TUMOUR BIOLOGY

Breast Cancer is most common malignancy among women worldwide and second most common cancer in Indian women. Although overall incidence of breast cancer in Indian women is not as high as in western countries, the incidence of early onset breast cancer cases (<40 years) does not show significant variations between population worldwide, suggesting that greater proportion of breast cancer in Indian population is due to early onset disease than in western population. The breast cancer susceptibility genes, BRCA1 and BRCA2 are thought to account for the majority of Breast/ Ovarian cancer families, and as much as 62% of inherited breast cancer . Data currently available on the mutation spectrum and frequency in these genes have primarily been derived from western population. The contribution of these high risk mutations to hereditary and overall breast cancer burden in the Indian Population has not yet been explored.

Total 152 cases of breast cancer have been registered for this study during the year under report among them 5 (3.2%) are male, 16 (10.52%) are Muslims and 34 (22.4%) have family history of breast/ovarian/other cancers. Study group also include 20 age matched normal controls. DNA from 100 breast cancer patients and five age matched controls has been screened for mutation in BRCA1 gene using exon specific primers (fig 1). Among these 100 patients 25 have family H/O breast/ovarian cancer. Incidence of familial cases was found high in both peaks viz <35 years (29.4%) and >45years(40%). Heteroduplex variants are noticed in 28 patients in exons 1,7,11,13,14,16, 17 and 18. Sequencing is currently under process to confirm the type of mutation/polymorphism present in these patients. Amongst these 28 patients, 8 pateints (28%) have family history of breast and ovarian cancer. Among 34 patients having family history, 8 cases (32%) showed presence of sequence variance in these genes.

Cancer prostate is the most common form of cancer detected among men worldwide and is the second leading cause of cancer deaths. Racial variability in prostate cancer is striking, highest rate being observed among African-American and lowest among Asians. The explanation for this behavior is unknown. The biological behavior of prostate cancer is variable ranging from preneoplastic changes to an innocuous tumor to rapidly disseminating and lethal disease. As far as biological markers for prostate cancer are concerned except serum PSA, no genetic or epigenetic marker has been reported correlating with progression of prostatic neoplasia and hormonal responsiveness. We have planned to study various genetic and molecular biomarkers in different stages of prostatic neoplasia to find out their diagnostic and prognostic value and behaviour of tumor.

(i) Role of p53 tumor suppressor gene in Apoptosis and Cellular proliferaton in neoplastic and pre neoplastic diseases of Prostate gland.

This work was carried under CSIR Senior Research Fellowship (1998-2001) awarded to Dr. Vaishali Gupta.

100 cases of prostate enlargement including adenocarcinomas (60), prostatic intraepithelial neoplasia (25), atypical adenomatous hyperplasia (5) and benign prostatic hyperplasia (10) were studied for immuno reactivity for p53 oncoprotein and correlation was done with proliferation index and apoptotic index. Proliferation index was calculated by studying PCNA expression in tumor cells and apoptotic index was calculated by counting number of apoptotic bodies in 1000 tumor cells using image acquisition software. Immunoreactivity for p53 was found in 36.7% cases of adenocarcinoma and in 20% cases of PIN. All the cases of AAH and BPH were negative.

This study showed significant increase in p53 immuno reactivity, proliferation index and apoptotic index as disease progressed from BPH to prostatic intraepithelial neoplasia (PIN) and then to prostatic cancer. Although significant increase in p53 immuno reactivity was noticed with increase in gleason score, PIN grade, stage and hormone resistance but it was found absent in stage A & B cancers with gleason score less than 3, suggesting that p53 has limited role in promotion of cancer but play significant role in initiation and progression and transformation to hormone resistant and metastatic phenotype.

No significant correlation was found between proliferation and Apoptotic index with Conventional markers viz clinical stage and gleason score, however apoptotic index was found significantly high in hormone resistant cases.

The results in study on role of p53 tumor suppressor gene in neoplastic tranformation of prostate gland have shown that in localized cancers some other molecular mechanism is responsible for posing barrier to proliferation as well as for its release other than p53 mutation. We planned to study the role of cyclin genes viz: p16, p21 waf1, mdm2, p34 cdc2 in progression of tumor cells from G1 to S phase of cell cycle.

(iii) Study of proapoptotic and antiapoptotic genses (p53 and bcl2) in preneoplastic and neoplastic conditions of prostate and their correlation with androgen receptor status and response to hormone therapy.

It has been seen that the mortality in prostate cancer in mainly due to metastasis and its conversion to hormone independent behavior. But the molecular events associated with conversion from an androgen dependent to independent behavior is poorly understood and further there are no definite molecular, cytogenetic and biochemical markers to identify the androgen independent cancer cells. But this change in behavior definitely reduces the disease free survival rates. It is said that during the involution of prostate induced by hormones by means of apoptosis, certain genes are induced and others are turned off. When these tumours acquire hormone independent activity, the hormone manipulations are not effective. Thus it is deemed appropriate to establish the genetic basis of apoptosis and its association with androgen independent behavior . This study has been undertaken to study propoptotic genes bax and caspase 3 amd antiapoptotic genes p53 and bcl2 in preneoplastic and neoplastic conditions of prostate and their correlation with androgen receptor status and response to hormone therapy.

Twenty cases of prostate cancer have been studied for expression of bcl2 (Fig 1), p53 and bax (Fig 2).

iv) Flow cytometric analysis of DNA ploidy from prostate cancer : implications for prognosis and response to endocrine therapy.

