug reactions. Among antibiotics and antiseptics, doxycycline, oxytetracycline, chloramphenicol and nitrofurazone ointment exhibited highest incidence of ADRs. Other antibiotics like streptopenicillin, Ciprofloxacin and Gentamicin also produced ADRs in considerable number of patients. Nystatin vaginal tablets caused ADRs in 10% cases. Among anthelmintics, diethylcarbamazine caused nausea and vomiting in 25% patients. Among antimalarials chloroquin was the only drug producing ADRs. Amongst anticonvulsants, phenytoin sodium, amongst antiemetics, stemetil and metoclopromide, amongst antiasthmatics, amino- phylline, amongst antihypertensives, atenolol, propranolol were the drugs causing ADRs. The adverse drug reactions with injectable iron preparations were seen in 12.5% patients and caused local irritation and necrosis. ADRs with i.v. infusion fluid (dextrose and saline) are probably due to impurities (pyrogens) present fit. The incidence of ADR with antimalignancy drugs (viz. bleomycin, methotrexate, mitomycin and endoxan) is significantly high i.e. 25%, 16.6%, 20% and 14.2% respectively. Amongst antispasmodics, isoxuprine, amongst antipyretic analegesics - analgin, amongst antinflammatory analgesics phenylubutazone and diclofenac were found to be more frequent ADR-causing drugs than others. Drugs which cause ADRs in significant number of treated patents are inj.Conray, (12.5%), inj. Dopamine (16.6%) tab.primiprol (12.5%), tabl Duoton (16.6%), tab Neocalmforte (25%) and tab. Xantinol nicotinate (14.2%). As noted in this study, type A - ADRs are less in incidence than type B-ADRs. It is contrary to what is reported in literature. It also shows that nowadays physicians are more cautious about doses of drugs prescribed because type A-ADRs are mainly dose related. Most of ADRs (75%) observed with different drugs were B type (allergic type). Therefore, prescribing physicians should be cautious about previous history of allergies, environmental or occupational exposures, previous drug reactions and ethnic origin, etc.



NEUROPHARMACOLOGICAL ACTIONS OF MYCOTOXINS FROM FUSARIUM SP 

Study was conducted to evaluate the neuropharmacological and neurochemi- cal activity of Fusarium (F. moniliforme and F. oxysporum) mycotoxins. The neurochemical changes induced by F.moniliforme and F.oxysporum were remarka- bly dissimilar. The non-specific and irreversible MAO inhibitory activity of former can be attributed to the presence of zearalenone in it. This substance has been reported to be absent in F.oxysporum. On the contrary,the neurochemical changes induced by latter can be correlated to the presence of fusaric acid, which has been reported to inhibit DA-B-hydroxylase. Fusaric acid is normally absent in F.moniliforme. Fusarial toxins were found to exhibit significant acute neurochemical and neuropharmacological effects which may contribute to their overall toxicity.



ISOLATION AND CHARACTERISATIONOF AN ANTICONVULSANT PRINCIPLEFROM A MOLLUSCAN SOURCE  

Isolation and characterization of an anticavuleant principle from a molluscon source. The study was carried out for isolation and charasterization of anticonsulvant principle from the shell of ceptalopoda molluscan and establish it pharmacological profiles. Sepia shells were collected dried and ground to fine powder and extracts were prepared. The extracts were tested in sw'ss albino mice for its anticanustlant properties in doses ranging from 100- 700 mg/kg body wt. The alkali soluble fraction of the shell (but not the aqueores and acid extract) was found to pri ocess anticanvulsant property when administrated intraperiteneally at a dose of 600 mg/kg. If also gave complete protection against pentylenetetrazole induced convulsions within 3 h. of its administration. The alkali dialysate also brought about behavioural and other changes in the motor activity and reserved the changes produced by chemically induced convulsions. At the cellular level the alkalidialysate prevented the onset of seizures by inhibiting the glycolytic pathway and enzyme glutamate decarboxylase and inhibiting the activity of GABA-T. It was found to have an antagonistic effect on glutaminase and prevented its synthesis and accumulation in the nerve endings, while enhancing the activity of glutamine synthetase and degraded the acetylcholine, by enhancing the activity of acetylcholine esterase. Like other anticonvulsants such as Diazepam and phenytoin the alkali extract of the sepia shell enhanced the release of serotonin and dopamine and produced its anitconvulsant effect through serotoninergic transmission rather than catecholaminergic transmission. The Ultra violet spectral studies of this alkali dislysate showed maximum absorption at 280 nm and bound to the Dextran associated with sephedex. In addition, it also possesed antidiabetic effect like sulfonylureas which are both anitconvulsant and antidiabetic agent indicated that the principle may contain an aromatic ring and associated with amine functional groups.



INVESTIGATION INTO THE MECHA- NISM(S) OF GENO-PROTECTIVE ACTION OF PROSTACYCLIN AGAINSTBENZO(A) PYRENE & CIS-PLANTIN-UM-INDUCED GENETIC DAMAGE  

The study was carried out to investigate the affect of prostacyclin (PGI2) on benzo(a)pyrene metabolism and on the bindings of benzo(a)pyrene and cis-platinum and or tis metabolites to DNA in order to know whether PGI2 can inhibit free radical generation and thus prevent genetic damage induced by benzo(a)pyrene and cis- platinum. Effects were also made to investigate the effects of benzo(a)pyrene and cis-platinum on free radical generation by PMA- stimulated neutrophils to find out whether PGI2 can influence PMN generation of free radicals by benzo(a)pyrene and cis-platinum. The PGI2 showed anti-mutagenic actions against benzo(a)pyrene, radiation, and cis-DDP. The genoprotective action of PGI2 was seen for 24 h which is longer than its half-life. The synthetic analogue of PGI2 viz carba-PGI2 also showed similar genoprotective action as that of the PGI2. But the stable metabolite of PGI2, viz 6-keto-PGF1 alpha did not show any genoprotective action against cis-DDP-induced genetic damage. Dibutyryl-cyclic AMP also did not show any genoprotective action against cis-DDP induced genetic damage in vivo in mice. PGI2 did not inhibit free radical generation in human leukocytes but it inhibited the formation of tumours in the 2 stage skin papilloma model wherin benzo(a)pyrene and croton oil were used as the initiator and promotor respectively. PGI2 also increased the formation of lipid peroxides in the skin of the benzo(a)pyrene and croton oil treated mouse skin in the 2 stage skin papilloma model, but did not influence the proliferaction index of the mouse epidermal cells treated with benzo(a)pyrene and croton oil. Both PGI2 and its analogue carba-PGI2 did not interfere with the cytotoxic action of cis-DDP on tumour cells both in vitro and in vivo, PGI2, insignificantly decreased the binding of radioactive benzo(a)pyrene to DNA (only by 12%), and decreased both PMA and cis-DDP induced chromosomal aberrations in human lymphocytes to a significant degree. PGI2 neither influenced the DNA unwinding nor unscheduled DNA synthesis. The results of the stydy suggest that the PGI2 has both anti-matagenic and anti-cancer properties.



PREPARATION OF A NEW ANTIFUNGAL ANTIBIOTIC FROM STREPTOMYCES KENAMYCETICUS & ITS CLINICAL STUDIES  

Streptomyces kanamyceticus is well known for its production of the broad spectrum kanamycin which is effective against various infectious diseases caused by Gram +ve, Gram -ve and acid-fast bacteria. But there is no report on the production of antifungal antibiotic by S. kanamyceticus or its mutants. In course of studies on the development of high kanamycin-yielding strains of S. kanamyceticus by induced mutation, a mutant S. kanamyceticus M was isolated and this was found to possess strong antifungal activity. The present report describes in detail the optimization conditions for production of antibiotic, utilisation of carbon and nitrogen sources by S. kanamyceticus for antibiotic production, isolation and purification of the antibiotic from the culture broth and study of some physicochemical properties of the active component. The mutant culture of S. kanamyceticus produced antibiotic complex which were separated into five fractions by thin-layer chromatography. These antibiotics contain non-amino acid substances besides amino acid residues. The structure of the peptide part of fraction II was studied. The antibiotic was found to be most active against a wide group of fungi and yeasts such as Aspergillus flavus, Epidermophyton floccosum, Micrasporum gypseum. Pharmacodynamic studies on mice show that it is free from toxicity. Studies will help in development of fermentation process for production of the antibiotic with great clinical potential.



STRESS & CENTRAL NEURO- MODULATORS/NEUROTRANSMITTERS IN RATS:INTERACTION BY ANTI- STRESS AGENTS (ADAPTOGENS & ANTI-ANXIETY DRUGS )  

Involvement of central neurotransmitters like 5-hydroxytryptamine and norepinephrine, in the process of activation fo the hypothalmo- hypophyseal-adrenal axis during restraint stress was studied by direct measurement of the monoamines in the whole brain or hypothalamus of healthy albino rats. These two monoamines are also implicated in stress- attenuating effects of adaptogen like Panax ginseng and anti-anxiety drug like diazepam, as far as their attnuating effect on stress-induced increase in plasma corticosterone (index of H-H-A axis function) is concerned. Involvement of central neuromodulator like prostaglandins in H-H-A axis activation during stress has been suggested, whether prostaglandins are involved per se or through modulation of some neurotransmitters is not clear. It may be suggested that prostaglandins are involved in H-H-A axis activation during stress (an possibly also in normal functioning of H-H-A axis) not directly but through the positive modulation of serotongergic system. Prostaglandins are also believed to be involved in stress attenuating effect of Panax ginseng and diazepam.



EVALUATION OF DEXAMETHASONE CYCLOPHOSPHAMIDE PLUSE THERAPYAS A CURATIVE TREATMENT FOR PEMPHIGUS  

Clinical trials were carried out between 1991-1996 in 251 pemphigus patients to evaluate the efficacy of dexamethasone-cyclophosphamide pulse (DCP) therapy. Patients with a confirmed diagnosis by Tzanck smears, histopathology, direct immunofluorescent (DIF) test on clinically uninvolved skin and indirect immunofluorescent (IIF) test for serum titres of intercellular antibodies, were included in the study. Dexamethasone (100mg) in 500 ml of 5 per cent dextrose was given intravenously(iv) on three consecutive days with 500 mg of cyclophosphamide iv preferably on day 2,followed by daily oral cyclophosphamide (50mg) on subsequent days. The higher doses of dexamethasene and cyclophosphamide were repeated at 4 weekly intervals till the patients achieved comple clinical remission (phaseI) and con-tinued to be in remission atleast for six months (phase II).Daily low oral doseof cyclophoosphamide was continued (phase III) and stopped after one ear and thepatients were followed up at 6 monthly intervals for as long as possible (phaseIV).Serum titres ofintercellular antibodies were determined at various phases ofthe study. Thirty eight patients could not complete the treatment, while five died during treatment. Of the remaining 208 patients, II were in phase I, 30 inphase II and 44 in phase III; 123 patients who had completed the treatment were in remission without any maintenance therapy in 1996. The duration of posttreat-ment follow up in these patients was more than 2 years in 34 patients and less than 2 years in 89 patients. The relapse rates were found to be 19 per cent (4 out of 21) in patients who received irregular treatment and 11 per cent (12 out of 132) in patients whoreceived regular treatment during phase II. These 16 patients were given a second course of DCP. Of these, 3 patients continued to have active disease (phase I), 6 were in remission but still had to complete the treatment schedule (phase (II and III). Seven patients were in phase IV; the duration of post- treatment follow up being more than 2 years in one patient and less than 2 yearsin 6 patients. There was a progressive reduction in the serum titres of intercellular antibodies as treatment progressed, but these changes were not uniform in all the patients. From the data generated during this study it may be concluded that DCP therapy regimen can cure pemphigus leading to quick recovery and low incidence of side effects. If DCP is not given strictly as per the schedule, patients arelikely to relapse requiring a second course of DCP.



STUDIES ON INTER-RELATIONSHIP BETWEEN CALCIUM DYNAMICS AND FREE RADICAL FORMATION IN NEUROTOXIC STRESS  

The study was carried out on male albino Wistar rats to dileneate the effect of diazepampn free radical mediated pro oxidant antioxidant system alongwith its interrelation to altered calcium functions of different segions of brain. Rats were given diazepam intraperitoneally (3 mg/kg body wt.) and scarificed after 1 hr to follow changes in the pro/ antioxidant status.The diazepam treated rats showed lowering of thiobarbituric acid reactive substance (TBARS) formation in all the brain regions in a dose dependent manner. However, at the subcellular level an ebhancement in the TBARS formation was found in the mitochondrial fraction from cerebellum and cerebral cortex. This effect was highest in brain stem with more than 2 fold enhancement as compared to controls. In the post mitochondrial fraction, cerebellum showed 49% enhancement whereas decreased formation of thiobarbituric acid reactive substances was observed in cerebral cortex and brain stem. Isozymes of superoxide dismutase showed a region dependent decrease in activity. Though, total thiols were not significantly altered free thiols showed depletion in cerebellum (39.8%) and brain stem (50%). Gluththione reductase activity was also decreased in the mitochondria of cerebellum and brain stem (72.41% and 59.1% of that of corresponding contriols).Similar trends were observed in the cytosolic fractions also where moderately significant (p<0.02) changes were observed in cerebral cortex and brain stem. A trend towards normalisation of antioxidant defenses was observed in rats killed 18 h after diazepam administration. Secondary antioxidant glucose-6-phosphate dehydrogenase was not affected by diazepam whereas membrane bound Ca2+ Mg2+ -ATPase activity was enhanced by 6.3 fold in cerebral cotex and 205 fold in cerebellums at a 5% LD 50 dose. In vitro studies showed diazepam reducing the prooxidant induced lipid peroxidation which was also observed after repeated daily dose of diazepam for 7 days. Calcium induced and oxidative swelling of mitochondria was also lowered by diazepam in vitro. Another intersting finding was greater turnover of total phospholipids in all the brain regions after diazepam treatment being nearly 204 fold higher in the cerebral cortex and 105 fold higher in the brain stem. After repeated administration of diazepam for 7 days, CuZnSOD showed significant enhancement in acitvity in a activity in cerebellums and brain stem whereas MnSOD did not show significant changes in this region. Glutathione reductase activity was enhanced in mitochondria but reduced in cytosol of cerebellum and brain stem whereas crebral cortex showed just the reversed trend. It was noted that changes in membrane phospholipid turnover and peroxidation of polyunstaurated fatty acids also affects calcium functions. The results thus indicate that diazepam causes free radical mediated stress and the modulatory response of antioxidant defenses appears to be not only region specific but also organelle specific.



TARGETING OF GLYCOSYLATED LIPOSOMES ENCAPSULATING METAL CHELATING DRUGS TO THE SITE OFTOXIC METAL STORAGE IN BIOLOGICAL SYSTEMS  

The study was carried out in male albino mice to achieve improved therapeutic and safety of metal chelating drug meso-2,3,dimeraptosuccinic acid (DMSA) in the management of cadmium toxicity by using glycosylatted liposomes as carriers for their targetted delivery. Glycosylated liposomes encapsulating DMSA were prepared involving coupling of glycosides to liposomes and their safety was evaluated in terms of blood and biochemical parameters and trace metal levels in tthe body, their stability and in vitro interaction of lipsomes with isolated hepatocytes. Cadmium toxicity was produced in the mice by injecting cadmium chloride (0.01 mmole/kg) intraperitoneally.The chelating drug DMSA was given as (i) DMSA encapsulated in glycosylated liposomes, (ii) DMSA via plain liposomes; and (iii) plain DMSA. The glycosylated liposomes encapsulating DMSA could be sucdessfully targetted to the intracellular site of cadmium accmulation i.e. hepatocytes (because of the presence of free terminal sugar galactose for which receptors exist on the hepatocyte cell surface). This resulted in the enhanced mobilization of cadmium from the body organs (lever, kidneys and spleen) and from hepatocytes. It also produced enhanced excretion of cadmium through urinary and fecal route. The efficacy of DMSA encapsulated in targettable liposomes was found to be better compared to that given via plain liposomes or in the form of free material. The DMSA given as free material and via plain liposomes resulted in lowering of biochemical (protein, glutathione, lipid peroxide levels) cadmium exposed mice, whereas the DMSA given through glycosylated liposomes was able to maintain the levels of these parameters to their normal values. The DMSA given via liposomes in the both the forms (plain liposomes and glycosylated liposomes) lowered the levels of copper and zinc enhanced by cadmium treatment, thereby preventing the disturbances in essential trace metal imblances. Also these liposomes when administered empty to normal animals did not show any toxicity to disqualify their use as carrier for metal detoxification.



LUNG SPECIFIC LIPOSOMES AS DRUG DELIVERY VEHICLES  

Lung specific liposomes as drug delivery vehicles. The study was carried out to formulated liposomes with increaased affinity for lung tissues and decreased uptake by liver and spleen and to evaluate the chemotherapeutic efficacy of antitubercular drugs encapsulated in these liposomes, against experimental tuberculosis in mice. Surface modification of stealth liposomes by tagging Ostearyl amylopectin (o-SAP) resulted in increased affinity towards lung tissue of mice. Liposomes containing egg phosphatidyl-choline (ePC) and cholesterol (CH) in a malor ratio of 2:1.5 were found to be stable in serum. Inclusion of dicetylphoshpate (DCP), O-SAAP and monosialoganglioside (GM)/ disterayl phosphatidylethanolamine-poly (ethyleneglycol) 2000 (DSPE-PEG 2000) in PC:CH liposomes further increased the stabilty in serum. Tissue distribution of different formulations of liposomes revealed that lung specific stealth liposomes (PC:ch:O-SAP:DCP:GM?DSPE-PEG 2000) accumulated more in the lungs than the reticuloendothelial system of mice. The preadministration of PC and CH(2:1.5) liposomes or injections of dextran sulphate (DS) before the administration of lung specific stealth liposomes further enhanced their uptake in the lungs. The behaviour of these liposomes in mice infected with Mycobaacterium tuberculosis H37Rv was similar to that in normal mice. In vivo stability of these liposomes demonstrated slow and controlled release of their encapsulated contents. Isoniazid and rifampicin encapsulated in liposomes were less toxic to peritoneal and alveolar macrophages compared to free drugs in spite uptake being equal in both the macrophages. Further, the encapsulated drugs, were less hepatotoxic as compared to free drugs. The chemotherapeutic efficacy of isoniazid and rifampicin entrapped in lung specific stealth liposomes was evaluated by injecting the liposome and free drugs intravenously twice a week, for six weeks to tuberculous mice. Liposome encapsulaated drugs at and below therapeutic concentrations were more effective than free drugs on the basis of colony forming units (CFUs). The organomegaly and histopathological studies also demonstrated that liposomal drugs cleared the mycobacteria more efficiently compared to free drugs. Liposomal drugs had marginal hepatotoxicity as seen by the levels of total bilirubin and serum glutamate pyruvate transaminase and alkaline phosphatase. The study thus demonstrated that lung specific stealth liposome encapsulated antitubercular drugs are more effective against tuberculous infection in mice and seems to be a promising therapeutic approach for the chemotherapy of tuberculosis.



POSSIBLE RECEPTOR-INTERACTION OF DECAVANADATE ON VASCULAR AND RESPIRATORY SMOOTH MUSCLES 

The study was carried out to establish the action of decavanadate on the vascular and smooth muscles of Wistar strain albino rats and guinea pigs. Vascular smooth muscles produced dose depedent contraction with decavanadate and metavanadate. The responses were mild and needed high concentration of vanadates in contrast to the highly effective phenylephrine (REFERENCE STANDARD). The decavanadate produced better response with lower (20% of the normal) and normal concentrations of Ca compared to high Ca concentration in the medical. The responses of phenylephrine were effectively blocked by phenoxybenzamine, phentolamine, and prazosin. But the contracitle response of decavandate were not blocked by phenooxybenzamine, phentolamine and prazosin but the contractile response of decavanadate were bbcked by phynoxy bozene phentolamy or prapzosin. The responses of phenylephrine were not significantly altered in the presence of lower contcentration of decavanadate. Similar responses of decavanadate were observed on blood pressure. Phenoxybenzamine, tolazoline, and prazosin effectively blocked the response of phenylephrine but not that decavanadate. Decavanadate produced prolonged and sustained effect for 4-10 min. The study of contractlie property of tracheal smooth muscles of rats and guinea pigs reverealed that work order of potency based on maxima of rat tracheal preparations was carbachol< decavanadate< metavanadate< vanadyl. Maxima of decavanadate and metavanadate were 84% and 82% of carbochol respectively/ Rank order of potency based on EDso was carbochol < decavanadate < metava Carbachol was found 42 and 170 folds were more potent than decavanadate and metavanadate respectively. Responses of carbochol were brisk and quick with a chorter duration while decavanadate produce slow and sluggish response. Sodium nitroprusside and isoprenaline provided 15% and 18% relaxation, respectively against carbachol induced tracheal spasm. However, they provided a complete relief of spasm. Atropine blocked the responses of carbachol but not that of decavanadate and metavanadate, whereas Verpami1 did not affect the responses of carbachol, decavanadate, and metavanadate. The guinea pig trachea produced similar result as produced by rat trachea. The rank order of potency based on maxima among the referance and test agonists was carbachol< histamine = decavanadate < metavanadate = va vanadyl. Rank order of potency based on ED50 was carbochol



CIRCUMVENTION OF DRUG RESISTANCE BY PHENOXAZINE MDR MODULATORS IN ANTIBACTERIAL CHEMOTHERAPY  

Examination of the MIC data has revealed that modulators 2,7,9,12 and 14, have potentiated the activity of kanamycin and streptomycin by 256 fold against E.coli.K12 (Table-III), 256 fold potentiated against E.coli HB101, (table IV) compounds 12,13, 14 17 have the activity of ampicillin and pencillin G by 8-16 fold against E.coli JM101.Modulators 2,7,9,12,13,14,17 have potentiated the activity of Kanamycin and Spectromycin 8-16 fold against E.coli. MTCC55. From among 20 phenoxazines examined, six modulators 1,2,7,9,12,14 have been found to be extremely potent in reversing the resistance of the bacteria. The present results suggests that a variety of E.Coli. plasmids are sensitive to the curing effect of phenoxazine. Three strains tested showed a loss of the resistance phenotype when treated with phenoxazine that can be attributed to the elimination of the coding plasmid, as can be proved by electrophoretic screening. In the case of E.coli JM101, 50 mue g/ ml phenoxazine modulators induce the loss of resistance from 8.3 to 31.8%. This could be the consequences of the high copy number of plasmid P UC18. The MDR reversal effect of the association of phenoxazine modulators and antibiotics on resistance strains which can be observed in the determination of MICs could be the consequences of a variety of possible events (a) The reduction in the number of plasmids with in the bacterial population (b) The inhibition of the expression of the resistant determinants by interference with plasmid transcription (c) a synergistic effect of phenoxazine modulators, which potentiates the antibacterial activity of the drugs. Therefore it appears that phenoxazine modulators is acting through the plasmid. None of these results give any indication about the mechanism of action of phenoxazine on plasmids. But its seems unlikely that it could be the result of inhibition of plasmid DNA replication.



PHARMACOLOGICAL ASPECTS OF GASTROINTESTINAL MOTILITY WITHSPECIAL REFERENCE TO DEVELOPMENT OF PROKINETIC DRUGS  

Study on the role of different neurotransmitters in rgulatiing the GI motility, indicates cholinergic involvement and partial role of prostaglandin and NO in the mechanism of atcion of black tea extract. Exepanol HCl (drug under development as a prokinetic agent) produced potentiation of GIT both in normal and hyperglycemic rat. Exepanol HCl ( 10 mue g- 100 mue g/ kg) reduced significantly indomethacin treated ulcerogenic rat. (upto 82%). Exepanol was equipotent as MCP by restoring the gastric emptying time in mechanism of prokinetic effect of Expenanol HCl. Now to extrapolate the data already generated in our laboratory as mentioned above, we are in the process of estimating NO in different parts of GI tract in control group as well as in experimental group treated graded dose of prokinetic drug, Exepanol HCl. Studies with ciprofloxacin (a, broad spectrum fluoroquinolone antibacterial agent) also indicates an involvement of cholinergic mechanism in the prokinetic effect of ciprofloxacin both in normal and in diabetic rat. It is suggested that systemic clinical studies may be initiated to evaluate the potential efficacy of ciprofloxacin in the treatment of GI motility disorders, specially in diabetic gastroporesis. During the course of investigation, some medicinal plants are evaluated for possible prokinetic and or antidiarrhoeal activity. The results so far indicates that edible mushroom shares prokinetic effect. Seed extract of albizzia lebbeck and root extract of psidium guajava exhibited marked loperamide, the clinically used antidiarrhoeal durg. Result indicate that the antidiarrhoeal actioin of the action of the extract may be due to the inhibition of prostaglandin synthesis and release.



RATIONAL APPROACH TO NEW REMEDIES FOR MYCOTIC INFECTIONS  

The prime objective of this project was to design and synthesize different prototype structrue of evaluate their antimycotic activity. We synthesized various prototype of compounds such as 4- chloropyrimidines (4,6,7) (imidazol-I-yl) pyrimidines (8), chloropyridines (10), monochloro and dichloroquinazolones (17), dibenzo (b,d) pyran (18) and (5-alky-pyrrol-I-yl)-2- carbodithioates (20). All prototype of compounds were screened for their antifungal activity against various pathogenic fungi. As it is evident form the screening profile only. Compounds of 4 chloropyrimidine series were found highly active followed by chloroquniazolones and pyrrole derrivates. One of our synthesized compound 7e displayed highly significant activity against Cn, Tm and Af and was found better than standard drug Ketoconazole and Amphotericin B in vitro test. Unfortunately this compound turned out to be inactive in invivo screen.



CHEMOTHERAPEUTIC POTENTIAL OF POLY(LACTIDE -CO-GLYCOLIDE) MICRO-PARTICLES AS CONTROLLED RELEASE DELIVERY SYSTEMS FOR ANTITUBERCULAR DRUGS AGAINST EXPERIMENTAL TUBERCULOSIS  

Tuberculosis is a serious health concern till date claiming two million lives across the globe. Poor patient compliance owing to a prolonged chemotherapy of 6-8 months with a daily intake of 3-4 drugs is a major culprit for therapeutic failure. Therefore the development of sustained release dosage forms is a need of the hour for reducing the dosage frequency of ATDS, The present study evaluates teh potential of PLG microparticles and nanoparticles as carriers for ATDs. Upon administration of PLG microparticles through subcutaneous/ oral route, INH and RIF were detectable upto 56 days/ 5 days in plasma. However, PL-NP showed a significant improvement in bioavailability of encapsulated ATDs on aerosolizaton. Further 10 doses of PLG-NP showed an equiefficacious chemotherapeutic efficacy as 46 doses of free drugs through aerosol route. Upon conjugation of PG-NP with WGA lectin, the bioavailability of ATDs was further enhanced and 5 doses of PLG-NP every 10 days were replaced with 3 fortnightly doses of WGA coated PLG-NP for undetectable mycobacterial cfus.



TOWARDS THE DEVELOPMENT OF THERAPEUTIC AGENTS USING INFORMATION FROM MALARIA IMMUNE PERSONS FROM ENDEMIC AREAS OF ORISSA  

A study on development of therapeutic agents uising information from malaria immune persons from endemic areas of Orissa was carried out. Repeated exposure to malaria leads to clinical immunityy to the disease in malaria endemic areas. It has been documented that antibodies present in the sera of such immune persons can transfer passive protection of non immune susceptible subjects. Through a differential immunoscreen of a cDNA expression library of the erythrocytic stages of P.falciparum with immune and patient sera from malaria endemic areas of Eastern India several putative protective epitopes were certified earlier. For the development of new candidates for vaccines or immunothorapeutic approaches it would be valuable to investigate protein candidates from the emerging genomics information on Plasmodium falciparum. For this propose several datamining tools based certain mathematically defined framework and principles of comparative genomics have been developed. Using this apprach malarial candidate proteins have been that decided may play a role in the disease pathogenesis process. Synthetic peptides were prepared and the properties of these peptides assessed in relation to immunity to the malarial parasite. The response of sera from malaria immune persons to these newly identified peptides showed reactives similar to that observed with protective peptides identified through defferntial expression screen. Whether the new candidates are protective or not need to be assessed idependently. Specific protective epitopes such PrpO protein epitopes. The Fab. Gene Gene fragments of the protetive mabs have been engineered into the phage display plasmid pCOMB3-SS, with which it is hoped to enhance the protein expressioin. However, the yield and the folding of the phage display Fab fragment were very poor. It was concluded that the monoclonal antibodies. Current attemts are to humanize the protective antibodies and increase the yield of the monoclonasis through various modification procedure. Overall monoclonal anthiodes were stated which are ablolutely specific for plasmodium



THERAPEUTIC EFFECTIVENESS OF PROPOLIS EXTRACT AND ITS ACTIVE PRINCIPLE AGAINST CARBON TETRACHLORIDE INDUCED LIVER DAMAGE IN RATS  

Present study was planned to evaluate propolis, a natural composite balsam for its hepatoprotective potential agent carbon tetrachloride induced toxicity in rats. It is produced by honeybees from the buds of various plants. Bees collct vegetal exudates and form pellets with their secretions. It contains more then 200 constitutents, mainly polyphenols and flavonoids, which are known for their antioxidative activity. In the present investigation, detailed study was conducted against acute and chronic exposure to CCl4. The entire investigation was divided into 6 sets of experiments. Whether prophylactic / curative treatment is effectiv e. To evaluate the dose dependent response. To observed the therapeutic efficacy of MED in DME. Whether extract recoupes LFT's after multiple administration of toxicant. Efficacy of propolis in chronic exposure (3- 5 months. First experiment deals with the selection of effective treatment. In curative study CCl4 was administered at a dose of 105 ml/kg, ip once only followed by single treatment of propolis. In prophylactic study propolis was administeredn at various doses followed by CCl4. Administration of CCl4 caused rise in the activities of AST, ALT and SALP. CCl4 also caused rise iin the level of LPO and decrease in reduced GSH. Treatment with propolis revealed that all three doses of propolis showed significant recovery in curative study when compared with prophylactic study. Thus furgher detailed investigations were conducted on curtive study. Second experiment deals with the selection of optimum dose of propolis extract. CCl4 was administrered to the animals at a dose of 1.5 ml/kg, ip followed by propolis therapy (50, 100, 200 and 400 mg/kg, po) after 24 hours. CCl4 caused a significant fall in glycogen and marginal rise in total protein in both liver and kidney. The enzymatic assay presented a significant fall in alkaline phosphatase, adenosine triphasphatase and succinic dehydrogenase activities. On the contrary there was an elevation in acid phosphatase activity. Results revealed dose dependent response with the treatment of propolis. Significant recovery was found in all paramenters at 200 mg/kg and 400 mg/kg doses. Administration of CCl4 showed enlargement in liveer with degeneration in the hepatocytes. Fatty degeneration and disturbed chord arrangement was also observed. propolis extract showed recovery at 200 and 400 mg/kg dose with well maintained hepatocytes and normal endoplasmic reticulum. 200 mg/kg and 400mg/kg doses of propolis exhibited almost same protective effect, so lowest effective dose ie 200 mg/kg taking drug metabolizing enzymes as targets. CCl4 and propolis was administered as in experiment no. 2. Microsomal drug metabolizing enzymes ie aniline hydroxylase and amidopyrine demethylase showed marked inhibition after CCl4 administration increased hexobarbitone induced sleeping time and bromosuplhpalein retention time. Propolis showed recovery in above mentioned biochemical parameters. No changes were observed in the cholerectic activity of liver by propolis. The rate of bile flow and bile solids showed insignificant changes with propolis therapy. These findings were also supported by histopathological studies. Ultra structural studies showed swelling in mitochondria, vacuolation and marked degeneration after CCl4 administration. Propolis showed recoupment when compared with experimental control. Forth experiment on acute study deals with multiple administriations of CCl4 and propolis. In this experiment CCl4 was administered orally at 0.5 ml/kg dose for 3 days. Propolis therapy was given to animals for 3 days after 24 hours of toxicant administration. Animals of all the group were sacrificed after 24 hours of last treatment. Multiple administrations of CCl4 caused alterations iin blood and tissue biochemical alterations. Propolis showed better dose dependent therapeutic efficacy by recouping liver markers and other enzmatic parameters when compared with experimental controls. Propolis extract revealed significant recovery at both 200 mg/kg and 400 mg/kg doses, thus it was confirmed that multiple administration of toxicant was also recouped by propolis. Experiments deal with the chronic exposure to carbon tetrachloride. In experiment 5A animals received toxicant (0.15ml/kg, ip) regularly for 12 weeks followed by therapy of 3 weeks. In experiment 5B animals were exposed to toxicant for one month followed by therapy of one week, like this the toxicant was given for 3 months followed by therapy for a week. Necropsy was performed after 48 hours of the last treatment. Significant alterations were seen in blood and tissue biochemical parameters after toxicant administration. Treatment with propolis clearly demonstrated recovery in GSH cycle Reduced GSH, GPx, GRx and G6PDH). Drug metabolizing enzymes showed recovery in their acitvies, which were diminished by the CCl4 administered. Micronodular cirosis was found in the liver after toxicant administration. Propolis treatment showed regeneration and improved liver anatomy towards normal. Last experiment deals with chronic carbon tetrachloride exposure for 5 months. CCl4 was administered orally at a dose of 0.15ml/kg daily for 5 days in a week. Propolis therapy was given to animals for 3 weeks after CCl4 exposure and necropsy was performed after 2 days of rest. Results depicted alteration in liver and kidney markers of serum. Serum triglycerides, cholesterol, glycogen and protein contents also showed altered values after toxicant exposure. CCl4 decreased antioxidant defence mechanism of liver thus significant reduction was observed in endoplasmic reticulum, swelling in mitochondria, fatty accumulations were seen in CCl4 treated rats. Propolis therapy showed recovery after 3 weeks treatment when compared with experimental controls. Well maintained chord arrangement with clear sinusoidal spaces and fatty degeneration was recouped with propolis therapy. Propolis showed recovery almost same as selymarin. Thus CCl4 administraion caused significant hepatic damage both in chronic and acute treatment. CCl4 is metabolized in liver to highly reactive CCl3 free radical. CCl3 rapidly interact with molecular oxygen and form CCl3O2 free radical. Both CCl3 and CCl3O2 radicals migh be involved in hydrogen abstraction, disproportion and cancellation reactions. These free radical disturb Ca2+ metabolism of heaptocytes, this may leads to impairment of Ca2+ ions transport. Excess Ca2+ recycling results in mitochondrial damage. These free radical carries lipid peroxidaton, this further results in drastic fall in enzymatic and nonenzymatic antioxidant defence system. Mictosomes of liver, orifinating from endoplasmic reticulum are also damaged and reducing the activities of aniline hydroxylase and amidopyhrine-N-methylase as seen in present investigation. This may further lead to prolongation of hexobarbitone induced sleep time, as hexobarbitone is metabolized by hepatic microsomal drug metabolizing enzymes (MDME). The increase in bromosulphalein retention in plasma clearly indicates the fall MDME and reduced glutathione contents. Formation of fatty liver is mainly due to the accumulation of triglyceride and cholesterol as is also seen in the results of present study. It was concluded from the above study that propolis possessses marked therapeutic potential in restoring the hepatocytes. This significant property may be due to presence of poly phenols and falvonoids found in it. It contains more then 200 polyphenolic compounds, which contribute to its therapeutic efficacy. Flavanoids can act at the inital stage of lipid peroxidation as scavengers, which may react with peroxy radicals of poly unsaturated fatty acids (PUFA's) and breaking chain reaction. Presence of caffeic acid phenethyln ester (CAPR) in propolis act as strong antioxidant thus preserving the integrity of liver cell membrane and there by protecting the hepatic drug metabolizing enzymes