Analysis of tumour DNA ploidy by flow Cytometry has been shown to provide additional independent information to prognostic assessment in a variety of malignancies. Several studies indicate that prostate cancer patients with DNA-aneuploid tumours have shorter disease-free and overall survival than those with DNA-diploid tumours. However, it is still controversial whether aneuploidy is an independent prognostic indicator. This study is undertaken to investigate the role of DNA ploidy in prognosis and response to endocrine therapy in prostatic malignancies.

In the management of transitional cell carcinoma of the bladder, chemotherapy and immunotherapy have been widely accepted as an adjuvant treatment to surgical resection. However, tumor recurrence is recorded in 30% of all cases even following adjuvant chemo and immunotherapy and progression is reported in 7% of cases indicating resistance to immunotherapy and chemotherapy in substantial number of tumors. In order to study tolerability, toxicity and therapeutic efficacy of these cytotoxic and immunomodulating drugs on an individual basis, we proposed to study an in vitro model of cultured tumor cells in each patient to be used to monitor the cytotoxicity produced by these drugs individually as well as in combination and utilization of this information in management of these patients.

A research proposal to investigate invitro monitoring of cytotoxicity produced by various chemotherapeutic agents viz doxorubicin, mitomycin C and Cisplatin on cultured cancer cells of patients having superficial TCC, individually as well as in combination of immunomodulating agents BCG and interferon alpha-2b, has been submitted in April 1999 to Department of Science & Technology for financial assistance. Project has been awarded in May 2001. Chemosensitivity has been studied in five cases.

Using an in vitro assay, the current study proposes to differentiate, if possible, those patients with acute leukemia who are likely to achieve complete remission after induction therapy from those who either achieve partial remission or fail to respond to current therapeutic regimes. As a preliminary work for this project, flow cytometric assays are being done for the diagnosis and detection of apoptosis in acute leukaemia (Fig 1-3). The objectives are:

1.Accurate diagnosis and classification of acute leukemia by combining immunophenotyping and flow

2.Determination of survival capacity of the leukemic cells by measuring viable and apoptotic cells.

3.Comparison of 7-AAD, propidium iodide (PI) and Annexin V staining to detect apoptotic cells.

Twenty two patients with diagnosis of acute leukemia by morphological criteria have so far been included in the study.

1.Immunocytochemistry, on air dried smears and flow cytometric based analysis, on fresh peripheral blood / bone marrow aspirates, is being done to differentiate acute myeloid and lymphoid leukaemia (Fig 4A-4B).

2. Survival capacity of the leukemic cells is assessed by flow cytometric determination of viable and apoptotic cell population using 7-AAD, Annexin V and PI staining.

Of 22 patients, 11 patients were diagnosed as acute myeloid and another 11 patients as acute lymphoid leukemia. For flow cytometry, 100 ml of whole blood is incubated with each fluorochrome-conjugated monoclonal antobody (CD 3, CD 5, CD 10, CD 19, CD 33) and analysed after addition of FACS lysing solution. 10,000 events are acquired and evaluated for each sample. Analysis is based on gating of subpopulation of cells by forward scatter versus side scatter. The apoptotic cell population is detected by combining Annexin V with Propidium Iodide staining using two-colour analysis. In addition, the 7-AAD method is also being used to distinguish the regions corresponding to live, early apoptotic and late apoptotic or dead cells.

Diagnostic uniformity has been found in most cases on flow cytometric analysis to distinguish acute myeloid and lymphoid leukemia. A comparison of 7-AAD and Annexin V + PI staining to detect apoptotic cells is being done. Fig (1-3) : Flow cytometric immunophenotyping of whole blood leucocytes in a patient with acute leukemia using FACS lysing solution and staining with flureochrome conjugated antibodies

A high prevalence (upto40%) of lower genital tract infections due to C trachomatis has been reported. There are reports which suggest that chlamydial infection of the uterine cervix can cause cervical dysplasia.Also PID due to C.trachomatis infection has been implicated in ovarian cancer. Control of chlamydial infection could potentially have impact on HIV transmission.There is a need to develop a sensitive and specific non culture diagnostic assay for early detection of C trachomatis infection which would be cost effective in developing countries and does not require strict storage and transport conditions.

This is a DBT funded project for collection of C trachomatis isolates and development of diagnostic assay for Chlamydia trachomatis. During this year, 175 endocervical swabs from symptomatic women with cervicitis/ PID and Infertility attending gynaecology out patient department of Safdarjung Hospital were collected. In addition, 65 urethral swabs and 45 conjunctival swabs were collected from STD clinic and Eye OPD at Safdarjang Hospital . Diagnosis of C. trachomats infection was performed by DFA on smear (Fig 5), cell culture in McCoy cell line(Fig 6) PCR using 517bp (fig 4) plasmid primers. % positivity of C. trachomatis infection in female patients was 45.3% by culture 35.2% by DFA on smear and 43.6% by PCR (fig. 1), in male patients % positivity was 36.3% in culture 31.8% on smear and 37.2% by PCR (fig. 2) and in occular infection % positivity was 27.2% by culture, 22.4% on smear and 28.5% by PCR (fig. 3). The clinical samples from female and male patients and patients with conjunctivitis were isolated passaged and stored in liquid nitrogen. Repository of C. trachomatis isolates is being maintained.