PHYSIOLOGY
COMPARATIVE EFFECTS OF FENUGREEK (TRIGONELLA FOENUM GRAECUM) SEED POLYPHENOLS AND (-) EPIGALLOCATECHIN GALLATE (EGCG) ON ETHANOL-INDUCED LIVER DAMAGE IN RATS  

Chronic alcohol consumption can cause damage to liver. Oxadative stress play a vital role in the pathogenesis of ethanol associated liver injury. Supplementation of antioxidants may have a hepatoprotective effect of aqueous extract of fenugreek seeds (Trigonella foenum graecum). The present study to evaluate the effect of polyphenolic extract of fenugreek seed on experimental ethanol toxicity in rats and in Chang liver cells in vitro. Fenugreek seeds have shown significant beneficial effect in experimental alcohol toxicity in this study. Fenugreek seed exteact (FPEt) at a dose of 200 mg/kg body weight exerted a significant hepatoprotective, antiperoxidative, antioxidant and hyperlipidemic effects in ethanol induced toxicity in rats. The exteact offered protection to Chang liver cells when exposed to ethanol. The result of the study show that EtOH induces toxicity of the liver and vivo and vitro in Chang liver cells. FPEt, EGCG and silymarin prevent cytotoxicity. Increased levels of llipids, lipid peroxidation, protein oxdation,altered cellular redox ratio and apoptosis were observed in EtOH treated cells in vitro. Upon treatment with FPEt/ EGCG/silymarin, ethanol induced liver damage was brought back to normal. The effect EGCG was found to be more effective than the others. In vitro radical scavenging activity of the seed polyphenols revealed the antioxidant potential of the extract. Further studies are required to understand the mechanisms of action of these substances.



MOUSE SUBMADIBULAR EPIDERMAL GROWTH FACTOR (MEGF) AS ULCER CURING FACTOR  

Study was carried out to investigate the role of submandibular gland secretion (which contains epidermal growth factor) in the prevention of gastric and duodenal ulcer. Mice of both the sexes supplied with normal ration of feed and water ad libitum did not show any ulcerogenic changes in the stomach and duodenum. Removal of salivary submandibular gland (Salivariadenectomy) caused ulcerogenic changes in the stomach and duodenum. A very remarkable observation was the formation of duodenal polyps in the salivariadenectomised mice. The removal of submandibular gland made the glandular mucosa (carpus) of the stomach very sensitive to the ulcerogenic agent, cysteamine- HC1. After the administration of cysteamine the mucosa of glandular stomach (carpus) of salivariadenectomised mice developed ulcers which were more severe unlike the control mice which were administered cysteamine. During ulcerogenic changes the carpus and Brunner's glands showed diminished Beta-glucuronidase activity, and loss of PAS-positive glycoprotein was also observed. Brunner's glands underlying the polyps showed much dilatation of the lumen of the secretory units. Histologic and histochemical changes in Brunner's glands of the salivariadenectomised mice were similar to that of cysteamine treated mice. The secretion of salivary submandibular gland (EGF) seems to play a very important role in protecting the gastroduodenal mucosa.



EFFECT OF ELECTRO-ACUPUNCTURE AND STRESS ON SOMATO-AUTONOMICRESPONSES EVOKED BY NOCICEPTIVE STIMULATION  

Study was undertaken with the objective to find out the effect of electroacupuncture (EAS) on somato-autonomic responses and compare EAS effects with those produced by stress stimulation (SS). It was also planned to investigate the possible mechanisms underlying EAS effects, and suggest some objective criteria for evaluation of pain and its relief. The study was conducted on 204 rats, 69 frogs and 84 human subjects (26 normal and 53 pain patients). Somatic responses to a nociceptive sti- mulation was evaluated by way of recording withdrawal reflex response time (WRRT) and nociceptive threshold (NT) in unanaesthetized freely moving frogs, and tail flick resposnes (TEL) in rats. Changes in autonomic responses, heart rate, blood pressure and respiratory rate following nociceptive stimulation were studied in anaesthetized animals and human subjects. Measurement of nociceptive threshold by TFL, WRRT and NT in unanaesh- tetized rats and frogs revealed variations due to age, sex, body weight and time of the day. Somato-motor responses to nociception have also been found sensitive to autonomic blockers like tolazoline, propranolol and atropine. Analgesic role of adrenaline as well as acetycholine (Ach) has emerged with Ach showing more potent effects. In anaesthetized rats effects of electroacupuncture stimulation (EAS) as well as foot shock stress stimulation (SS) have revealed EAS having stabilizing effect on BP and some tilt towards parasympathetic dominance whereas SS tends to potentiate sympathetic effects. A similarity in actions of EAS to opioid and SS to catecholamine, suggests possiblility different underlying neural/neurotransmitter mecha- nisms involved in these interactions. In chronic pain patients altered autonomic status with parasympathetic preponderance appears to exist particularly in cancer and neuralgia patients. Patients treated with acupuncture show a clear trend of increase in BP and pulse rate following therapy. By contrast, acute pain patients responded by showing a decrease in BP and pulse rate. The rise in BP following EAS therapy in the former and fall in the latter group could be correlated with the initial low BP in the first and higher BP in the second case. Thus it may be concluded with reasonable certainity that EAS tends to restore autonomic balance irrespective of the initial altered autonomic status (e.g. high or low BP and/or pulse rate). The study further suggests the utility of monitoring pulse rate, BP and otehr autonomic parameters before, during and after EAS which seem to be good inidicators of the effectiveness of the therapy.



PHYSIOLOGICAL STUDIES IN YOGA AND BODY WEIGHT CONTROL  

Studies were taken up to explore, using yoga, the possibilities of exploiting synergistic covariance between genotype and environment for controlling energy expenditure of involuntary activities and acquiring control over body weight in the process. Objectives of the study included assessment of the changes in body weight through experiments of underfeeding and overfeeding before, during and after yoga training,study if Yoga affects the efficiency of energy utilisation through energy cost of involuntary, habitual activities and standard exercise and for assessment of the effect of yogic exercise on parameters such as haemoglobin, serum cholesterol, fasting blood sugar and serum protein.



COMPARATIVE STUDY OF RESPIRA- TORY MINUTE VOLUME, FUNCTIONALRESIDUAL CAPACITY & LUNG DIFF-USION IN PERMANENT & TEMPORARYRESIDENTS OF HIGH ALTITUDE*  

Study was undertaken in 186 healthy school children (96 boys and 90 girls) aged 10-17 years with body height 132-179 and weight 23-73 kg. Lung volumes/capacities [Vital Capacity (VC), Inspiratory Capacity (IC), Expiratory Reserve Volumes (ERV), Inspiratory Reserve Volumes (IRV), Functional Residual Capacity (FRC), Residual Volume (RV) and Total Lung Capacity (TLC)], mechanics [Forced Vital Capacity (FVC), Forced Expiratory Volume for First Second (FEV1), Peak Expiratory Flow Volume (PEFR), Maximum Mid Expiratory Flow (MMEF) 25-75% Vital Capacity (VC) and Maximum Voluntary Ventilation (MVV)] and diffusion (DLco) were determined by spirographic, helium dilution and carbon monoxide single breath techniques. All the indices of lung volumes/capacities and mechanics increased possibly non-linearly with increasing body height, age and surface area of the subjects but the best correlation was with body height. All values except RV were significantly higher in boys as compared to girls from the body height of 148 cm and upwards. Majority of indices recorded significantly higher values in boys as compared to girls from the age of 14 years and above. There was highly significant increase in ratio FRC/TLC with body height. The percentage for FEV1/FVC (90 + OR - 6.2%) was independent of the height whereas MMEF 25-75% VC/FVC tended to decrease with increase in height suggesting that there was larger airway patency in smaller than in taller children. Single breath carbon monoxide lung diffusion (DLco) values increased linearly with increased TLC. DLco/TLC ratio decreased with the growth of the children suggesting a larger alveolar gas exchange in the lungs of smaller children.



PATHOPHYSIOLOGICAL IMPLICATIONS OF COARSE CEREALS 

The study was carried out on healthy volunteers and non-insulin dependent diabetics of either sex to define the effects of a few cereals on serum lipoprotein profile and glucose tolerance. The effect of a mixture of wheat, barley and Bengal gram was also studied. In response to maize, none of the variables examined were signifi- cantly different as compared to white bread. The glycaemic response to bajra was significantly lower than that to white bread in healthy subjects, but the two responses were indistinguishable in patients of non-insulin dependent diabetes mellitus (NIDDM). The insulinaemic response to bajra and white bread were not significantly different in either group of subjects. These characteristics do not make maize and bajra very suitable for prevention or treatment of diabetes. The glycaemic response to barley was significantly lower than that to white bread in both groups of subjects. However, the insulinaemic response to barley was significantly lower than that to white bread only in healthy subjects. In NIDDM patients, there was a tendency for the response to barley to be higher than that to white bread 0.5 h after ingestion. Barley, with a low glycaemic index (68.7 in healthy subejcts 53.4 in NIDDM patients) and a high insulinaemic index (105.2) in NIDDM patients seems to mobilize insulin in NIDDM. The glycaemic index of the wheat-barley-Bengal gram mixture was 68.6 and 64.9 and the insulinaemic index 88.1 and 66.0 in healthy and NIDDM subjects respectively. The long-term effects of both barley and the wheat-barley-Bengal gram mixture were an increase in HDL cholesterol, a fall in LDL cholesterol, an increase in HDL/total cholesterol ratio, and a marginal improvement in glucose toterance. Although barley had favourable physiological effects, its acceptability is likely to be low because of its poor organoleptic properties. Whereas the mixture of wheat-barley-Bengal gram has much better organoleptic properties than barley alone, and may be more acceptable for the prevention and treatment of diabetes mellitus.



CATECHOLAMINERGIC MECHANISMS OF MEDIAL PREOPTIC AREAS IN THE CNS REGULATION OF MALE SEXBEHAVIOUR IN RATS  

The study was carried out to develop a method of quantifying sex behavior in Wistar male strain albino rats using a IBM compatiable PC and to investigate the role of catecholominergic (CA) fermirals and the adneric mechonism in the medinal preoplic area (mPOA) in the elaboration of male sex behaviour as also sexual arorisal and copulatory performance respectively. The effect of administration of -Hydroxydopamine (8 Mue g in 1.2 Mue g saline containing 5% ascorbic acid) at the mPOA, with and without pretreatment of desrrethylimpramine (DMI, 25 mg/kgintraperiteneally), on copulatary behaviour was studied in six sets of rats. The destruction of CA fibres was confirmed by monoamine histofluorescence using glyoxylic acid staining technique. Sex behaviour of rats which received pretreatment of DMI was scored on 4th, 8th and 12th day. In four sets of rats, without pretreatment of DMI, sex behaviour was scored on different days from 2nd to 16th day after the destruction of CA terminals. One set of rats, which received saline injection, served as control. DMI treated group showed significant decrease in sex drive score (SDS) till 4th day after the infusion of neurotoxin (6-OHDA) at the mPOA. The decrease was not significant on 8th day, and it reached near normal level by 12 day. Injection of 6-OHDA, without DMI pretreatment, produced significant decrease in SDS till 8th day. But rats also showed recovery by 12th day. The time difference in recovery in SDS and other parameters confirm the involvement of noradrenergic fibres in the mPOA regulation of male sex behaviour. Recovery of of SDS by 12th day could be attributed to the compensatory activity of remaining neurons and terminals or neural plasticity. The role adrenergic mechanisms in the mPOA in sexual arousal and copulatary performance was studied by administration of adrenergic agonist and antagonists at the mPOA. Saline, norepinephrine (NE), phenoxybenzamine (PBZ) and propranolol (PROP) were injected into the mPOA in different groups of rats. NE application (3 Mue g) facilitated the male sex behaviour by increasing sexual arousal and copulatary performance. Onthe other hand application of PROP and PBZ produced inhibition of male sex behaaviour in a dosedependent manner. Effects produced by PROP were more prominent than PBZ. The results thus suggest that mPOA beta adrenergic mechanism is important in the elaboration of male sex behavior. It is thus concluded that the noradrenergic (NE) terminals from ventral noradrenergic bundle, innervating the mPOA is critical for execution of male sex behaviour, and this action is primarily mediated through beta adrenergic mechanism.



STUDIES ON THE EFFECT OF FUROSEMIDE AND THE MECHANISM INVOLVED IN GASTRIC ACID SECRETION  

Studies on the effect of furosemide and the mechanism involved in gastric acidd aecretion. The study was carried out on CF strain of rats and frogs to elucidate the mechanism of secretagogue stimulated gastric acic secretion on the application of furosemide, a potential antiulcer drug. Gastric lesions were induced in rats using nonsteriodal anti-inflammatory drugs. Thereafter normal and experimental rats were treated with oral furosemide at the rate of 2 mg/kg body wt/day for six consecutive days. Increase in the release of defensive factors like hexosamine and mucin with reduction in the gastric acid and pepsin secretion, were observed. There was an enhancement in the rate of DNA and protein synthesis. Furosemide inhibited histamine stimulated H+ transport from the secretory side of frog's gastric mucoso by releasing K+ ion to the medium. The inhibition was rapidly reserved by an increase in K+ in the secretory solution replacing equimolar Na+ from the medium. This suggested that the effect of furosemide occurred at or near the secretory membrane and induced a conformational change in K+/H+ exchange system resulting in the vectorial transport of H+ across the membrane. Scanning electron microscopy also revealed the partial interaction of furosemide and gastric parietal cell, resulting in partial inhibition of gastric acid secretion without much alteration in the classical intracellular pathway of acid secretion without much alteration in the classical interacellular pathway of acid seretion. Furosemide at 1 mM concentration partially inhibited (50%) K+ stimulated H+, K+-ATPase activity. Gastric microsomes pretreated with fursemide also exhibited an inhibition of H+, K+-ATPase activity. These findings suggest that furosemide is likely to act as an antisecretory/ antiulcer agent ie both as a gastric cytoprotective agent as well as in the therapy of gastric ulcer.



INTERACTION OF MELATONIN AND CIRCADIAN RHYTHMICITY IN ANTARCTIC CONDITIONS  

Interaction of melatonin and ciradian rhythmicity in Antarctic conditions The study was carried out to evaluate the coupling patterrn of melatonin and other circadian rhythmicity in the human subjects in Antarctic conditions and on rats under simulated Antarctic exposure of diversifying lighis and dark cycle. }The experimental study was carried ouut on 42 male Wistar strain rats (200-300 g) divided into 3 groups viz normal (control without surgical intervention): sham operated; and superior cervical ganglionectomized. The human study was conducted on eight volunteers feom the 12th Indian scientific expedition to Antarctica (Antarctica winter extended dark condition) and six volunteers from the 13th expedition (Antarctic summer extended light conditions). The human volunteers divided into two groups were given either beta adrenergic blocker (atenolol) or placebo. The results of both the animal and human studies showed that under the influence of constant dark and light, sympathetic innervation to the pineal primarily has tonic inhibitory influence on the suprachiasmatic nucleus, the biological clock, which alters the amplitude and mean level of various rhythms to keep the synchrony and coupling of various physiological and hormonal rhythms. The rat study also provided convincing data for the presence of extra sympathetic inputs (probably neuropeptide Yergic and cholinergic) to the pineal which are stimulated only if melatonin level is severel depressed as in cases of absence of sympathetic innvervation or on exposure to continuous light.




RADIOLOGY
TO INVESTIGATE NEUROCHEMICAL REGULATION OF REM SLEEP AND CELLULAR CHANGES AFTER REM SLEEP DEPRIVATION  

The results support our hypothesis that GABA in PPT modulates REM sleep however, their detailed projection and interactions with norepinephirine, etc need further extensive study. It was observed that synaptosomal membrane lipid peroxidation was decreased after REM sleep deprivation, which was mediated byh increased NE due to REM sleep deprivatino and the action was mediated through the alpha-1 adrenecptors. Further, the result also suggest that there is a role of calcium in REM sleep deprivation induced decreased lipid peroxidatioin, however, the detailed mechanism for such action needs to be studied. There was significant decrease in phosphorylated form of synaptosomal Na-K ATPase after REM sleep deprivation, which was again mediated by NE acting through the alpha-1 adreneceptor. The finding confirms our earlier proposition that REM sleep deprivation increases the Na-K ATPase activity by dephosphorylation of the enzyme. It was also observed that REM sleep deprivation slso increases the transcription of alpha-3, beta-1 and beta-3 subunits; the molecular mechanism of sucvh transcription need further study. The study showed that after REM sleep deprivation there was an increase in the level of phosphatidylethanolamine, however, it was not mediated by NE.




TUBERCULOSIS & CHEST DISEASES
EFFECTS OF X-RADIATION ON STRUCTURAL AND TRACE ELEMENT CONTENTS IN INTEGUMENTARY SYSTEM  

The study was carried out on CF strain albino rats to determine the histo- logical and histochemical changes in the integumentary system and estimate the changes in trace element contents in tissues caused by chronic low dose and acute exposure to X-irradiation. An attempt was also made to determine the occupational radiation hazards to radiographers. For chronic exposure rats wereexposed to chronic intermittent low dose X-radiation at the dose rate of 0.015 cGy/sec/day (7 days irradiation alternating with 7 days rest) for periods of 90,180, 270, 360, 540 and 720 days. For acute effect the rats were exposed to 7Gy X-radiation (1.211 Gy/minute). Structural (transmission electron microscopy, scanning electron microscopy and light microscopy) and trace metal (atomic absorption spectrometry) studies revealed that chronic low dose X-radiation affected the structural integrity of the skin and induced imbalance in the trace metal homeo-stasis of the inegumentary system. Transmission electron microscopy revealed increase in apoptosis in the epidermal cells and giant fibrocytes and hyaliniza- tion of collagen which started after 6 months of irradiation and became exten- sive after 24 months. Atomic absorption spectometry revealed depletion in magnesium and zinc levels and zinc to iron ratio and increase in iron and copperconcentrations. Acute exposure study reconfirmed the damaging effects of chronic low dose exposure to X-irradiation. However, the trace metals did not show any definite pattern of change in their tissue concentrations over the postirradiation period. Study of human scalp hair and nail samples of medical radiographers and non-radiographers (controls) matched for age and food habits, revealed increase in zinc, copper and iron concentrations along with varied types of cuticular damage in the radiographers compared to non-radiographers. These findings possibly confirm the hazardous effects of long-term exposure to X-rays. It alsoindicate that the integumentary system (scalp hair and nails) may act as an useful biogenic indicator of radiation hazard.



FIELD TRIAL OF SHORT TERM INTERMITTENT CHEMOTHERAPY AGAINST TUBERCULOSIS  

The aim of the study was to see if in a specific area, namely in the district of Wardha, effective containment of tuberculosis by using bacteri- cidal drugs like Rifampicin could be achieved. The efficacy (bactericidal activity and sterilising activity) of short term intermittent chemotherapy was tested in field conditions. The impact on prevalence of tuberculosis was assessed after administration of short term intermittent chemoterapy under field conditions. The scheme broadly had two main components:- i) Survey : for detection of sputum positive pulmonary tuberculosis cases; ii) Treatment and follow- up, for assessing the efficacy of the regimens under trial. The scheme covered the whole of Wardha district with its 10 lakh popula- tion, and when the survey ended on 31st March, 1988, 7.6 lakh population had been covered. Both the urban and the rural population of Wardha dist- rict was surveyed. Attempt was made to achieve maximum coverage as per the 1981 census. For analysis, only the population of age 5 years and above has been taken into consideration. After recruitment the investigators were given one months training at the Institute. The data were collected by house to house survey on the basis of a pretested and precoded proforma. Attempt was made to collect two sputum samples from every symptomatic, one on the spot and the other of overnight collection. The samples were all subjected to dirct microscopy by Ziehl Neelsen staining and were then con- centrated by Petroff's method and cultured on two slopes of Lowenstein Jensen medium. Drug resistance tests for streptomycin, isoniazid, rifampicin and pyrazinamide were also done. Amongst the biweekly regimens, given in new cases, the regimen (NR) 2H2R2Z2/4H2R2 is recommended as its efficacy was best under operational con- ditions 71.34%. Among the patients who were regular in their intake of drugs the efficacy was good as 96.46%. This regimen should be given for 6 months. Prolongation of maintenance phase by another 2 months gave no added advantage. Mode of administration of this regimen is also simple. It is an oral regimen and no injection need to be given. The supply of drugs at the doorstep of the patients for domiciliary self administration is recommended as it ensures better drug compliance and there are less defaulters. The 3 gm dosage of pyrazinamide led to side effects resulting in many defaults. It should be reduced to the minimum possible on the basis of weight of the patients. The maximum relapse rate in all the regimens was within in the first 6 months after the end of treatment. Hence follow up should be strict atleast for the first six months of completion of treatment. Among the old cases, biweekly regimens OUI and ORI were found less effec- tive than the regimen MR(O) which had 3 drugs given 5 days a week during the intensive phase of 2 months and 2 drugs biweekly during the continuation phase of 4 months. Its efficacy under field conditions in old patients on this regimen was 72% and in regular patients with 80% or more drug intake it was 89.80%. Therefore, the regimen MR(O) is recommended in all chronic cases. The door to door survey, irrespective of X-ray, was successful in picking up cases of early tuberculosis as is shown by 50% of the cases being culture positive (majority with less than 20 colonies) and smear negative. In 25% of cases culture was positive and the X-ray was normal. Methodology for providing culture facilities to the peripheral centres should be worked out, cost consideration should be carefully weighed against the importance of early detection, prompt treatment and better response in these patients. This would go a long way in the control of tuberculosis. High risk groups such as contacts and families with positive history of pulmonary tuberculosis should be identified and given special attention. Primary drug resistance to rifampicin alone and in combination with INH in this area was fairly high. The picture may be true even in other areas where these studies have not been done. Judicious programmes for early and comprehensive coverage with these effective drugs should be worked out without loss of time.



TUBERCULAR LYMPHADENITIS : INCIDENCE, LABORATORY DIAGNOSIS AND ROLE OF SHORT TERM CHEMOTHERAPHY *  

The study on field survey of 23229 population with 7900 children in the age group 0-14 years in 35 villages of district Wardha, Maharastra, envisaged to find the prevalence of lymphadenopathy and tubercular lymphadenitis in the study group.It also tried to evaluate the routine laboratory techniques in diagnosis of tubercular lymphadenitis. The sex ratio (M/F * 100) was 113 and BCG scar was found in 72.69% children examined. Prevalence of lymphadenopathy in the examined children was found to be 27.21 + - 3.66 per thousand children examined with 95% confidence limits. The prevalence of lymphadenopathy increased with age and the differences in the three groups was staistically highly sigificant (p<0.001). No significant difference was observed in the prevalence of lymphadenopatghy among male and female children. Out of the 215 children detected with enlarded lymph nodes, in 105(48.84%) lymph nodes regressed without ATT; in 22 they were presistent while 11 were lost to follow up. The remaining 77 were biopsied and investigated for tuberculosis. The prevelence of tubercular lymphadenitis was 3.31+-1.3 per thousand children with 95% confidence limit. The corrected prevalence after includiing the clinically suspected but unbiopsied presistent lymph nodes (22) was 4.2 per thousand. Calculating further for 11 subjects lost to follow up the projected prevalence was 4.43+- 1.49 per thousand i.e. range 2-6/1000 with 95 % CL. There was no statistically significant difference found in TBLN in children belonging to difference castes. TBLN cases were found only in children living in K kacha houses. TBNL was more in children living in jount families. No difference in the positivity of TBNL was observed in children with respect to per capita income per month. A total of 26 lymph nodes were positive for TBNL by any of the three methods; smear' culture and histopathology (23 from 64 biopsies and 3 from 13 FNAC's). Positivity by histopathology was 14.06% (9/64), by culture 20.31% (13/64) and by smear 32.81% (21/64) The sensitivity of smear and culture with HP was 100% and 77.8% respectively while sensitivity of HP in culture positive cases was 53.84% (7/13) and that in smear 42.85% (9/21). Together the two microbiology investigations detected 17 extra cases of TBLN as compared to HP; giving an additional (22.07%) positive yield of TBLN. Ziehl Neelsens staining was positive in 18 (85.7%) of the 21 cases positive for tuberculosis by fluorescent microscopy. there was no case positive by ZN which was negative on fluorescent microscopy. Fluorescent microscopy was false positive for tuberculosis in 7 cases; 3 nocardia, 2 fungus and 2 which were negative by all parameters used in the study. All culture isolates for tuberculosis were indentified as M.tuberculosis. No BCG or M bovis was isolated. Routine culture were all sterile except in two cases that grew Staphylococcus epidermidis (skin contaminant).



ALLERGY EVALUATION BY CUTANEOUS SENSITIVITY AND BRONCHIAL CHALLENGE OF NEWLY IDENTIFIED AEROSPORA  

During an ICMR funded aerobiological survey of Kerala between 1983 to 1986, large number of pollengrains and fungal spores were identified some of them have never been evaluated for human allergy. In this study eleven of these pollen and two fungal spores were subjected to detailed study for allergenicity. The study consisted of three aspects i.e. botanical, biochemical and clinical. In the botanical part the plants and pollens were collected and the following were prepared, herbarium, reference slides by acetolyated and unacetolyated methods. On the biochemical side the pollen were purified, dried, defatted and extracted. The extracts were dialysed, sterilised by biological filtration, sterility and animal testing and standardisation were done and dispensed for clinical testing. The clinical evaluation was done by skin testing and bronchial challenge in asthmatic patients. The hypothesis tested was that these newly identified pollen and fungal spores are allergenic to human beings. A sample size of 190 was calculated by fixing the type I error at 5% selection bias was prevented by fixing inclusion and exclusion criteria. The patients were made their own controls. The measurement bias was prevented by assigning numbers to these angigens and mixing them with usually used antigens for intradermal testing to disquise them. The number of patients tested were fixed much above the sample size to increase the power of the study. 500 patients were tested with nine pollen grains and 200 patients with 2 pollen and 2 fungal spores. The pollen tested and reactions obtained (in brackets) are given below. Areca catechu (4/500-0.8%), Cassurina equaestifolia (20/500-4%) Heteropogon contortus (23/500-4.6%), Oreodoxa regia (6/500-1.2%) Peltoforum ferruginium (19/500-3.8%), Phoenix dactylifera (30/500-6%), Sweitenia mahogani (16/500-3.2% ), Eleis guinensis (10/500-3.2%), Spithodea campanulam (18/500-3.6%) Dimeria sp (8/200-4%), Eulalia sp (0/200-0%). The fungal spores tested were cephelosporium (22/200-11%)and clorida (0/200-0%). Only ' 2plus ' reactions and above were considered positive. Those who were positive had bronchial challenge testing. More than 50% of all those skin tested positive has bronchial challenge reaction with a 20% or more fall of FEW1. The percentages were as follows in the order of pollens given above. 75%, 65%, 73.9%, 66.7%, 78.9%, 66.6%, 50%, 75%, 55.6%, 50%, 0%, 36.4% and 0%. Thus this observational study showed that most of these pollen grains and fungal spores are potentially allergenic to human beings. Isolation of the allergenic material in these will from the subject of next study."



DIAGNOSTIC POTENTIALTIES OF ELISA IN PULMONARY TUBERCULOSIS USING PURIFIED MYCOBACTERIAL GLYCOLIPID ANTIGENS  

Study was carried out with the aim to develop ELISA for the detection of circulating antibodies to glycolipids (PIMs), mannophosphoinositide and antigens in circulating immune complexes (CIC) and in sera of tuberculous patients. These assays proved to be quite specific and sensitive. A simple and economical dot ELISA for the detection of mannophosphoinositide antigen in sputum samples of tuberculosis patients has also been developed using affinity purified antibodies. This test detects free as well as bound anti- gen. An overall sensitivity and specificity of 89% and 93% respectively was obtained with this test.



EFFECT OF CORTICOSTEROID TREATMENT ON VARIOUS SEROLOGICAL & BRONCHOALVEOLAR LAVAGE (BAL) ABNORMALITIES IN PATIENTS WITH SARCOIDOSIS  

It was aimed to study various immunologic abnormalities in serum and bronchoalveolar lavage fluid (BALF) in patients with sarcoidosis. Relationship, if any, between these immunologic abnormalities and clinical,physiologic and radiologic parameters was established in the patients. Further studies on effect of corticosteroid treatment on various immunologic abnormalities was also carried out. Twenty nine patients with sacoidosis were studied (17 male & 12 females). Twenty five of 29 patients with sarcoidosis were non-smokers. (mean age 39.5 Yrs). All of them had a clinical picture consistent with the diagnosis of sarcoidosis along with the biopsy evidence of noncaseating, epithelioid cell granulomas from two bopsy sites. None of the patients had any evidence of mycobacterial,fungal or parasitic infection or history of exposure to organic or inorganic material known to cause granulomatous lung disease. Of these patients, cough was present in 13, fever in 7, dyspnoea in 9 and arthralgia in 8. Patients were staged by the traditional roentgenographic criteria as follows : Stage I, bilateral hilar lymphadenopathy without parenchymal infiltrates; stage II : bilateral hilar lymphadenopathy with parenchymal infiltrates and stage III: parenchymal infiltrates without hilar lymphadenopathy. (4 patients had stage I, 22 patients had stage II and 3 had stage III disease). Study showed that patients with sarcoidosis had increased total cell count, the proportion of lymphocytes in the bronchoalveolaer lavage and the proportion of helper/inducer cells (CD4+) was increased in the BAL fluid. Pretreatment CD4+/CD8+ ratio was higher in the BAL fluid in patients with sarcoidosis. Pretreatment absolute lymphocyte count and the proportion of lymphocytes were significantly decreased in the peripheral blood in sarcoidosis. Proportion of helper/inducer (CD4+) lymphocytes was also low in the peripheral blood in sarcoidosis. Pretreat- ment immunoglobulins and whole complement (CH5O), C3 and C4 levels as well as circulating immune complexes (IgG, IgA and IgM) were significantly increased in both peripheral blood and BAL fluid in patients with sarcoidosis. The proportion of CD3+ lymphocytes and CH5O levels in the BAL fluid was significantly higher in the responder group. Complement C4 and CIC levels were significantly higher in the peripheral blood in the responder group. A significantly positive correlation was observed between BAL lymphocytes and BAL IgM levels. With prednisolone treatment, there was a significant fall in the toatal cell count and the proportion of lymphocytes in the BAL fluid. The proportion of CD+, helper /inducer (CD4+) lymphocytes and B cells also decreased significantly after treatment (CD4+/CD8+ ratio also decreased with prednisolone treatment. With prednisolone treatment, although the proportion of lymphocytes in the peripheral blood did not increase significantly, however,absolute lymphocyte count showed a significant increase after treatment. The proportion of CD4+ lymphocytes showed a significant increase with treatment. the proportion of B lymphocytes fell significantly on treatment. Immunoglobulin IgC IgA and IgM CH5O, C3 and C4 levels and CICs decreased significantly in peripheral blood and BAL fluid with prednisolone treatment. FVC and pulmonary diffusive capacity (DLCO) increased significantly.



RESTRICTION FRAGMENT LENGTH POLYMORPHISM ANALYSIS OF CLINICAL ISOLATES OF MYCOBACTERIUM TUBERCULOSIS DNA - A PRELIMINARY STUDY  

Genomic diversity of 100 clinical isolates of Mycobacterium tuberculosis isolated from sputum samples of tuberculosis patients was evaluated to study the nucliec acid diversity (rRNA gene restriction patterns) and to look for a res- triction enzyme (or a combination of such enzymes) displaying polymorphism. M. tuberculosis H37 Rv (TMC 102) was used as standard to compare RFLP patterns of different isolates. High molecular weight DNA was isolated in sufficient quantities (yield 1.2 to 2 mg/g wet cell pellet) by using cesium chloride to break lipid-rich mycobacterial cell wall and guanidium isothiocynate for extraction. The rRNA probe was made free of poly adenylated RNA by passing the lysatesof the mycobacterial cells through poly (U)-Sepharose 4 B column. RFLPs were studied among the clinical isolates of M.tuberculosis using 4 different endonucleases ie EcoRI, BamHI, Pstl and EcoRV to test their ability inproducing cleavage of the test DNA and hence discriminate clinical isolates. Among the 100 clinical isolates of M. tuberculosis, EcoRI produced 6 different ribotypes with 8 to 13 fragments; 32 per cent of the isolates had 9 fragments and 2 per cent had 13 fragments. All isolates tested shared the high molecular weight fragment of 21.0 kb except isolate 5. Also all the isolates tested shared at least one fragment with the reference strain. BamHI produced 4 different ribotypes with 6 to 9 fragments; 60 per cent of the isolates had 6 fragments, 10 per cent each had 7 and 8 fragments and 20 per cent had 9 fragments. These patterns shared at least a high molecular size fragment (21.0 kb) and a low molecular size fragment (3.8 or 3.6 kb). PstI produced 3 different ribotypes with 4 to 8 fragments; 50 per cent of the isolates had 6 fragments and 33 per cent 8 fragments. These patterns sharedat least one fragment of same molecular size among them. EcoRV produced 6 different ribotypes with 7 to 12 fragments; 30 per cent of the isolates had 7 fragments and only 10 per cent had 12 fragments. Further evidence of a close genetic relatedness among the clinical isolateswas produced by the pairwise comparison of all the isolates by determining the fraction of restriction fragments containing rDNA that were of the same size. It was clear that restriction endonuclease EcoRV showed the greatest variability among the clinical isolates followed by EcoRI. PstI was the least discriminative. It is concluded that the RFLP of rRNA gene can be used as a rapid and definitive technique for strain differentiation of M. tuberculosis.