To know prevalent serovar in our region genotyping is being done using PCR and RFLP. For this DNA amplification is being done with MOMP primers i.e., 871 bp and 1100 bp (fig 7) covering variable region of MOMP gene. For production of monoclonal antibody isolates are being passaged to increase infectivity of isolates and Protein estimation is being done. Tumour cell line (SP2O) is also being maintained. Later Balb/C mice will be injected in tail vein with 50 ug/ml of antigen and fusion of spleenocytes with myleoma cell line will be done.

In addition to developing a diagnostic method for C.trachomatis, FISH is being used to detect C.trachomatis in McCoy cell culture and in clinical specimens. For this, flurescein-labelled probes targeting rRNA sequences are being examined under a fluorescence microscope after in situ hybridzation. Samples are being reported as positive when apple green fluorescent Ebs or inclusion bodies (Fig.1) are seen. The specificity of FISH for detection of C.trachomatis is being determined by varying the hybridization and pest hybridization washing temperature. Attempts will also be made to determine the sensitivity of this technique using different fluorophores.

The immunopathogenesis of chlamydial salpingitis in infertile women is incompletely understood and requires a greater understanding of immunologically mediated response for control of chlamydial infection. Cytokine synthesis by infected cells together with persistent antigen synthesis contributes to chronic inflammation, tissue damage and immunopathology. There is a need for better dignostic marker for understanding the mechanisms involved in pathogenesis of scarring during chlamydial salpingitis/infertility.

During the year TH1/TH2 cytokines responses in lower and upper genital tract of chlamydial infertility were studied and compared with those patients who were negative the infection. For this study, endocervical swabs, cervical secretions, tubal aspirates and blood was collected from 12 infertile women undergoing laparoscopy to study cytokines locally and systemic cytokines studied were gIFN IL-2, IL-6, IL-8, IL-10,and IL12. ELISA was done using Opt-EIA Human Cytokine kit (BD Pharmingen, USA). RT-PCR is being standardized on cells from cervical swabs. In addition, combined intracellular staining for cytokines and surface staining for CD4 and CD8 cells was done and cells were analysed on flow cytometry (fig. 1,2,3). Stained cells were viewed on confocal laser scanning microscope (fig. 4). IL-10 was more often present in CT +ve infertile women than CT -ve women locally in cervix (mean, 8.3 pg/ml) and in fallopian tube (mean 8.9 pg/ml) than sera (fig5). Cut off value was 7.8 pg/ml. IFN-n IL-2 were not present in CT +ve infertile women (mean 1.7 pg/ml and 5.9 pg/ml), thereby indicating a failure of protective response due to C. trachomatis infection in infertile women. However, IL-8 and IL-12 which are inflammatory cytokines were present in both CT+ve and CT-ve infertile women locally and in sera. High levels_of_IL-6 were present (21.5 pg/ml )locally in one CT+ve infertile women which may be indication of fibrosis. This study is in progress.

Sexually transmitted diseases pose a significant problem to maternal, foetal and prenatal health. Chlamydia trachomatis infection in pregnancy has been implicated in the aetiology of low birth weight (LBW), premature delivery (PMD) and pregnancy loss. Further treatment of chlamydial infection during pregnancy could reduce the impact of the infection on pregnancy outcome.

Prevalence of C. trachomatis infection in 350 pregnant women was studied by DFA and positivity was found to be 18.8% (fig. 1). Coinfection with other STD pathogens viz.; bacterial vaginosis (BV), Trichomonas vaginalis (TV) and Candida (CA) spp. in the endocervix was found to be 1.7%, 1.7% and 2.0% respectively. VDRL was positive in 0.5% pregnant women

Treatment was given to 17 Chlamydia-positive women and their partners and data on obsteric outcome was recorded in Chlamydia-positive, treated and - negative cases. The mean PMD and LBW in CT+ve treated and CT-ve pregnant women were 33.1 weeks, 2113.3 gms ; 35.5 weeks, 2200.0 gms and 34.4 weeks, and 2250.0 gms (figs. 2&3) respectively. The percentage occurence of still births in C. trachomatis infected pregnant women was 11.5, however, no still birth was found in patients who had taken antichlamydial treatment (fig. 4).

Salpingitis is one of the most significant complications of chlamydial genital tract infection because of its impact on fertility. However, immunopathology of chlamydial salpingitis/infertility in women is poorly documented because of the need of invasive techniques. In this regard, animal models offer substantial advantages. So far, mouse models have been mainly inoculated with MoPn biovar of C. trachomatis, which differs largely from human isolates. There is, therefore, a need to develop a mouse model of chlamydial salpingitis/infertility that is inoculated with a human serovar of C. trachomatis for studies on pathogenesis and basic immune functions.

During the year under report, this project was given clearance by Animal Ethics Committee. Further, C. trachomatis was isolated from the endocervix of a patient with chlamydial cervicitis. It was passed serially in McCoy cells to increase infectivity. After passaging, chlamydiae were suspended in sucrose-phosphate medium containing 10% foetal calf serum and stored in liquid nitrogen for use in these experiments.