INDUCTION OF RESISTANCE TO EXPERIMENTAL TUBERCULOSIS WITHCELL WALL AND CELL WALL PLUS RNA OF MYCOBACTERIA  

Although BCG is used for several decades as vaccine against tuberculosis, recent trials with BCG vaccine have revealed its efficacy to vary from 0-80%. Studies have recently been focussed on immunoprotective agents alternative to the BCG. Seveial components of the mycobacteria cell wall, lipid fractions ribosomes and RNA have been shown to be immunogenic against experimental tuberculosis Among these cell wall and RNA vaccines are the best because immunity produced by these components is equivalent to that of BCG. However,the combined effect of mycobacterial cell wall and myco RNA has not been examined. Therefore, a detailed study is planned with these components (antigens) to study their role in affording protection against tuberculosis and to study the mechanism of resistance.



ABDOMINAL TUBERCULOSIS : EVALUATION OF NEWER DIAGNOSTICMODALITIES AND THEIR COMPARISON  

Pathogenesis and diagnosis of abdominal tuberculosis In patients with abdominal tuberculosis the virulence of mycobacteria isolated from diseased tissue was assessed as also the cell mediated immune response in patients of peritoneal tuberculosis. The diagnostic potential of soluble antigen fluorescent antibody (SAFA) test and ELISA was assessed for abdominal tuberculosis and usefulness of laparoscopy and its diagnostic accuracy in peritoneal tuberculosis was evaluated. The index of virulence evaluated in specimens from a small sample of patients indicated that abdominal tuberculosis was coused by low virulence Mycobacterium tuberculosis. The delayed hypersensitivity to recall antigen PPD was intact in patients of peritoneal tuberculosis. Similarly the leukocyte migration inhibition (LMI) test was also positive in most of the patients. In contrast, the patients responded poorly to DNCB. The degree of sensitivity in most of the DNCB positive patients was 2+ needing a higher dose of DBCB as compared to controls. ELISA with mycobacterial salinextract had a sensitivity of 81 percent and specificity of 88 percent in patients of abdominal tuberculosis. ELISA showed a strong correlation with SAFA test. ELISA was less time consuming and simple compared to SAFA. These serological tests were helpful in confirming the diagnosis. Laparoscopy was found to be useful in diagnosing exudative ascites particularly due to peritoneal tuberculosis. A laparoscopic visual diagnosis was more accurate than histology or bacteriology. Laparoscopic appearance could be classified into 3 types thickened peritonium with miliary tubercles, thickened peritonium alone and fibroadhesive pattern. These appearances were highly suggestive of tuberculosis in 95 percent patients. In comparaison histology. AFB on histologic slides and culture and guineapig inoculation of biopsy material confirmed the diagnosis only in 82.3 and 37.5 percent patients respectively.



STUDIES ON OXYGEN FREE RADICALDEPENDENT KILLING OF PATHOGENIC, ATTENUATED AND NON-PATHOGENIC MYCOBACTERIA  

Study was undertaken to investigate the macrophage component which could effectively kill the pathogenic strain of mycobacteria i.e. M. tuberculosis, the causative agent of tuberculosis. For this purpose in vitro exposure of M. smegmatis and M. tuberculosis H37Rv to ROS generated in vitro, helped in controlling the quantum of these cytotoxic entities required to kill specific number of bacilli. It was found that M.tuberculo- sis H37Rv ws resistant to killing by ROS than M. smegmatis since 4.5 times higher quantum of ROS was required to cause significant killing of M. tuebrculosis. ROS operated through mycobacterial cell wass and membrane lipid peroxidation to cause cell death. It was postulated that decreased activity of membrane bound ATPase causes imbalance of ions in the cells which lead to mycobacterial death. DNA of mycobacteria was another target of ROS as it caused mycobacterial DNA strand scissions. This study helped in delineating the role of ROS in mycobacterial killing and their mechanism of action.



AN ENQUIRY INTO A POSSIBLE LINK BETWEEN SMOKING AND PULMONARY TUBERCULOSIS  

The study was carried out in Municipal Wards 10 and 11 of the old city of Delhi (approximate population 40,000) to determine whether the prevalence of pulmonary tuberculosis among adult smokers in a general population was significantly higher than in a corresponding group of non-smoking adults. A complete census of the entire population of the area was carried out on specially designed census registers. Besides the identifying particulars, information on age, sex and smoking habits and nature of smoking were also collected. Persons in the age group of 15 years and above were subjected to 70 mm chest X ray. Those with radiographic abnor- malities were subjected to bacteriological examination including sputum/ laryngeal swab examination by direct microscopy and culture. Where indicated, repeat bacteriological and radiological examinations were carried out to establish a diagnosis. Of the 24064 persons in the age group of 15 years and above only 19399 (80.6%); 10057 males and 9342 females, could be studied. Overall 26.0 per cent of males were smokers, 2.4 per cent ex-smokers and 71.6 per cent non-smokers. As the prevalence of smokers among females was only 0.6 per cent, further analysis was confined only to males. The proportion and intensity of smoking increased with increase of age. Among the 10057 males, 162 (1.61%) patients with active pulmonary tuberculosis, of whom 54 (0.54%) were bacteriologically proven, were found. The overall prevalence of active pulmonary tuberculosis in smokers, ex-smokers and non-smokers was 3.26, 3.29 and 0.96 per cent respectively. The corresponding rates for bacteriologically proven pulmonary tuberculosis were 1.9, 1.23 and 0.28 per cent. After standardi- sation for age it was found that 2.43 per cent of the smokers were suffering from pulmonary tuberculosis (1.03% of theses being bacillary cases). The corresponding figures for non-smokers being 1.07 per cent (0.32% bacillary cases). The differences are statistically significant. A 'trend test' also revealed that the prevalence of pulmonary tuberculosis increased with the intensity of the smoking.



EFFECT OF CHEMOTHERAPY ON HISTOPATHOLOGICAL CHANGES AND BACTERIOLOGY OF GASTROINTES- TINAL TUBERCULOSIS  

The study was conducted to determine the prevalence of antibodies to hepatitis C virus (HCV) in patients of acute and chronic liver diseases, blood donors and recipients of multiple blood transfusions using second generation anti HCV ELISA. Only individuals who were serologically negative for hepatitis B virus (HBV) and hepatitis A virus (HAV) infection were studied. These included 100 patients of acute viral hepatitis (AVH), 12 of subacute hepatic failure (SAHF), 43 of acute hepatic failue (AHF), 70 of cirrhosis of the liver, 11 with chronic active hepatitis (CAH), 10 with hepatocellular carcinoma (HCC), 550 blood donors and 63 recipients of multiple transfusions. In 35 of the 100 AVH patients, the disease was due to sporadic NANB infection. Of these, three were positive for anti-HCV. Among 48 patients of AVH whose blood samples were taken only in the acute phase, 20 had NANB infection, but none was found to be positive for anti-HCV. In 52 patients tested serially upto 12 weeks, 15 were found to have NANB infection and three of these were positive for anti- HCV. Five of the 12 patients of SAHF and 23 of the 43 patients of AHF were found to have NANB infection but none of these were found to be anti-HCV positive. Nineteen of the 70 patients of cirrhosis of liver had NANB infection and 2 of these 19 were found to be positive for anti-HCV. Six of the 11 patients of CAH and four of the 10 patients of HCC had NANB infection and of these, 2 patients each were found to be positive for anti-HCV. Of the 550 blood donors, 494 did not show evidence of HAV and HBV and of these only 11 were positive for anti-HCVV. Of the 63 patients of multiple blood transfusions, 46 did not show HAV and HBV infection. Of these, 24 were found to be positive for anti-HCV. It is thus concluded that in patients of acute liver diseases, screening of HCV antibody should be done serially upto 12 weeks. Patients with cryptogenic chronic liver disease and recipients of multiple blood transfusions should be evaluated for HCV infection.



THE ROLE OF FINE NEEDLE ASPIRATION AS AN EFFECTIVE DIAGNOSTIC TOOL IN LYMPHADENITIS AFFECTING CHILDREN  

Study was conducted to determine the aetiopathogenesis of lymphadenitis in children and to evaluate the accuracy of fine needle aspiration technique as a diagnostic aid in lymphadenitis. Children below the age of 5 years having lymphadenitis were also screened for HIV infection by ELISA technique. 200 children (132 males, 68 females) all below the age of 12 years with lymphadenopathy were subjected to aspiration biopsy cytology. 120 children were further subjected to a surgical biopsy of the same lymphnode from which aspiration biopsy cytology was done. The diagnosis made by aspiration cytology was then compared with the histopathological diagnosis. Maximum children were seen in the age group of 4-6 years (35%), followed by 7-9 yrs (32%). Presenting symptoms were loss of appetite and weight (73%), fever (60%), swelling in neck (55%), cough (36%), progressive pallor (2%) and bleeding manifestation/rash in 2% of the cases. Fever was seen in 60%, pallor in 38%, hepatomegaly in 18%, splenomegaly in 16% and respiratory findings in 16% of the cases. Among the lymphnode groups involved, cervical node (66%) group was the commonest group followed by axillary node (10%) group. Involvement of cer- vical and axillary lymphnodes was seen in 13% and generalized lymphadeno- pathy in 8% of the cases. Anaemia was seen in 70%, raised ESR in 50%, positive tuberculin test in 15%,accelerated BCG diagnostic test in 10% and radiological findings in 18.5% of the cases. Sputum/gastric aspirate for AFB was positive in 2% of the cases of tuberculous lymphadenitis. Overall accuracy rate of aspiration biopsy cytology was 86.6%. Accuracy rate of cytodiagnosis in reactive hyperplasia was 100%, in tuberculous lymphadenitis 71.4% and in lymphoma 55.5%. Reactive hyperplasia was diagnosed in 61.7%, tuberculosis in 23.3% and lymphoma in 15% of the cases by histopathology. 70 children below the age of 5 years were screened for HIV infection by ELISA technique and were found to be negative. No notable complications were seen following this procedure. The study establishes the utility of aspiration biopsy as a diagnostic procedure for lymphadenopathy.



TO STUDY THE INTERACTION OF THE 65 KD PROTEIN WITH OTHER MACROMOLECULES & ITS USE IN THE DEVELOPMENT OF AN IMMUNO- ASSAY FOR TUBERCULOSIS  

The main aim of the project was to study the interaction of 65 kD mycobacterial protein with other mycobacterial macromolecules. Initial studies were carried out using the 65kD protein of M. smegmatis and later M. tuberculosis. The objectives of the study enclosed identification of proteins and other macromolecules which can bind to the 65 kD protein characterisation of the identified macromolecules and study of the effect of binding on the antigenicity of both the 65kD protein and the bound macromolecule. The 65 KD protein complex was partially purified from the cell cytosolic extract using ammonium sulphate precipitation and anion exchange column chromatography. the 65 kD protein was found to be associated with phospholipids as well as some ethidium bromide sensitive material. The phospholipids associated with the M. smegmatis 65 kD protein complex were identified as phosphatidyl inositol mannosides, phosphatidyl ethanolamine, and cardiolipin by thin layer chromatography and by the standards that were run alongside.



ANALYSIS OF VARIOUS PREDICTIVEFACTORS FOR HEPATOTOXICITY OF RIFAMPICIN AND ISONIAZID COMBINATION  

Analysis of various predictive factors for hepatotoxicity of rifompicin and isoniazid combinations. Prospective and case control studies were carried out to find at the risk of hepatotoxic reactions due to rifompicin and isoniazid combination in antitubercules therapy and to ascertain the association between hepatotoxicity among patients and certain pertitive risk factors viz age, sex, extent of disease, mutitional status, alcohol intake, prior history of hepatitis and acetylator status. Efforts were also made to delineate the clinical and biochemical patterns of anti tubercular theropy induced hepatitis. The study samples for prospective study consisted of 479 consecutive newly diagonosed adult patients with perlmanary tuberculosis. The drug segimens used were rifompicin, isoniazid, ethambutol, pyrazinamide (RHEZ) for 140 patients and RHE for 399 patients. For case control study, 86 patients (including 28 from the prospective study) were recruited aas cases prospectively who had developed hepatotoxic jaundice while seacising rifompicin and isoniazid combination. A total of 406 patients diagnosed and treated for the full dutation of chemotherapy with regular follow up and did not develop clinical or biochemical hepatitis, formed control group. The risk of developing hepatitis was significant in the patients of pulmonary tuberculosis therapy constining rifompicin and soniazid. The risk of developing drug induced hepatotoxicity (DIH) with regimen containing RHE was formal to be 5.31%(18/339) compared to 9.29(13/140) in patients on RHZE regimen. Of the varuous risk factors analyzed DIH was mere frequent among those patients receiving pyrazinamide in addition to rifompicin and isoniazid. The riks of developing hepatitis was higher if patients were older malnaurished and had derangement of baseline liver function. Also the risk of hepatitis was greater in slow acetylators.



SERUM CONCENTRATION OF ISONIAZID, RIFAMPICIN AND PYRAZINAMIDE IN ELDERLY PATIENTS  

Serum concentration of isoniazid rfifompicin and pyrazinamlde in elderly patients. The study carried out an 10 confirmed elderly (age 49-59 years) male patients of pulmonary tuberculasis and 10 younger paitents (24-35 years of age) matched for weight (controls) to find out the serum level of isoniazid, fifompicin and pyrazinamide. All the patients in both the groups were given isoniazid (300 mg/day) rifompicin (10 mg/kg body wt.) and pyrazinamide 30mg/kg body wt). The drugs were given under supervision and serum levels of drogs were estimated 1,2,3,4 and after the intake of the drugs. It was observed that there was no significant difference in the absorption and serum peak concentration of all the free drugs in elderly and younger patients. However, all the drugs were refained in higher concentration in the plasma in elderly patients suggensting that the clearance of these drugs is slowed down with increasing age resulting in higher toxicity of drugs in older patitents. The study need to be repeated on a large sample, before any definite conclusion.



EVALUATION OF INTERSITIAL LUNG DISEASE BY HIGH RESOLUTION COMPUTED TOMOGRAPHY 

The study was carried out to elucidate the pattern of involvement of pulmonary intersitium with < high resolution computed temography (HRCT) in biopsyproven cases of intersitial lung disease (ILD) and to correlate the specific findings to their underlying etiology. Efforts were also made to evaluate the HRCT findings in clinically diagnosed ILD patients and determine the accuracy ofHRCT in making a specific diagnosis. In the first part of the study fifty ILD patients (26 males and 24 females in the age group of 20 to 77 years) proven on basis of clinical history, pulmo- nary function tests, laboratory data and transbbronchial lung biopsy were sub- jected to chest radiography and HRCT. There 50 patients included 24 with idiopathic pulmonary fibrosis (IPF), 15 with sarcoidosis, 7 with collagen vabcular disease (CVD), 2 with extrinsix allergic pneumonitis and one each of silicosis and post radiation fibrosis. HRCT was performed by taking 2 millimeter axial sections at 20 millimeters table increments in the supine position. The HRCT findings were categorised making use of the three compart- ment model where the interstitium was divvided into axial, middle and the peripheral compartments. The involvement of the axial compartment was seen in the form of peribron- chovascular thickening and abnormal interfaces between the bronchi and adjoininglung parenchyma on HRCT. Ground glass haze and nodularity were the abnormalities in the middle compartment involvement, whereas the peripheral compartment involvement was in the form of thickening of interlobular sepate, pleura and fissures, nodules and honeycombing. Patients with IPF showed a predominant peripheral compartment involvement in 95.83 per cent of cases with lower and middle zone predominance. Sarcoidosispatients showed involvement in all compartments middle and upper zone involve- ment seen in most of the cases with nodularity being the most common abnormalityon HRCT. Nodular thickening of the fissures was an important feature not seen in any other condition. All patients with CVD also showed a peripheral compart-ment involvement especially involving the middle and lower zones. These appearances were indistinguishable from those seen in IPF. The patients of extrinsic allergic pneumonitis showed a peripheral and axial compartment involvement; nodularity being an important abnormality on HRCT. Silicosis patients showed diffuse involvement involving axial and peripheal compartments with no zonal predominance. The post radiaation fibrosis patients showed a middle and axial compartment involvement with ground glass naze being the main abnormality on HRCT. HRCT was found to be more sensitive in detecting areas of ground glass haze suggestive of a middle compartment involvement compared to chest radio- graphy. HRCT was also able to delineate axial compartment involvement in the form of bronchovascular thickening which was not seen on radiographs. The nodular, reticular and mixed opacities on chest radiography correlate well with similar abnormalities on HRCT. In the second part of the study 22 clinically confirmed ILD patients were subjected to HRCT to assess its accuracy in diagnosis of underlying etiology based on the findings of the earlier study. Transbronchial being biopsy was 5 patients of sarcoidosis, 11 patients of IPF and 6 cases of CVD. IPF was correctly diagnosed in all eleven patients as the first choice diagnosis based on a peripheral compartment involvement. Four of the 5 patientsof sarcoidosis were accurately diagnosed as first choice diagnosis based on involvement of aall three compartments in 1 patient and nodularity and nodular thickening of fissures in another 3 cases. In one patient only a aperipheral compartment involvement was seen, and sarcoidosis was kept as a differential of the. Two cases of CVD showed a middle and a peripheral compartment involvement and the other 4 cases aa peripheral compartment involvement. They were all accurately kept as a differential of three based on the HRCT findings. It is thus concluded that HRCT is more sensitive in detecting areas of ground glass haze and bronchovascular thickening compared to chest radiography. Nodular shadows and reticular opacities suggesting interlobular septal thickening were picked up equally well by chest radiography and HRCT. Although CT provided useful information in the detection of abnormalities in patients with silicosis, hypersensitive pneumonitis and post radiation fibrosis, no definite conclusion regarding specific distribution pattern could be drawn due to the very small number of patients with these diseases in the present study.