C3H mice, clinically free from infectious or contagious diseases were purchased from Cancer Research Institute, Mumbai. After standardization of operative techniques, female mice, 6-8 weeks old (n=8) were divided into 2 groups, viz.: group-I (n=4)-Ia, control & Ib-d, experimental and group-II (n=4)-IIa, control & IIb-d, experimental, respectively for studying the development of chlamydia induced salpingitis in mice. All mice were given a single dose of sub-cutaneous injection of medroxyprogesterone acetate (Depo-Provera, Pharmacia Upjohn), 7 days before intra-uterine (iu) or intra-bursal (ib) inoculation with chlamydiae to synchronize all mice in a state of anestrous. Under Ketamine (Themis chemicals) anesthesia given intra-muscularly, the inoculum was introduced either, directly into the uterus, thus infecting the lower genital tract, or, under the ovarian bursa causing primary infection of the upper genital tract .

For iu inoculation, the uterus was exposed through a small lateral incision over the right ovarian fat pad and the ovary and oviduct were exteriorized. The inoculum (viz: 100 ml of chlamydial suspension or sucrose-phosphate medium) was introduced into the uterine cavity through a 30-gauge needle. The peritoneum was closed with 5-0 absorbable surgical sutures (chromic Catgut) and the skin with a fine silk suture. For ib inoculation, a similar incision exposed the right ovarian fat pad. 10 ml of chlamydial suspension or sucrose - phosphate medium was injected through the fat pad and under the ovarian bursa. Mice of group I(b-d) were given 5x10 ⁵ IFU, 4x10 ⁵IFU and 10⁵ IFU into the right uterine horn while group II (b-d) mice were given 5x10⁴ IFU, 4x10⁴ IFU and 10⁴ IFU under the right ovarian bursa. Control mice of groups-I and II, viz.: Ia and IIa received 100 ml and 10 ml of sucrose-phosphate medium via the iu and ib routes, respectively.

Cervico-vaginal swabs were taken at sequential intervals after inoculation to confirm the presence of C. trachomatis infection in the lower genital tract of mice. These are being screened for chlamydial shedding by EIA. Subsequently, samples of ovary, oviduct and uterus will be collected from both inoculated and uninoculated sides at necropsy for histological comparison of the severity of salpingitis. The study is in progress.

Archival biopsy material from 145 PKDL patients are available at the Institute of Pathology. Study has been planned on 50 cases of PKDL skin lesions. The causative agent L. donovani could be detected is only 50% of cases in routine H&E sections. The aim of this study were chiefly 1) to identify the causative agent Leishmania donovani (amastigote form) in the tissue (2) to characterize the cell population in PKDL skin lesions by immunophenotyping , using appropriate monoclonal antibodies (3) to study the cytokine profile Th1/Th2 at the tissue level with special reference to IL-10, IL-12, at the tissue level with special reference to IL-10, IL-12, TNF-a, IFN-n by immunohistochemical staining procedures.

The amastigote antigen detection in 50 cases have been completed. Immunohistochemical staining (IHC) technique were done on PKDL skin lesions and monoclonal antibody G2D10 (raised against amastigote antigen of lesishmania gerbelli) was used. The amastigotes were seen in 80% of cases (40/50) using IHC stains as against 50%(25/50) positivity with H & E stains (graphs 1). The amastigotes were seen as dark brown spherules of 3-5m size both within and outside histocytes (Fig 1) . Fourteen cases were from pure macular lesions of PKDL. Nine out of 14 cases showed amastigotes with the IHC as against none with H&E stains. The technique however proved useful in cases with a low parasite load. Papular and nodular lesions also showed a greater degree of positivity with IHC stains (Graph 2).

Immunophenotyping of the cellular infiltrate has been done and the results are being analyzed. Identification of the cytokine profile at the lesional level is in progress.

The protozoan parasites of the genus Leishmania are the causative agents of a group of diseases called Leishmaniasis, endemic in more than 80 countries worldwide. Considerable morbidity and mortality occurs in the visceral infection termed kala-azar (KA), which is a significant infectious disease in the developing world and of late in the developed world because of increased international travel and HIV infection. KA is a symptomatic infection of the liver, spleen and bone marrow caused by organisms of Leishmania donovani complex. PKDL is an unusual dermatosis that develops as a sequel of KA, producing gross cutaneous lesions in the form of hypopigmented macules, erythema and nodules. The need to search for cases of PKDL and treat them as a part of kala-azar control programs has been emphasized since PKDL provides the only known reservoir for the parasite in India. Current diagnostic methods for KA and PKDL based on parasite detection (stained smears, culture and histopathology) and immunological methods (DAT, ELISA etc.) have several limitations including low sensitivity and specificity. Diagnostic procedures that are simple, sensitive and specific need to be developed.

1. Evaluation of ELISA for diagnosis of PKDL using crude and recombinant k39 antigen

We have evaluated the potential of ELISA as a diagnostic tool for PKDL. Antigen prepared from promastigotes and axenic amastigotes using parasite isolates that were derived from skin lesions of a PKDL patient were tested in ELISA. In 88 PKDL cases examined, the sensitivity was 86% and 92% respectively with promastigote and amastigote antigens while specificity was approximately 90% with either antigen. Use of rK39 antigen gave a higher sensitivity (94.5%) and specificity (93.%). Fig 1 shows comparison of mean ELISA values with 3 different antigens. End point titers of ELISA with recombinant K39 antigen and crude promastigote antigen were also compared (Fig 2).

Fig 1. Evaluation of ELISA for diagnosis of PKDL using crude and recombinant k39

antigen. Sera were used in 1:200 dilution with k39 and 1:100 for promastigote and

The utility of dipstick test using immuno-chromatographic strips pre-coated with rK39 antigen was evaluated for PKDLdiagnosis. The results showed that this could provide a rapid and accurate method for field diagnosis of PKDL. A total of 89 serum samples of PKDL patients were tested along with 125 controls. The controls included 46 patients with skin diseases such as leprosy and vitiligo, 29 patients with common infections such as malaria and tuberculosis, and 50 healthy persons of whom 20 comes from the endemic area of Bihar. Immunochromatographic nitrocellulose strips, pre coated with recombinant K39 antigen were evaluated for the detection of circulating antibodies to leishmanial K39 in all these serum samples. A drop of serum applied to the strip followed by buffer led to the development of two visible bands indicating presence of anti K39 IgG (fig3). The test was able to detect cases of PKDL with 91% sensitivity while all of the 125 controls examined were negative.

3. Nested PCR for detection of L. donovani in clinical samples of Kala-azar and PKDL

A specific and sensitive PCR assay capable of detecting cases of both KA and PKDL has been developed by us. To explore the possibility of further improvement in sensitivity and/or specificity of the test, attempts are currently ongoing to develop a nested PCR assay.

4. Evaluation of DAT based on antigen derived from axenically grown amastigotes

Direct agglutination tests attempted so far are based on promastigote antigen. We propose to examine the potential of whole amastigote antigen as a tool for DAT for KA diagnosis. Further we plan to develop DAT for PKDL diagnosis using parasites isolated from PKDL patients.

From the epidemiological point of view, the parasite pathogenic to human needs to be identified and their geographic distribution and transmission determined. In an endemic more than one pathogenic species or subspecies may exist. Parasite identification is important for the diagnosis of disease and for the evaluation of new therapeutic regimens. It is proposed to characterize parasite from KA patients originating from hyper endemic and low endemic regions of Bihar by biochemical and immunological methods. These studies will help in better understanding of nucleotide divergence and designing new diagnostic/therapeutic tools.

The isolates from different regions of Bihar were collected amd maintained in in vitro cultures. Isozyme profile and immunological characterization using species specific monoclonal antibody for L.donovani showed that cultures are of same species. DNA was isolated and subjected to species specific PCR based on kDNA region of the parasite . The parasites were characterized as L. donovani . Genotyping studies based on AP-PCR were undertaken and indicated polymorphism within the isolates. Restriction fragment length polymorphism studies using Nco1 and Mva1 enzymes using P-13 (cloned from 28s rRNA) as probe, are underway to examine polymorphism at this locus.

Studies have been initiated to identify and characterize genes that have stage-specific expression in L. donovani. Conventional approaches using differential DNA library screening tend to overlook low copy messages, which have often been shown to code for essential biological functions. It is proposed to utilize arbitrarily primed PCR to identify sequences that are differentially expressed in promastigote and amastigote stages of the parasite. Differentially expressed genes will be cloned and sequenced and there putative function deduced by sequence comparison with known genes. Based on this, genes will be selected for altering their expression using gene disruption in order to understand their biological function.

In order to identify genes that control growth, we have isolated for the first time in the order Kinetoplastida, a gene encoding for centrin for Leishmania donovani. Centrin is a calcium binding cytoskeletal protein essential for the centrosome duplication or segregation. Protein sequence similarity and immuno-reactivity confirmed that Leishmania centrin is a homolog of human centrin 2. Last year we have reported details of cloning and expression of Centrin gene from L donovani. Here we give results of further studies with centrin. The copy number of centrin was determined as I (fig 4a).To explore the possibility of using centrin gene as a tool to differentiate strains/species of Leishmania we carried out Southern Blot analysis using genomic DNA from different

strains of Leishmania digested with NcoI or StuI enzymes and probed with centrin gene (fig 4b). The results showed that DNA from L. major has a NcoI restriction length polymorphism. Fig 5 shows the evolutionary tree from clustal alignment of Leishmania Centrin. Immunolocalization of Centrin showed it to be localized near the flagellar pocket in both the stages ( Fig 6).

Antibodies from Chlamydomonas centrin and human centrins were tested for cross reactivity with either recombinant Leishmania centrin or cell lysates (fig 7) by western blot analysis. Recombinant Leishmania centrin cross reacted with human centrin 2 antibodies but not with other antibodies tested. In cell lysates Human centrin 2 antibody cross reacted with additional centrin band of 25 kDa besides the 17Kda protein. Human centrin 3 and Chlamydomonas centrin 1 antibodies cross reacted with different sizes of centrins in Leishmania cell lysates. Results indicate the existence of more than one centrin isotypes in L. donovani. The implications of the molecular heterogeneity in Leishmania centrin protein remain to be determined.

The level of expression of both centrin mRNA and protein was measured during the different stages of promastigotes and axenic amastigote growth. Northern and Western blot analysis (fig 8) indicated that the level of mRNA and protein, in both the stages, was maximum in the log phase. The levels steadily declined as the parasites progressed from late log to stationary phase cultures. This confirms that the expression of centrin coincides with the growth of L. donovani in vitro.

Further studies are on going for the functional analysis of centrin gene to get an insight into its role in Leishmania growth. Efforts are on to disrupt gene expression in the parasite to provide means to attenuate parasite growth. Such attenuated parasites will be then evaluated as vaccine candidates.