ROLE OF NATURAL KILLER CELLS IN TUBERCULOSIS  

The study was a comparision of NK cells numbers between healthy normal volunteers, patient contacts and untreated active pulmonary tuberculosis patients. Comparison of NK Activity between normals contacts and patients. Enumeration of conjugates. Mechanism of defect in NK activity of TB patients. Separation of single cells suspensions of M. tuberculosis. Effect of NK cells on macrophages infected with Mycobacteria. NK cell number among different groups remains the same. NK activity significantly lower in TB patients compared to Normal/ contact groups. 77% of TB patients showed improvement in Natural killer activity after treatment. Novel flowcytometric method standardised. Defect is not at the recognition/binding stage but at later steps of cytotoxicity. Apoptosis observed during cytotoxici IFN-gama, TNF alpha and GM-CSF are produced to a lesser extent by LAK cells of TB patients as compared to normal individuals in response to stimulation with target cells. Novel procedure for obtaining single cell suspensions of Mycobacteria standardised. Enriched and cytokine stimulated NK cells bring about reduction in the growth rate of Mycobacterium tuberculosis. Further studies are required to fully elucidate the role of NK cells and NK activating cytokines in the in the host resistance to tuberculosis.



PLASMID PROFILING OF MYCOBACTERIUM AVIUM - INTRACELLULARE ISOLATES CAUSING PULMONARY MYCOBACTERIOSIS  

This study presents the first report of the presence of plasmid and characterzation of one of the plasmids from Mycobacterium avium intracellulare complex (MAC) isolates obtained form Indian patients having mycobacterial infection. 90 isolates were analysed biochemically and then were subjected to plasmid. 22 isolates showed the presence of the plasmid, out of which 2 gave multiple bands but were lost on repeated subculturing. One isolate gave consistently a plasmid band that was purified using the Qiagen column kit. The purified DNA was subjected to transmission electron microscopy that endorsed the circular nature of the plasmid. Evidence was also giver, by arc like band in 2 dimentional gel electrophoresis. From restriction digestion using EcoRI, HindIII, Pstl, EcoRV, BamHI and double digests (BamHI and Pstl) the exact sizes of individual fragments generated were calculated and the size of the intact plasmid was found to be 24kb. To localize the origin of replication, a 37 mer oligoprobe was hybridized using the ECL non radiolabeled direct labeling and detection system. Signals were obtained with two fragments of 4kb each of BamHI and Pstl double digest plasmid DNA blotted on nylon membrane. Based on these observations, a tenttative restriction map of the isolated plasmid has been sonstructed. This study has established the existence of plasmid in MAC isolated from Indian patients. The methodology standardized for plasmid isolation may be used to screen large number of indigenous MAC isolates for further studies. Probes developed form these plasmids could be of much use in the epidemiology of MCA in India. It paves the way for the development of a cloning/expression vector and thus open up a whole new chapter in muycobacterial diagnostics and vaccines in the face of the Indian scenario. standardization of plasmid screening was done using MAC isolated in our labouratory. Repeated attempts at purification of the crude plasmid DNA by cesium chloride ethidium density gradient centrifugation met with consistent failure. Attempts at restriction digestion of these preparations did not yield distinct restriction fragments on agarose gel. Probably the contaminants in the lysate interfered with the separatin of the plasmid DNA on CsCl gradient. The crude lysate was then subjected to purification by commercial Qiagen column with some modifications. The crude DNA was treated with RNAase and proteinase K and subjected to exhaustive (3-4 times) phenol-chloroform extraction. the aqueous phase was passed through the Qiagen resin based column. The purified plasmid DNA thus obtained gave reproducible results during restriction degestion. Electron microscopy of the purified plasmid DNA from MAC isolated confirmed its circular nature. Electron microscopy has been used earlier to show the circular nature of the plasmid isolated from MAC 2 dimensional gen electrophoresis was undertaken to observe the mobility pattern of plasmid isolated from MAC because circular replicating forms of extrachromosomal DNA have been reported to give a characteristic are like appearance in 2 D gel electrophoresis while the linear lemda DNA gave a band the plasmid isolated from MAC showed a characteristic arc like pattern further giving an evidence that the isolated plasmid was having a conformation different from linear lemda DNA.



CYTOKINE RELEASE IN TUBERCULOSIS : EFFECT OF DRUG TREATMENT  

One hundread and six HIV negative patients with various forms of tuberculosis mean age 29.9 years 79 males, 24 normal controls mean age 29 years, 20 males and 13 patients with non tuberculosis pleural effusion mean age 51 years, 10 males were included in the study. Antituberculosis treatment resulted in significant increase in the body weight, body mass index, triceps skin fold tickness and mid arm circumference in patients with tuberculosis. Antituberculosis treatment resulted in significant rise in the absolute lymphocyte count, percentage of lymphocytes ad a fall in the ESR in patients wit tuberculosis. Total serum proteins and serum proteins and serum albumin values also showed a significant rise with antituberculosis treatment. With antituberculosis treatment, IL-2 receptor level (estimated in 16 patients with MDR- TB) (mean 3046.9 pg/ml; SD 2219.6) showed a decrease compared to the pretreatment levels (mean 2556.3 pg/ml; SD 1535.6). However, this difference did not attain statistical significance. Patients with tuberculosis pleural effusion had significantly igher CD4+/CD8+ raito in the pleural fluid compared to those with non tuberculosis pleural effusion(p,0.05). However, the ratio in the peripheral blood in tuberculosis and non tuberculosis pleural effusions was not significanlty different. There was no significant diffenencein the levels of various cytokines in the peripheral blood of all patients with various forms of tuberculosis included in this group and normal controls. Patients with DTB/MTB had significantly higher IL-4 levels in the blood compared to patients with other forms of tuberculosis and normal control subjects (p<0.001). Patients with tuberculosis had significantly higher IL-6 levels in the BAL fluid compared to normal controls(p<0.01). There was no significant difference in the other cytokine levels in the BAL fluid of patients with tuberculosis and normal controls. Patients with tuberculosis had higher levels of IFN- gama in the blood compared with BAL fluid (p<0.001). There was no significant difference in the other cytokine levels in the blood and BAL fluid of patients with tuberculosis. Patients with tuberculosis pleural effucion had significantly higher levels of TNF- alpha, IFN- gama, IL- 1, IL-2, IL-6 levels in the pleural fluid as compared to those with non-tuberculosis pleural effusion. There was no statistically significant difference in the IL-10 levels in the pleural fluid between the two groups. The mean IL-4 level in the pleural fluid of tuberculosis patients was 8.4 pg/ml, whereas, the level was not detectable in the pleural fluid of non-tuberculosis etiology. The lowest detection limit of IL-4 assay was 6pg/ml. Patients with tuberculosis pleural effusion had significantly higher IFN- gama and significantly lower IL-10 levels in the blood compared with non tuberculosis pleural effusion. There was no significant difference in the levels of other cytokines. Patients with tuberculosis had higher pleural fluid levels of IFN-gama, IL-6 and IL-10 levels compared to blood. The levels of the remaining cytokines were comparable in the blood and pleural fluid. Sub group analysis revealed that the patients with severe pulmonary tuberculosis had higher TNF- alfa levels (mean 19.4, SD 9.9, n=7) in the blood compared with those with moderately severe disease (mean 6.4, SD 9.9, n=12)(p<0.05). Hwoever, no significant difference was observed between the TNF-alfa levels in the blood between those with severe or mild forms of pulmonary tuberculosis. The levels of other cytokines were comparable in patients with mild, moderate and severe forms of pulmonary tuberculosis. Similarly, the cytokine levels in mild, moderate and severe forms of multidrug resistant tuberculosis and pleural effusion were comparable. The cytokine levels in the blood and bronchoalvelolar fluid and pleural fluid (in patients with tuberculosis pleural effusion) in patients with and without fever, with and without anorexi and with and without weight loss, initial erythrocyte sedimentation rate (ESR>/=20 mm at the end of first hour and <20 mm at the end of first hour,hody mass index (kg/m**2) 18 and nonreactive Mantoux test(5TU) (10mm) were comparable. Patients w2ith tuberculosis pleural effusion who were non reactors to Mantoux test had lower pleural fluid levels of IFN-gama (mean 5.0, SD 7.1, n=10) compared with those who were Mantoux reactors (mean 41.9, SD 84.0, n=18)(p<0.01). Patients with tuberculosis pleural effusion who were non reactors to Mantoux test also had lower pleural fluid IL-1 (mean 8.2, SD 21.4, n=10) compared with those who were Mantoux reactors (mean 35.1, SD 78.4, n=18) (p<0.01)



STUDIES ON MUTATION OF TGFB AND IFNY GENES IN THE PATHOGENESIS OF PULMONARY FIBROSIS  

The study was carried out in 25 proven patients of Idiopathic pulmonary fibrosis (diagnosis based on clinical, radiological, and biopsy findings.). No significant differences were found in the distribution of frequencies of genotypes iin TGF- beta 1 and IFN- gama between patients and controls. Elevated levels of TGF beta 1 mRNA. sera and culture supernatants were observed in IF patients while IFN- gama from same sources was higher iin controls than in patients. These cytokine levels might serve as a useful marker for diagnosis of IPF speically in patients who can not undergo more invasive proceure like TBLB or OLB. A statistically significant difference was seen in Pulmonary function test (PFT), IFN-gama, TGF- beta values at bseline between IPF and control group. The PFT and IFN-gama were significantly low while TGF-beta were high in IPF patients when compared to the controls. Linear correlation was found between FVC and INF-gama and an inverse correlation was found between FVC and TGF- beta among patients during their serial follow up. Anti-cytokine therapeutic approaches may be of value in treating patients IP. However, genetic factors and the stage of the disease may prove to be crucial element in determining if the cytokine modifying therapy is effective in individual patients.



STUDIES ON THE ROLE OF virS GENE IN THE PATHOGENESIS OF MYCOBACTERIUM TUBERCULOSIS  

We had earlier identified a gene of M. tuberculosis ( encoding a 38kDa protein having homology with virulence regulating proteins from several other bacteria) and had shown its presence exclusively in the members of MBT complex (now assigned the name virS(Rv3082c). Based on our earlier work, the VirS proteins has been described as a putative virulence regulating protein. The present study was aimed at evaluating the role of VirS in the pathogenesis of .tuberculosis. Activity of VirS promoter was monitored in the culture medium and inside the mouse macrophages by employing a gfp based promoter probe vector. The virs mutant of M. tuberculosis (Mtb delta virS) was developed by homologous recombination and disruption of virS was confirmed by southern hybridization and immunoblot analysis. Furthermore, we observed that disruption of virS resulted in altered colony morphology and cell wall ultrastructure of M. tuberculosis. Another proof of involvement of cell envelope emerged from the observation that when exposed to SDS detergent, the virS mutant exhibited much more pronounced effect on the survival as compared to the parental strain. Studies on the role of virS as transcriptional regulator showed that it positively regulating its own synthesis. Besides, the induction of mymA operon at acidic pH and in macrophages as well as the acidic tolerance response exhibited by . tuberculosis is dependent on the presence of VirS. The disruption of virS impairs the ability of M. tuberculosis to survive in activated macrophages but not in resting macrophages suggesting the importance of VirS in protecting the bacterium against harsher conditions. Infection of guinea pigs with virs mutant and the parental strain as compared to the parental strain in spleens but not in the lungs of animals at 20 weeks post infection. It was further show that virS mutant of M. tuberculosis exhibit reduced content and altered composition of mycolic acids along with the accumulatiin of saturated c24 and c26 fatty acids as compared to the parental strain. The alteratioins in the cell surface of Mtb delta virS strain was furterh substantiated by the HPCL profiles of mycolic acids from the mutant and the parental strains. Furthermore, mutant produced less mycolic acids in comparison to the parental strain as analyxzed by TLC. These finding suggest that the observed alterations in the cell cell wall ultrastructure results from teh altered mycolic acid compostion. On exposure to acidic pH, the reduction in mycolic acids synthesis was markedly more prominent in the Mtbdelta virS strain in comparison to the parental strain. The accumulation of fatty acids (C24:0/C26:0) at acidic ph was also observed to be higher in the mutants as compared to the parental strain. The emergence of new mass peaks corresponding to C88-C92 chain length of mycolic acids(1328,1356 and 1384) in the synthesis of these mycolic acids at acidic pH. The involvement of VirS in the persistence of M. tuberculosis together with its role in maintaining appropriate structure of cell envelope to resist antitubercular drugs indicate that precise targeting of virS or mymA operon gene products may increase the the effectiveness of combination chemotherapy and impede the mechanisms involved in the persistence of M. tuberculosis.




UROLOGY
INSIGHT INTO PATHOGENIC MECHANISMS IN ANTITUBERCULOSISDRUGS (ATD) INDUCED HEPATOTOXICITY  

The important observation of sur study were occurrence of increased oxidative stess in the liver as well as in its mitochondrial fraction, associated with mitochondrial premeability alterations and increased apoptosis of the hepatocytes was an important mode of liver cell injury in INH-RMP induced hepatotoxicity and increased cell death could be modified by altering the degree of oxidative stress in the liver particularly in the mitochondria. Pronounced oxidative stress within the mitochondria in liver of mice treated with INH-RMP caused increase non protein and protein thiols oxidation. The mitochondruial pool of GSH is critical to maintain the functional competency of the organelle and for cell survial {13}. A decrease or increase of mGSH sensitizes or protects cells from oxidant mediated cell death in the absence of cystosolic GHS changes (14,15). Results of our present study had shown incereased GHS deplection as well as oxidation in the liver of mice, co-treated with INH-RMP. Such alteration of hepatic GSH under these circumstances could be either due to its consumptive utilization by the drugs reactive metabolities or an inability of the GSH synthetic mechanery in the liver to cope up with the increased demand of the synthesis or both, resulting in the observed imbalance in GSH hemeostaisis. Alteration of hepatic HS content either by methionine or hporone supports the importance of GSH in preventing INH-RMP hepatotoxicity. An important event in initiation as well as prepetuation of mitochondrail injury is MPT. Altered MPT due to AT drugs as demonstrated in the present study is correlated with induction of oxidative stress in the mitochondria. Augmentation of mitochondrial GSH by methioine pretreatment pretreatment prevent the onset of MPT and thus protects the liver cell from injury in INH/RMP hepatotoxicity. Mictochondria regulate cell death either by apoptosis or necrosis. We found in the present study of INH-RMP hepatotoxicity that significant increase in apoptosis of hepatocytes occurred along with mitochondrial changes that we have mentioned here. We also demonstrated that phorone treatment before INH-RMP administration caused increased coourrence of hepatic necrosis. Phorone caused profound GSH deplection, as reflected by a marked decrease in hepatic as well as mitochondrial GSH. Profound GSH depletion is known to be lethal by impairing the denense against endogenous H2O2 produced in mitochondria leading to necrotic cell death (16). Moreover caspase activation also depends on a threshold GSH level within the tissue(17). In summary, we demonstrate a critical role for mitochondrial preturbations oxadative stress permeabilit transition that underlie hepatocyte injury in INH-RMP hepatotoxicity. These changes were modifiable by modulating hepatic GSH levels and were more pronounced with hydrazine, suggesting the phenomenon to be a metabolite mediated event.