Identification of AP-PCR fragment which has higher expression in amastigotes

Genomic DNAs from different species of Leishmania viz. L. donovani Indian, L. donovani Sudanese, L. infantum, L.major, L.donovani Ethiopia along with our recent lab isolates f L.donovani India were used in analysis using AP-PCR primer 9 (fig 9A). A fragment of 1.4Kb size distinctly amplified in the lab isolate, was cloned in PCR–TOPO vector and used in Northern blot analysis with total RNA isolated from promastigotes and amastigotes (fig 9B). The probe hybridized with a RNA of 1.6 Kb size and the level of this RNA was significantly higher in the axenic amastigotes than in promastigotes. The cloning of full length gene from this fragment is underway for its characterization.

PKDL is a dermatosis in which the causative organism Leishmania donovani resides in dermal lesions. Although KA and PKDL are both caused by L. donovani they differ considerably in their site of predilection. The cause of altered site of predilection could be due to altered immune status of the human host or due to intrinsic differences in the parasite or both. There are no reports on Molecular studies of the parasites of PKDL origin mainly because of difficulties in isolating parasite of PKDL origin. We have developed methodology to isolate and propagate parasites from dermal regions of PKDL patients and have initiated study to characterize PKDL isolates at biochemical and molecular level.

Biochemical characterization of PKDL isolates was carried out in a few by classical approaches such as isozyme profile and reaction with specific monoclonal antibodies. These studies have led to characterization of these isolates as L. donovani. Such studies are ongoing with more isolates. In addition, we propose to carry out sensitive molecular studies to detect polymorphism at DNA level using sensitive probes

It is not known how the biology of parasite of PKDL origin differs from that of L. donovani of KA origin. A correlation between differentiation of Leishmania from promastigote to amastigote form and expression of a variety of genes has been established in several species of the parasite, but never in parasite of PKDL origin. We propose to exploit AP-PCR technique for identification of differentially expressed genes in PKDL isolates on the basis of their expression in different stages of the parasite and comparative expression in PKDL isolates versus KA isolates.

Several isolates of L. donovani were setup and propagated from skin lesions of PKDL patients. These isolates were characterized as L. donovani by a species- specific PCR assay. Studies on molecular analysis of these isolates have been initiated using various AP primers to compare DNA fingerprinting pattern of PKDL isolates with KA isolates and other geographical isolates of L. donovani with a view to identify unique gene fragments in PKDL isolates.

The presence of Estrogen and Progesterone receptors (ER/ PR) has been demonstrated earlier in the testes of rat, monkey and human. However their presence in the testicular spermatozoa were not reported. Our preliminary studies demonstrated the presence of both estrogen and progesterone receptors in the ejaculated human spermatozoa.

Investigations were undertaken in young adult male mice to understand at what stages of maturation the spermatozoa acquire the Estrogen and Progesterone Receptors. Studies with spermatozoa collected from different segments of the epididymis and vas deferens and evaluating the same by fluorescent labeling, indicated that ER and PR could be detected in the sperm head and mid piece of mouse spermatozoa with a higher concentration in the post acrosomal region (Fig.1-3.) of spermatozoa moderate concentrations in the acrosomal region and least or absent in the tail.

The studies revealed that in contrast to ER, the progesterone receptors are present in the entire sperm head . The presence of membrane bound ER/PR was also reconfirmed.

Previous studies carried out in the laboratory indicated the presence of Estrogen receptor (ER) human in spermatozoa, that visible expression of ER was less or abscent in some of the infertile subjects and that there is significant positive correlation between the seminal plasma estradiol concentrations and spermatozoan motility, especially in oligo spermic individuals. Since the samples were obtained from subjects attending infertility clinic of the Safdarjang Hospital, attempts were made to collect the base line data from the spermatozoa of fertile men

Serum and semen samples, from forty subjects whose wives are currently pregnant were collected during their first visit from the women attending ante-natal clinic of the Safdarjang Hospital. The seminal smears were stained for immuno histochemical localization of Estrogen Receptor (ER) and Progesterone Receptors (PR) using specific monoclonal antibodies. The immuno complexes were conjugated with Fluroscene and examined under Confocal microscope. The control smears stained either by following omission of the primary antibody or by staining with non-ER/ PR monoclonal antibodies, did not show any luminescence in any part of spermatozoa. However following immunostaining with specific antihuman ER monoclonal antibodies, the ER response was observed to vary in different spermatozoa, some showing visibly uniform response throughout the head. The ER response was more on the post acrosomal region.

The Progesterone Receptor response was more uniform with nearly all the spermatozoa showing more luminance in the post acrosome region. Comparatively lesser response was observed in the acrosomal region with tail of failing to show any PR response.

It is known that estrogens induces their responses through ER receptors by activations a number of genes / proteins at the different target organs. The presence of different estrogen modulated proteins like PS2, HSP-70, Ubiquitin, Cathepsin-D etc. on the spermatozoa from fertile subjects were evaluated. The evaluation is incomplete at present and will be completed in and their significance / relationship, if any with ER and fertility need to be evaluated subsequently.

Our earlier studies using immunohistochemical techniques indicated very little or absence of estrogen receptors (ER) in the spermatozoa obtained from infertile individuals. The observations were also supported by two other investigators in their preliminary studies indicating the absence of ER/PR in infertile subjects. There are also recent reports suggesting the existence of isotypes for estrogen and progesterone receptors.

In view of these, it may be useful to develop molecular biology probes for understanding the splice variants, if any, in the population and to evaluate their role in diagnosing infertility.

The RNA isolation from spermatozoa and RT-PCR methodologies has been standardized using oligo markers. Further studies using specific primers will be undertaken to evaluate their possible role in the diagnosis of male infertility.