SCREENING FOR EARLY DETECTION OF ASYMPTOMATIC, RENAL DISEASEIN SCHOOL CHILDREN  

Urine samples of 17391 asymptomatic school children (9580 boys and 7811 girls) aged 5-14 years were analysed for detection of proteinuria, haematuria, pyuria and aminoaciduria. Attempts were also made to detect asymptomatic renal disease through ultrasonography, urine culture, etc. Children who had proteinuria, haematuria, pyuria, etc. in the first urine analysis were evaluatedat monthly interval for 2 successive months. The general incidence of asymptomatic proteinuria was 4.2 per cent on firsttest, 1.77 per cent on the second and 1.61 per cent on the third examination; the incidence in boys and girls being 3.57, 1.45, 1.39 and 4.97, 2.16 and 1.88 per cent respectively on first, second and third examination. Proteinuria was more than traces in 0.99, 0.46 and 0.58 per cent of boys and 1.81, 0.93 and 0.70 per cent of girls in each test respectively. The degree of proteinuria was more in older age group children. The urinary protein/creatin- ine ratio showed significant proteinuria in 150 (53.57%) and massive proteinuriain 5(1.79%) of proteinuric children. Seventeen children showed significant pyuria on the first and 3 each on the second and third examinations. Urine culture was found positive in 4 of the 17391 children; of these three were posi-tive for Escherichia coli and one for Klebsiella. Five children had microscopic haematuria on the first, one on the second and two on the third examinations. Two children were found positive for alkaptonuria. Renal ultrasonography showed abnormality in 11.41 per cent of asmyptomatic proteinuric children. The abnormalities were renal parenchymal changes in 3, hydronephrosis in 4, calycial dilation in 10, renal calculi in 2, renal cyst in 1, duplex kidney in 1, mega pelvis in 2 and foetal lobulation was seen in 5 children. Two children had a contracted kidney with posterior urethral valve and multiple renal calculi and bilateral parenchymal changes in one each. It is concluded that screening for asymptomatic renal disorders is easy and cost-benefit ratio is very high. It can thus be included as a routine test for all school children during their annual medical test.



EVALUATION OF CITRATE THERAPY IN MANAGEMENT OF OXALATE LITHIASIS WITH SPECIAL REFERENCE TO RENAL TUBULAR ACIDOSIS (RTA)  

Evaluation of citrate therapy in the management of oxalate lithiasis with special reference to renal tubular acidosis. A total of 181 (164 male and 17 female) radiologicaly proven renal stone formers (SF) and 30 normal subjects with no history of renal disorders were evaluated for total distal renal tublar acidosis (dRTA) defects by acid challange (150 mg ammonium chloride/kg body weight) test. Potassium citrate therapy in the management of stone formers with dRTA defect and recurrent calcium oxalate stone formers with hypocitraturic defect, was also evaluated. Nephrolithiasis had occurred for the first time in 120 patients while in 61 it was recurrent. dRTA defect was found in 3.3 percent normals and in 30.3 percent SF; only one SF had complete dRTA defect. The prevalence of dRTA was 28.6 and 47.1 percent in males and females respectively, in the first episode SF, dRTA was found in 29.0 percent males and 61.5 percent females while in recurrent SF, dRTA was found only in males (28.1%). In normals and SF without dRTA defect a progressive fall in the urinary pH was seen after acid (ammonium chloride) loading. The initial pH in SF was slightly higher than in normal subjects. In SF with incomplete dRTA defect, the pre-acid load pH was higher than in those without dRTA defect and it did not fall below 5.0 after acid load. In the only patient with complete dRTA the urine did not become acidic even after acid load. No difference was observed in urinary creatinine excretion in normals and various groups of the SF. However, calcium excretion increased after acid load in both group of subjects; the calciuric effect of acid load being greater in SF without dRTA defect. A progressive decrease in the excretion of citric acid after acid load was obsered in normals and SF without dRTA defect. Comparatively the hypocitraturic effect was milder in SF with incomplete dRTA defect. No significant differences was observed in pre-load ammonium excretion in normals and different groups of SF. Increasing pattern of ammonium excretion after acid load was observed in all the subjects. No significant differences were observed in the acid base status of normals and SF and in patients with the first and recurrent episodes of nephrolithiasis. The base excess tended to be lower in recurrent SF. After acid load the bicarbonate, base status and base excess of extra cellular fluids reduced in SF with and without dRTA defect. Potassium citrate therapy for the management of renal stones was evaluated in 58 stone formers (32 first episode and 26 recurrent). Urine chemistry (24 h sample) was studied pre therapy and 1,3,6 and 12 months after therapy. Urinary pH increased progressively during therapy. Calcium excretion decreased significantly and reached normal levels after one year in all hypercalciuric patients. Hypocitraturia was also corrected in all the stone formers. Twenty four h urine output, creatinine, magnesium, inorganic phosphorus, uric acid and oxalic acid remained unaltered during this period. Serum chemistry and acid base status also remained unaffected. Recurrence of the disease was not observed in any of the patients during one year of therapy. The results of the study thus indicate that dRTA defect is a significant factor in the etiopathogenesis of urinary calculi. Recurrence does not appear to be related to this defect. Potassium citrate therapy was found to be an effective remedy for aleviating hypercalciuris and hypocitraturia.




VECTOR BIOLOGY AND CONTROL
ASSESSMENT OF RISK FACTORS IN UROLITHIASIS  

A hospital based case control study was carried out on 300 patients (222 males and 78 females with a mean age of 37.7 year) suffering from urolithiasis and an equal number of normal healthy individuals mathched for age. Sex and socio-economic status to assess the risk factors associated with urolithiasis. Patients of urolithiasis with any other chronic disease were excluded from the study. A semi structured questionnaire was administered to all study subjects to elicit information on social factors (religion, occupation, education, income, residence) and personal (smoking and drinking) and dietary habits. Eighty one (27%) patients were from rural and 219(73%) from urban areas. Sixty five, 32.7 and 19.3% patients suffered from kidney, ureter and bladder stones, respectively. Of the 300 patients, 19.3% had a histroy of repeated urinary tract infections, 18.6% had past history of urinary tract infections and 13% had a family history of urolithiasis. One hundred fifteen (38.3%) patients were vegatrian, 163(54.4%) non vegetarian and 22(7.4%) eggitarian. The analysis of the data by calculating odds ratio ( an estimate of relative risk) indicated that the risk for urolithiasis was about 2 tmes higher for smokers. Similarly a 3 fold risk was observed for those who consumed alcohol. It was observed that the patients who consumed eggs had nearly 2 fold risk of urolithiasis than those who did not consume eggs. Similarly a 2 fold risk of urolithiasis was observed in patients who consumed flesh food twice or even less than twice per week compared to vegetarian. Consumption of rajma more than twice a week also resulted in 2 fold higher risk of urolithiasis. It was thus concluded that urinary tract infections, smoking, alcohol intake and consumption of egg, non vegetarian food items as well as rajma were associated with increased risk of urolithiasis.



MOSQUITO CONTROL PROGRAMME IN RURAL AREAS OF DEHRADUN VALLEY : RECONNAISSANCE, HOST-SELECTION PATTERN & BIOLOGICALCONTROL  

Study was conducted to make a survey of the occurrence of anopheline species around peri-urban areas of Doiwala and Sahaspur in Dehra Dun valley to make a survey of the mosquito breeding sites alongwith its physico- chemical characteristics to study the feeding efficiency of certain larvi- vorous fishes under different ecological conditions and to study the biolog- ical control strategies by using larvivorous fishes nymphs and water bugs. 1. A survey on Anopheline mosquito fauna of the valley revealed the availability of 13 species of 'Anopheles' viz. 'An. culicifacies, An. subpictus, An.annularis, An.fluviatilis, An.stephensi, An.aconitus, An.gigas, An.vagus, An.maculatus, An.splendidus, An.pulcherrimus, An.jeyporensis and An.minimus. 2. Highest combine density of Anophelines recorded from cattle sheds and An.culcifacies showed low prevalence in the human dwellings. 3. Abiotic factors such as temperature, wind velocity and rainfall have pronounced effect on the pattern of seasonal fluctuation in the population density of Anophelines. 4. Evaluation of Premise Index, Bretau Index, Larval density Index, Receptacle Index and Inspected Receptacle Index in the treated and untreated areas of the Valley. 5. Water temperature and pH were found to be the potent factors which fluctuate the density of mosquito larvae. 6. Culture of 'An.culcifacies, An.stephensi' and 'An.annularis' was performed under laboratory conditions. Breeding of 'An. stephensi' was found in peak numbers in association with 'An. subpictus' under natural conditions at Doiwala and the breeding of 'An.fluviatilis' was found in association with 'An. subpictus' at Sahaspur. 7. Fish fauna of Doon Valley revealed the availability of 25 species of fishes. Laboratory experiments on the larvivorosity tests of indigenous fishes with those of standard anti-larval fishes under different ecological conditions suggest that the rate of feeding was higher or more or less equal in 'O.goromy' and 'B.rerio' with those of 'G.affinis' and 'P.reticula- ta.' 8. Mass culture of the Notonectid bug 'Anisops' sp. was perforemd under laboratory conditions. The experiments showed that in a heavily larval infested environment the bug could only reduce the population of larvae but not completely control them. 9. Laboratory experiments on the larvivorosity tests on odonate larvae ('servilia' and 'Bradinopyga' sp.) suggest that the larvae can feed on an average of 100 IV instar larvae and pupae per hour in 'C.servilia' and




VENEREOLOGY
SUSCEPTIBILITY OF CULEX MOSQUITOES IN MYSORE CITY TO PYRETHROIDS  

The study was carried out to test the susceptibility of Japanese encephalitis (JE) vectors of Karnataka to synthetic pyrethroids, organo- phosphate and organochlorine insecticides. Both adult and larval population of Culex tritaeniorhynchus, Cx. geli- dus and Cx. fuscocephala, from Mysore and Mandya districts of Karnataka were tested for their susceptibility to insecticies. Adult susceptibility tests were conducted with propoxur (0.1%), DDT (4%), malathion (5%), deltamethrin (0.025%) and cyfluthrin (0.05%). Deltamethrin was found to be the most effective adulticide as it produced 100 per cent mortality in 15 min. DDT required 120 min to kill over 90 per cent mosquitoes compared to 30 min by malathion and cyfluthrin. Cx. tritaeniorhynchus was found to be more tolerant compared to the other two species. The larval tests revealed differential tolerance of the vectors with regard the LC50 values of insecticides tested. LC50 values for cypermethrin, deltamethrin, fenthion, fenitrothion, temephos and malathion against larvae of Cx. tritaeniorhynchus were 0.000254, 0.000424, 0.039472, 0.075500, 0.025930 and 0.061908 respectively, compared to 0.000063, 0.000063,0.002343, 0.011647, 0.001868 and 0.044426 respectively against larvae of Cx. fusco- cephala. Results revealed high tolerance against insecticides in Cx. tri- taeniorhynchus during larval stage also. Experiments conducted to compare the effects of continuous and inter- mittent exposures with cypermethrin showed that multipulse exposures with cypermethrin showed that multipulse exposure of two, 1 h duration exposures with a 6 h insecticide free period was more effective compared to continu- ous 2 h exposure in all 3 vector species tested.




VIRUS DISEASES
ROLE OF CHLAMYDIA TRACHOMATIS IN PELVIC INFLAMMATORY DISEASEDETECTION OF ANTIGEN AND ANTIBODY BY ENZYME IMMUNOASSAY 

Out of the 335 patients of PID, C.trachomatisantigen was present in 35.22% of cases and chlamydial antibodies were detected in 56.11% of cases. An increase in the titre of antibodies was observed in 40%; of the cases. 80.43 patientscould be treated with Doxycycline. Out of the 19.57% failures. 17.12% could be treated by the second line of therapy. Amongst the 225 infertile patients chlamydial antigens was present in 22.66% and antibodies were present in 34.22% Rising titre of antibodies was seen in 41.66%. ERadication of the organism was achieved in 83.33% by therapy. Two of the infertile patients conceived within eight to thirteen months of therapy. Considering the number of years that these patients underwent treatment for infertility, pregancy was a very rewarding outcome. It was sen that 6% of the controls harboured the antigen and 30 of controls had antibodies in their sera but the antibodies were neither in a high titre nor did they show a rising titre. This study therefore highlights the fact that a large number of cases of PID and infertility in Delhi are caused by C.trachomatis. Early diagnosis and treatment can prevent these sequelae in a large majority of cases. Hence detection of chlamydia should be included in the routine investigations of all cases suffering from lower genital tract infection.



HBsAg SCREENING IN BLOOD DONORS - A HEALTH SERVICE  

Different centres were assigned different responsibilities. Centres at AIIMS was given responsibility of training personnel from other centres in MICRO ELISA test, taking care of quality control needs and also to procure- ment and supply kits required to carry out the study. The centre in Bombay was assigned the responsibility of creating a panel of approximately 3000 sera and undertake an evaluation of ELISA and various RPHA tests by blood banks for HBSAg screening. The three centres at Hyderabad, Madras and Trivandrum were given the responsibility for creating a network for blood banks who will send the sera for HBSAg screening to the centres and to establish a viable system of reporting the testing results within 24 to 48 hours after receiving the blood. The centre at Delhi completed all the assigned tasks of training, procurement and supply of kits on demand to centres and quality control for the ongoing work in other centers. The centre's work clearly demonstra- ted the feasibility and advantages of using such a centralised system for operationalising the screening of donated blood in India. IIH Bombay undertook the comparison of different RPHA kits currently being used in Bombay blood banks for screening blood donors with Abbott's ELISA and RIA for HBSAg. It is evident from this evaluation that the Abbott's ELISA kit was the best as far as specificity and and sensitivity are concerned. None of the RPHA were as sensitive and specific as the ELISA. As part of the HIV screening programme several of the blood banks had been provided with ELISA readers and hence it might be possible for these centres to shift over to ELISA test for HBSAg testing also. Hyderabad, Trivandrum and Madras centres had no problem in standardi- sation of the tests in the laboratory and doing HBSAg testing of samples received in the laboratory. However inspite of repeated efforts it was not possible to ensure that all the blood banks sent the blood samples for screening to the laboratory in time and waited for the report before releas- ing the blood to the recepient. Thus the project demonstrated the feasibility and cost effectiveness of this system. It is noteworthy that this approach has been successfully used to operationalise screening of donated blood for HIV infection.



DEVELOPMENT OF RAPID TESTS FORDETECTION OF ENTERIC VIRUSES IN CONTAMINATED WATER  

Contamination of drinking water with enteric viruses is a major public health concern in our country. Improvement in water quality warrants development of methods to rapidly monitor viruses at low doses. A simple and rapid method for elution and reconcentration of viruses ha s been developed. The method has been used to concentrate upto 50 L of water and reduced sample volumes 10(4) folds using negatively charged membrane filters and urea-arginine phosphate buffer. The method provides higher yields (89-95%) for polio and coxsackie viruses as compared to currently available standard Methods (40%) and thus enhances the sensitivity of virus detection technique for use in environmental samples. A nitrocellulose enzyme immunoassay (NC-EIA) has been developed for detection of polio virus Type I and Coxsackie Virus B1. The procedure detects 200-500 PFU of virus in 10-12 hours as compared to 8-12 days in conventional methods. A luminol - peroxidase chemiluminescent reaction was adapted to NC-EIA with the aim to provide automation for virus quantitation by chemiluminescent enzyme immunoassay (C-EIA) using a Luminescence Photometer developed indigenously at NEERI. The detection limit for luminol was observed to be 25 pmol at pH 10.5 and 25 deg. C. The methodology developed will prove useful for rapid detection of enteric viruses in contaminated water.



CLINICAL SEQUELAE OF JAPANESE ENCEPHALITIS IN CHILDREN AND PERSISTENCE OF JAPANESE ENCEPHALITIS VIRUS INFECTION IN HUMAN SUBJECTS  

Children presenting to the King George's Medical College & Hospital, Lucknow with acute encephalitis like illness were subjected to virological investigation for JE. These included viral isolation from CSF (by intra cerebral inoculation into infant mice) and Haemagglutination inhibition (HAI) test in paired sera. The diagnosis of Japanese encephalitis was considered highly suggestive by viral isolation and / or 4 fold or higher rise in HAI antibody titres in paired sera. Patients in whom the diagnosis of JE was confirmed and were discharged from hospital were followed up at regular intervals by postal appointment. A complete clinical and neurological examination was done at each visit. IQ and psychiatric evaluation was done by standard methods. EEG and audiometry was done in selected cases. In addition, blood was collected for antibody level and reactivation experiments. A total of 68 patients of confirmed JE have been followed up. The period of follow up is variable (1 to 4 years). It was found that 47% had major sequelae in the form of frank mental retardation, frank motor deficits and/or convulsions. 23.5% had minor sequelae such as scholastic backwardness, mild behavioral problems and/or subtle neurological signs only. The remaining 29.4% were completely normal. Japanese encephalitis infection is, therefore not a benign illness but has a high mortality and high rate of disabling sequelae. The 2nd part of the study related to latency, persistence and reactivation of the virus in human subjects. Evidence already exists for the latency & persistence of this virus in mice. The present study provided evidence that the virus may also persist in a latent form in human subjects and can be reactivated by co-cultivation of peripheral blood mononuclear cells with primary mouse embryo fibroblast cultures. The virus could be reactivated in 3 out of 8 asymptomatic children 8 months after primary infection and also in 3 children with recurrent symptoms.



RISK FACTORS FOR HEPATITIS B INFECTION IN PATIENTS WITH CANCER  

330 cancer patients were studied to find the risk factors for hepatitis B infection. Out of 200 patients in the cross sectional study 43 cases were positive for HBsAg and 46 cases were positive for antibody. Maximum number of HBV marker positives were among malignancy of haemopoeitic system, carcinoma breast and carcinoma cervix. Statistically significant factors for the transmission of hepatitis B among these patients were antitumour chemotherapy, blood transfusion and surgery. In the case control study 65 cancer patients with acute hepatitis B were studied along with sex and duration matched controls, ie patients without history of hepatitis B infection. Significant risk factors in the case cont- rol study included antitumour chemotherapy, blood transfusion, surgery and injections with reusable needle. The study shows that apart from the immunosuppressed state, other risk factors also play an important role in the transmission of hepatitis B among cancer patients. Since the burden of illness is high among cancer patients, vaccination of high risk groups (like those preparing for antitumour chemotherapy, those receiving blood transfusions, patients undergoing surgery, and those receiving multiple injections) is supposed to reduce the high incidence of hepatitis B. The other precautions like use of safe disposable syringes, screening of blood for HBV markers before transfusion and proper sterilization of instruments will reduce the high incidence. Early vaccination is the only measure to prevent hepatitis B and its consequences among cancer patients.



PREVALENCE OF HIV INFECTION INPREGNANT WOMEN  

The study was done to determine the prevalence of HIV infection in unselected pregnant subjects. Samples were collected from 3000 subjects. Majority of subjects (80%0 were between 21- 30 years of age and were either primigavidae or second graviade. Amongst the distribution of subjects according to place of residence, it was seen that nearly 75% of the urban subjects were from Chandigarh and 60% of subjects were from rural areas of Punjab. Though Chandigarh has significant rural population, subjects did not seek the care form this hospital. Illiterate subjects were 13.2% in this study and only half this number had illiterate husbands. The subjects were asked about their contraceptive practice prior to planning the pregnancy. Nearly 83% had not used any form of contraception. This could be due to the fact that 43% of these subjects were primigravidae. Most of the subjects who were gravida 3 or more had previous pregnancy losses or less number of livinig children and hance did not use any contraception. Possible risk factors that existed in these unselected group of women were looked for. There was history of blood transfussion in 1.43% of 3000 subjects. Screening was done only in 2780 subjects and in this the history of blood transfusion was present in 29 subjects (1.04%) and only one of these subjects was seropositive (3.44%). In an ICMR report, 1.5% of the seropositive subjects had history of transfusion of blood or blood products. Amongest the 2780 samples tested for HIV antibodies, seropositivity was confirmed in only one (0.036%). This is lower than what has been reported. this study was initiated to determine the prevalance rate in the North India population and whether a routine screening of presence of HIV anitbodies should be recommended. With this low prevalence rate, perhaps a routine screening will not be cost effective at the present moment.



A PROSPECTIVE STUDY OF THE PREVALENCE OF HEPATITIS C VIRUS IN ACUTE AND CHRONIC LIVER DISEASES AND BLOOD DONORS AND RECIPIENTS  

The study was conducted to determine the prevalence of antibodies to hepatitis C virus (HCV) in patients of acute and chronic liver diseases, blood donors and recipients of multiple blood transfusions using second generation anti- HCV ELISA. Only individuals who were serologically negative for hepatitis B virus (HBV) and hepatitis A virus (HAV) infection were studied. These included 100 patients of acute viral hepatitis (AVH), 12 of subacute hepatic failure (SAHF), 43 of acute hepatic failure (AHF), 70 of cirrhosis of the liver, 11 with chronic active hepatitis (CAH), 10 with hepatocellular carcinoma (HCC), 550 blood donors and 63 recipients of multiple transfusions. In 35 of the 100 AVH patients, the disease was due to sporadic NANB infection. Of these, three were positive for anti-HCV. Among 48 patients of AVH whose blood samples were taken only in the acute phase, 20 had NANB infection, but none was found to be positive for anti-HCV. In 52 patients tested serially upto 12 weeks, 15 were found to have NANB infection and three of these were positive for anti-HCV. Five of the 12 patients of SAHF and 23 of the 43 patients of AHF were found to have NANB infection but none of these were found to be anti-HCV positive. Nineteen of the 70 patients of cirrhosis of liver had NANB infection and 2 of these 19 were found to be positive for anti-HCV. Six of the 11 patients of CAH and four of the 10 patients of HCC had NANB infection and of these, 2 patients each were found to be positive for anti-HCV. Of the 550 blood donors, 494 did not show evidence of HAV and HBV and of these only 11 were positive for anti-HCVV. Of the 63 recipients of multiple blood transfusions, 46 did not show HAV and HBV infection. Of these, 24 were found to be positive for anti-HCV. It is thus concluded that in patients of acute liver diseases, screen- ing of HCV antibody should be done serially upto 12 weeks. Patients with cryptogenic chronic liver disease and recipients of multiple blood transfusions should be evaluated for HCV infection.



PREVALENCE OF HEPATITIS C AMONG BLOOD DPONORS AND PERSONS WITH CHRONIC LIVER DISEASE  

The prevalence of hepatitis C Virus (HCV) was assessed in 100 consecutive voluntary blood donors, 100 patients with chronic liver disease and 12 patients with hepatocellular carcinoma. Six per cent of blood donors had anti-HCV. Foruteen blood donors had alanine amino transferase (ALT) levels more than 45 IU/1; of these 5 were anti-HCV positive, indicating a correlation between high ALT and anti-HCV positive. Three of the blood donors were HBsAg positive; one of whom was also anti-HCV positive. Prevalence of anti-HCV in patients with chronic liver disease was 17 per cent. Ninteen patients were positive for HBsAg; of these 3 were anti-HCV positive as well. A total of 35 patients gave history of previous blood transfusions; among them 7 were anti-HCV positive. Of the 47 cirrhotic patients with history of significant alcohol consumption, 8 were positive for anti-HCV. There were 4 cases of Wilson's disease; none of whom had anti-HCV antibodies. One patient of haemochromatosis with previous history of blood tranfusion was positive for anti-HCV. Three of 18 patients with chronic liver disease with no known cause, were anti-HCV positive. One of the 12 patients with hepatocellular carcinoma was anti-HCV positive; neither of the 2 HBsAg positve patients of hepatocellular carcinoma had anti-HCV antibodies. The results suggest that anti-HCV testing combined with donor screening will substantially reduce the risk of post-transfusion HCV infection.



SEROSURVEILLANCE FOR HIV INFECTION IN PREGNANT WOMEN  

The study population was mainly yound women Para O or Para 1 in their 205. Most couples were educated upto secondary shcool, most of the men were in skilled or semiskilled jobs. About 1/4 of the women were gainfully employed outside the house. Sexual history espicially history of extra or premarital sex was not taken as it is not a part of routine antenatal care. It is also difficult to get reliable history on these points from antenatal women. However Hbs Ag and VDRL were used as markers to assess prevalence of other STD in this population. The prevalence of both were lower in this study group than other Indian reports. It is therefore not surprising that there were no HIV seropositive individual in this group. The study population in very small and therefore a defenite conclusion could n not be drawn. However, it is possible that the middle income group urban educated segment which formed most the study population are mostly in stable mutually faithful monogamous relationship with their spouses. The practice of casual sex may not be very common in this group as accounted for by a low prevalence of all three STD Hbs Ag. VDRL and HIV. If we can confirm this low STD rates in this segment of the population in a larger study it will be worthwhile to educate this population to prevent spread of HIV. The stable family relationship in this population should be further fostered. Casual sex could be discouraged. Other sagety measures like universal use of aseptic and anti septic precautions during health care measures, use of properly sterilised needles, syringes and instruments for any health care need etc should be fiven wide publicity to this literate and aware population.



A PROSPECTIVE STUDY ON THE NATURAL HISTORY OF HIV AND AIDS AMONG IVDUs AT MANIPUR  

The main objective of the stydy was to investigate factors which are related to unsafe injecting and sexual parctices among injecting drug users(IDU). Focus group discussions were conducted with different community leaders (belonging to different ethnic groups) and church groups in the project area for the community assessmet. Initial assessment of IDUs was conducted in 16 municipality wards of Churachandpur town. One IDU was randomly selected form each ward and intensively interviewed. Qualitative and ethnographic frame of reference was used for the data interpreetation and analysis. According to an available estimate there were about 400 HIV positive HDUs reported in the locality, yet the community leaders feel that HIV infection may not be a significant problem but in the near future it might turn out to be so. They felt that different HIV prevention programmes should run for different risk groups. Awareness programme in the community has to be strenghthened to make substiantial behavioural changes. They emphasise the need for an information centre supported by counsellors in the locality. They were ambiguous about the possiblity of beiong infected from hospital settings and particularly inadequacy of blood transfusion and testing facilities. According to some of the respondents people who are treating facilities. According to some of the respondents people who are treating and caring for the AIDS patients are also at risk of getting the infection. They were not aware of bleach as a cleaning material for injecing equipments. Condom usage according to them, should be promoted and distributed only to selected persons like commercial sex workers. Distributions should be done at a low profilem since some of the redpondents felt that it might promote free sex. They felt that there should be more stress on HIV prevention messages. There should be message to promote use of disposale syrines, test blood before transfusion etc. Regarding distribution of HIV prevention material, most respondents felt that it should be through philanthropic youth organisation, teachers and women associations and voluntary agencies. Lastly about reaching IDUs the community leaders mentioned that initial contact could be made at detox or rehailitation centres and then they could be follow up in the community. Individual appreach through volunteers who could be an ex addict, might be a better approach, the respondents thought. Awareness and knowledge about HIV/ AIDS and its mode of transmission was higher among IDUs than other respondents. They knew a few friends who were already infected. Instead of harassing the IDUs the police should controlled drug peddling, IDUs felt. They also want better access to sterile/ disposable injecting equipment and were quite open about the use of bleach for cleaning injection equipment.



CLINICAL STUDIES ON HIV INFECTION  

The clinical studies showed that the weight loss, fever and diarrhoea were the most common symptons, with tuberculosis as the most prominent secondary infectious disease. Opportunistic infections other than oral candidiasis were infrequent. Reduced lymphocyte cunts, particularly the CD4 subset, anemia, hypoalbuminemia and elevated liver enzymes were frequent laboratory findings. Clinical profile of AIDS patients was found to be not different from the common picture of patients with low socioeconomic and poor hygiene standards presenting with tuberculosis. The epidemiological studies showed that the AIDS epidemic was in the ascending phase with an estimated doubling time of 12 to 14 months. The mean age of men was 33 years and for women 31 years. Among men the primary mode of infectin was h Hertosexual contact with female comercial sex workers(CSW). Among women, the most common source of infection was their husbands. A majorityy of the subjects belonged to low socioeconomic classes. The infection is wide spread, both in urban and rural areas. Regular condom use was reported in less than 8% of subjects. The prevalence among the CSW in Vellore region increased from 30/1000 in 1986 to 500/1000 in 1997. The number of HIV-2 infected persons detected with or withour HIV-I, increased form 2 in 1990 to 24 in 1997. Immunological studies carried out on the CSW have shown that uninfected CSW themselves had abnormal profiles. Flow cytometric investigations done on HIV infected persons and controls showed wide range of results, with low CD4 and high CD8 counts. Among the various subsets B, CD11+56+, CD8+28+ cell counts correlated with progression. Decline in p24 and p17 antibodies was found to be a useful but inexpensive marker for identifying clinical AIDS. The predictors of progression and markers of Th1 and Th2 participation that have been investigated were serum beta-2 microglobulins, IFN-gama, IL-2, IL-4, IL-10 and sIL-2 receptors. The usefulness of the presence of pruritic papular eruptions (PPE) as a marker of HIV infection has also been investigated. Clinical and immunological studies done on children born to seropositive mothers have shown a good correlation between their clinical status and CD4 counts. Presence of PCP among 2 children was documented. In summary, the data generated through the above investigation provided an insight into the clinical, epidemiolog and immunological aspects of HIV related disease in India. Investigations on cildren and non progressors are still in progress. Further detailed studies on immunological and virological aspects are urgently required to understand better the epidemiology and natural history of HIV related disease in our coutry.



DETECTION AND CHARACTERIZATIONOF ROTAVIRUS BY POLYMERASE CHAIN REACTION  

The study carried out on 400 stool samples collected from children (< 5 years of age) with aute diarrhea to determine the senwitivity and specificity of polymerase chain reaction (PCR) for detection of rotavirus and compare it with ELISA. Effects were also made to characterize rotavirus into G genotypes by PCR of gene segment 9 and digonucleotide hybridization and study the intratypic variations within G genotypes by restriction enzymes analysis. The sensitivity of RT- PCR was found to be 100 times higher as compared to ELSIA. The quantitative determination of sensitivity showed that the minimum quantity of genomic RNA of rotavirus detected by RT-PCR was 500 fg which equivalent to 2 * 10**4 virus particles. The presence of inhibitory substances hampared the amplification by RT-PCR. It is therefore necessary to remove these inhibitory substances during extraction of RNA prior to RT-PCR. In the present study CF11 cellulose was found to be highly effective in removing the nonsepcific inhibitors of RT-PCR. G genotyping by RT-PCR followed by oligonucleotide hybridization showed that 86% rotavirus strains form fecal samples could be typed. All the typeable strains belonged to either of the four major ( G1, G2, G3 and g4)/ genotypes. Majority of the strains had G1 followed by G2 genotype; G3 and G4 genotype strains being in very less numbers strains. A few samples had mixed genotype possibly due to mixed infection. The study of Intratypic variations of G1- G4 genotype strains by restriction enzyme analysis revealed that G1 and G3 strains had intratypic variations. However, these variations within the same genotype may not reflect the antigenic variabliity of the strains unless these variations affect the amino acid constitution of one or more of the six variable regions of VP7 protein, known to determine the antigenic specificity of the strain. The intratypic variations found in G genotypes in the present study reflected the variation ovserved in 532-824 bp region of vp7 gene and are based on the restriction enzyme used. It is thus concluded that the use of RT-PCR for detection of ratavirus from fecal samples is very useful in situations where the sample size is very small or the sample obtained eate in the illness when nunber of virus particles shed in stool are very less . Intratypic variations within G genotype may arise due to mutations and these variations may affect the neutralizing epitopes and thus are important to study.



FORCASTING THE ADVENT OF JAPANESE ENCEPHALITIES  

The present study was aied at find out a way to forecast the appearance of the JE antibody in the domestic birds as well as in mosquitoes. Optimum level of viraemia to transmit infection from one to another susceptable host has een experimientally depicted here. The seroconversion in sentinel chicks were studied for consecutive two years. The survivality rate was 65% (156/240) and 88.33% (212/240) seropositivity rate was 3.85% (6/156) and 12.26% (26/212), maximum seropositivity was observed during the month of April for both the year as 10% and 15% in the Ist and 2nd year respectively. The result clearly shows that in the sentinel chicks survivality rate may differ from year to year and seroconversion rate varies widely in consecutive two years, although the time of occurance of maximum s Aeropositivity in sentinel chick is similar. In addition to that the minor peak also correspond to the consecutive year of study. The seropositivity was 2.22% and 6.06% in the rainy season, 6.45% and 25.76% in the winter season and 3.75% and 6.25% in the summer season during Ist and 2nd years of study respectively. The relsut shows clearly that the maximum seroconversion in sentinel chicks are found during the winter season and minimum in the rainy season with a moderate in the summer months. Although other worker differs. The seropositivity was observed during the six months of a year starting from September, peak in October and continued up to May. The level of antibody titre reached upto 1:80 during September, October and November whereas rest of the months exhibited 1:20 titre level including 1:40 titre level in some months. The result thus shows that in addition to the seropositivity the higher level of antibody titre corresponds with human over case incidence. The vector mosquitoes of West Bengal i.e. Culex vishnui was 57(7.12 pmd), 50(6.25pmd) and 61(7.62pmd) during the rainy, the winter and teh summer season respectively. Other related vectors of India were ranged between 2pmd and 5.87pmd. So the maximum vector density is in the summer and minimum is in the winter, indicating that seasonwise analysis of vector density will be of less important than of considering in monthly basis. During the rainy the winter and the summer season the overt cases of JE were 1 and 1, 3 and26, 0 during 1st and 2nd years of study respectively. The study indicating that, maximum prevalence of cases are observed during the winter season and moderate in the rainy but no cases are reported during the summer months. Vector density peaked in the march and in the August. Similarl with peak in vector density in the month of April and September seroconversion was found, interestingly cases were found during September and onwards not during April onwards. So not only the seropositivity but its higher rate and also the level of antibody titre are equally important factors for the occurance of JE cases in an endemic area. The seroconversion and overt cases are positively correlated, similarly seroconversion and vector density are positively correlated and even if the other two i.e. vector and overt cases are also positively correlated. Out of 8 pools inoculated with 0.5 to 6.4 dex unit dosage of antigen, it was found that 0.8 dex was the minimum inoculation dosage for detection of mosquito infection and even if 4.5 dex was required for 100% infectivity of mosquitoes after incubation for certain times of interval. The results shows clearly that experimental viraemia is possible in the case of sentinel chicks. The vector mosquito is able to get infection with certain level of viaemia in chicks.