A proposal to develop a Toxicology laboratory at IOP was presented to the Technical Advisory Group on Toxicology, Pharmacology and Traditional Medicine of ICMR. The group has recommended utilization of histopathology expertise of the Institute for Toxicology Studies.

A project proposal entitled “Establishment of a Reference Laboratory for undertaking hematological, Clinical Biochemical, Histopathological and Immunopathological evaluation of Pre-Clinical and Clinical Toxicological Studies” has been prepared and has been submitted to ICMR – TAG for extra mural funding.

INDIAN CHILDHOOD CIRRHOSIS

EXPERIMENTAL MODEL OF ICC

Back

Scientific staff : Dr. S. Sriramachari

Dr. Jagjit Kaur Sindhu

Dr. A. K. Jain

Duration : 1994 - 2002

BACKGROUND

This project had been initiated as a follow-up study of ICMR National Multi-Centric Collaborative Study on ICC. Trace Element Studies in ICC as part of INSA Senior Scientist Award work by Dr. S. Sriramachari revealed significant elevation of not only hepatic copper but also hepatic zinc levels. It was hypothesized that such an elevation was a result of Metallothionein response as a consequence of iatrogenic treatment received by mother at home. Accordingly attempts are being made to develop an experimental model of ICC, employing some of the indigenous herbal constituents as previously reported.

WORK DONE

During the year under report, the role of “polysulfides of garlic and asafoetida” and “polyphenols of katha” was investigated in mice maintained at 5-6% level of proteins. At the end of one month, five animals were sacrificed. While the livers were normal in three, one showed hepto-cellular degeneration and another a picture of sub-acute/chronic hepatitis without fibrosis.

In a second group of 4 mice of same age and a slightly higher protein level of 8.8%, two animals were discarded, as they were found dead. The remaining two were sacrificed after 45 days. Specific hepatocellular damage was seen associated with centri-lobular intra-cellular hyaline in one and confluent hyaline masses in the other. Occasionally there was polymorphonuclear infiltration but no eosinophils. However, there was no mesenchymal reaction with mononucleus nor-fibrosis.

Back

AN IMMUNO-HISTOCHEMICAL STUDY ON ICC

Back

Scientific staff : Dr. Jagjit Kaur Sindhu

Dr. A. K. Jain

Dr. K. R. Beena

Dr. S. Sriramachari

Duration : 1999 - 2002

BACKGROUND

This project also had been initiated as a follow-up study of ICMR National Multi-Centric Collaborative Study on ICC and Trace Element Studies in ICC where we found significant elevation of not only hepatic copper but also hepatic zinc levels probably due to Metallothionein response. Last year we demonstrated presence of metallothionein in archival sections of autopsy and some available biopsy samples of ICC and livers samples of rat from our studies on experimental model of ICC.

WORK DONE

No further progress was possible with clinical material during the year under report, since all the available ICC liver biopsy specimens has been exhausted. However, during the year, a few samples of liver tissue from Experimental mice were studied for Metallothionein reaction. It was found to be positive in 2 out of the 7 mice livers.

ENVIRONMENTAL BIOLOGY

USE OF PLACENTAL TISSUE FOR HUMAN ENVIRONMENTAL BIOMONITORING OF INORGANIC AND ORGANIC POLLUTIONS

Back

Scientific Staff : Dr. Arun Kumar Jain

Dr. Jagjit Kaur Sindhu

Dr. Rama Jayasundar*

Dr. Sudha Salhan**

Dr. S. Sriramachari

In Collaboration With : * Department Of Nmr, Aiims, New Delhi And

** Dept. Of Obstetrics And Gynaecology, Sjh, N. Delhi

Technical Staff : Mr. Manoj Sajewal

Duration : 1999 - 2004

BACKGROUND

In view of increasing awareness about changes in environment and resulting environmental pollution due to exploitation of benefits of science and technology and other human activities, this project had been planned to monitor the environmental changes using human placental tissues. Last meeting of Scientific Advisory Committee had approved the project and it was submitted to Ministry of environment in the month of January 2000.

WORK DONE

During the year under report, attempts were made to regularly follow-up the progress regarding funding of the project by the Ministry of Environment and Forest. Around July 2001, it revealed we were informed that the project had been further forwarded to WHO which refused to sanction the funds for the project since the project did not directly involve medical biotechnology. Later it was discussed with the Secretary of the Ministry of Environment and Forest since the scope of the project fell directly under the purview of Ministry of Environment. Based on his suggestion, the project has been resubmitted to the Ministry of Environment and Forest in August 2001. Simultaneously part of the project relating to detection of organic pollutants has been written as another project and submitted to Environment Cell of the Department of Bio-Technology for funding.

Back

MORPHOLOGICAL CHANGES OF PLACENTA IN TOBACCO USERS AND NON TOBACCO USERS

Back

Scientific Staff : Dr. Gayatri Rath*

Dr. Arun Kumar Jain

Dr. Ashok Mukherjee

Mr. Banjeet Bastia

In Collaboration With : *Department Of Anatomy

Lady Hardinge Medical College, N. Delhi

Duration : 1999 - 2002

BACKGROUND

Several studies have demonstrated that nicotine content of tobacco acts like a stimulant for post-ganglionic neurons of sympathetic and para-sympathetic systems resulting in strong sympathetic vasoconstrictions in the abdominal organs and limbs. It has also been noticed intake of nicotine in the form of cigarettes increases the level of carbon monoxide in blood leading to inactivation of foetal and maternal haemoglobin. Further there are reports disorders like pre-eclampsia, decreased placental and foetal weights, increased abortion and rate of neonatal mortality in case of chain smokers.

In India, bidi and hukka smoking chewing of tobacco are quite popular amongst women in rural areas as well as those belonging to lower income group. On the other hand in urban elite and socialite circles there has been in increasing tendency towards cigarette smoking and consumption of pan masala and related products. The middle class and most other category of non-smoker women are mostly exposed to the tobacco smoke indirectly as passive smokers due to smoking habits of their near relations etc.

There is almost no information available on the effects of tobacco exposure on morphological changes in human placenta at microscopic and electron microscopic level in Indian women. Therefore this project has been initiated as an extramural project of ICMR in collaboration with Lady Hardinge Medical College to study the changes in trophoblasts / foetal capillaries and placental barrier in mothers exposed to tobacco and compare the structure with non-smoker mothers

WORK DONE

During the year under report, we have collected a total of 115 samples of placenta (65 from non-tobacco consumers, 10 from active smokers, 25 from passive smokers and remaining 15 from women actively chewing tobacco). All the one hundred and fifteen samples were processed were light microscopy and stained with H& E and Mason-Trichrome Stain. Fifteen controls and five each from three groups of tobacco consumers were processed for electron microscopy and 1µm thick semithin sections were cut and stained with toluidine blue. Only tertiary villi were considered during the year under report.

Light microscopic examination of control cases showed normal architecture of placental villi consisting of syncytiotrophoblast, occasional cytotrophoblast, connective tissue stroma, and the capillaries lined by endothelial cells. Degenerative changes and syncytial necrosis along with increased number of syncytial knots was observed in syncytiotrophoblast of passive smokers. Under EM the important changes seen were as follows:

Active Smoker: Reduction in cytoplasmic organelles, viz., lysosomes, pinocytic vesicles, mitochondria etc. in the apical part of the syncytiotrophoblast. Presence of microfilaments, free ribosomes and rough endoplasmic reticulum in the basal part of the syncytiotrophoblast. Although reported in literature, cytotrophoblastic hyperplasia was observed in only few cases of active tobacco smokers. Irregular thickening of basement membrane could be noticed just below the cytotrophoblasts. The capillaries were constricted and decreased in number. Endothelial cells lining the capillary lumen were observed to be projecting into the lumen.

Passive Smoker: Similar findings were observed in passive smokers also, however, increased number of dilated endoplasmic reticulum in the cytoplasm of syncytiotrophoblast was seen in this group. This group further revealed prominent syncytial as well as stromal necrosis and significantly thickened trophoblastic basement membrane. The endothelial cells were found to be prominent and protruding into capillary lumen giving it a beaded appearance.

The project was aimed at the development of methodology for cultivation of differentiated epidermis from human keratinocyte culture and application of the epidermal sheets in burns patients as autologous graft material.

We undertook a pilot study wherein the growth conditions for in vitro cultivation of human differentiated epidermis were standardized. The standardization work was divided into three phases. They are isolation of keratinocytes, cultivation of epidermal sheets and expansion into secondary cultures.

The isolation of keratinocytes basically consisted of (a) Epidermal separation from dermis and (b) preparation of single cell suspension from the isolated epidermal sheets. The skin biopsy, obtained from patients undergoing plastic surgery, was cut into small bits that were incubated in thermolysin, trypsin and Dispase at 37^o C at various concentrations. The epidermal separation was performed at various incubation time points with two pairs of fine forceps. It was found that incubation at 0.1% trypsin resulted in easy separation of epidermis at our lab conditions. The resultant epidermal sheets were pooled and incubated further in trypsin (0.05%)-EDTA (0.1%) solution followed by mechanical shredding and mincing resulted in cell suspension. The viable cells were counted in a Neubauer chamber after vital staining with trypan blue. With the above conditions a cell yield of 9.4 X 10⁵ ± 2.3 x 10⁵ was obtained per cm² of skin biopsy.

The epidermal cells were plated onto the mitomycin treated mouse fibroblasts (NIH 3T3 cells) at various cell densities. The 3T3 cells were grown in culture conditions as described by Navasaria et al (1994). First of all, an optimal cell density of 3T3 and an optimal concentration of mitomycin C were worked out by plating 3T3 cells at various cell densities and treating the cells with mitomycin C at various concentrations. After the treatment the cells were continued in culture to find out the survival time of the treated cells. It was found that a cell density of 30,000 cells per cm² and a mitomycin C concentration of 2.5 X 10^-4 % (w/v) were optimal to maintain 3T3 for 30 days without further cell proliferation. A freshly treated 3T3 cells were used as feeder layer for plating epidermal cells at 40,000 cells per cm². The culture conditions were similar to the reported method (Green, et al. 1979). The multi-layered differentiated epidermis (Fig.1 & 2) was formed within 8 days depending on the seeding densities.

The cultured epidermis was histologically verified using paraffin embedded &-Hematoxylin-Eosin stained 5 µm section (Fig 3) and Plastic embedded & Toluidine blue stained semithin sections (Fig. 4) by light microscopy.

Figure 1: A differentiated Figure 2: A detached keratinocyte colony (2.5X) epidermal sheet grown in a 25 cm² flask.

Figure 3: A differentiated keratinocyte Figure 4. A Semi-thin section of