Determination of proteins secreted by growing rat decidual cells in vitro.

Proteins secreted by rat decidual cells while growing in culture medium were monitored on gel electrophoresis. It was done by assuming that outgrowing cells in vitro mimic differentiated endometrial stromal tissue in vivo following attachment of embryo. The decidual cells were obtained from implantation swellings of day 10 pregnant rats, maintained in serum free medium and the cell dependent protein profile was analysed after 48 hr on single dimensional polyacrylamide gel electrophoresis (SDS-PAGE). The proteins identified were in molecular weight range of 29, 43 and 66 kDa respectively. The result indicated involvement of the differentiated stromal cells in secreting these low molecular weight proteins.

The 65 kDa protein of Mycobacterium habana and its putative role in immunity against experimental tuberculosis.

Mycobacteria including Mycobacterium tuberculosis and Mycobacterium leprae possess multiple antigens some of which inhibit other anti-mycobacterial immune responses. Whole cell vaccines are not free from these suppressive molecules and may adversely affect the immunogenic response(s). Purified protein components having only immunogenic properties should prove to be superior vaccine(s). Mycobacterium habana, a candidate vaccine for mycobacterial infections has been dissected for analysing its antigenic myriad. A 65 kDa protein of this mycobacterium has been isolated and characterized for its protective and cell mediated immune responses. The protein was isolated in pure form using an isotachophoresis (SDS-PAGE filtration) technique and identified with low molecular weight markers along with mAb using the immunoblot technique. Mab IIH9 has identified a 65 kDa protein in M. habana. This protein has been found to be immunoprotective in mice against M. tuberculosis H37Rv infection. It generates high levels of DTH responses in mice against M. tuberculosis and M. leprae antigens and inhibits migration of sensitized cells under the antigenic influence of homologous and heterologous origin. Possibilities of developing this protein as a subunit vaccine are discussed in this report.

Effect of centchroman and tamoxifen on protein patterns of fallopian tubes of rhesus monkeys, Macaca mulatta.

To study the effect of centchroman and tamoxifen on estrogen-dependent proteins of fallopian tubes of rhesus monkey, these antiestrogens were given with and without estradiol to ovariectomized monkeys. In absence of estradiol, both the compounds induced the synthesis of 130 and 95 K proteins. Concentration of 85 K protein was also increased markedly. These compounds, however, suppressed the estrogen stimulated synthesis of 130 K protein when administered with estradiol. The results show that both centchroman and tamoxifen possess estrogen agonistic as well as antagonistic properties and 130 K protein can be used as a marker protein to study estrogen action and for screening of antiestrogenic compounds in a primate model.

Ancylostoma ceylanicum infection in female hamsters: an observation on altered reproductive function.

Reproductive performances of female hamsters were investigated during Ancylostoma ceylanicum (hookworm) infection. Animals having the highest levels of infection (34.96 +/- 1.11 worms) showed degenerative changes in the reproductive system. Ovaries of infected animals contained a few primary or secondary follicles. On cocaging with males of proven fertility, only 7-8% (80% in controls) of the infected females mated but did not conceive as evidenced by the absence of corpora lutea or implantation sites on day 10 postcoitum. Animals with low worm burdens (5.94 +/- 0.65 worms), however, showed almost normal fertility. The uterine weight bioassay and compensatory ovarian hypertrophy suggest strong suppression of pituitary gonadotrophin contents in infected females. Resorptive effects on the pregnancy outcome of infected female hamsters were also recorded.

Oviductal fluid protein patterns in the rhesus monkey (Macaca mulatta) during the menstrual cycle.

Oviducts were obtained from monkeys on Days 8, 14, 19 and 25 of the menstrual cycle and changes in the pattern of luminal fluid proteins were examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Densitometric analysis after periodic acid Schiff's reagent (PAS) and coomassie blue staining of the gels revealed 85 and 95 kDa proteins only up to Day 14 whereas a 130 kDa glycoprotein persisted up to Day 19 and reached a nadir at mid-menstrual cycle (Day 14). The absence of the 130 kDa glycoprotein in the serum and its presence in cytosolic preparations up to Day 19 suggest that it is of oviductal origin. The 130 kDa glycoprotein is of particular interest since it was present in the oviductal fluid during mid cycle, a period when the oviduct participates in gamete transport, fertilization and embryo development. The conclusion drawn from this study is that the protein profile of monkey oviductal fluid changes during the menstrual cycle.

An improved PVC strip ML-ELISA technique for diagnosis of recent cases of amoebiasis.

Conventional plate ML-ELISA technique for diagnosis of recent cases of amoebiasis was converted into a simple polyvinyl chloride strip multilayer-enzyme linked immunosorbent assay (PVC strip ML-ELISA). This report details the development and preliminary characterization of the PVC strip ML-ELISA method for Entamoeba histolytica antigen detection in human fecal samples. In addition, we evaluated the relative efficacies of the PVC strip ML-ELISA and the conventional plate ML-ELISA methods by screening different categories of amoebiasis patients and other intestinal parasites. A significant correlation was found (P less than 0.01) between the two assay methods with reference to sensitivity and specificity of detecting E. histolytica antigens. However, with reference to operational case and economy, the PVC strip ML-ELISA may be performed even by paramedical technical personnel under field conditions.

Ancylostoma ceylanicum: immunological consequences after anthelmintics therapy in hamsters (Mesocricetus auratus).

Alterations in immunological response before and after chemotherapy were investigated in hamsters infected with A. ceylanicum. Four reference anthelmintics mebendazole, albendazole, levamisole and pyrantel pamoate and one newly synthesized anthelmintic compound 81-470 were used. Drugs in curative doses were administered on day 30 post infection and the humoral response was assessed by counter immunoelectrophoresis and ELISA and cell mediated immunity by delayed type of hypersensitivity reaction. In infected untreated animals the precipitins appeared on day 30 and remained prominent till day 250 post infection. However with ELISA the antibodies could be demonstrated as early as day 3 post infection and peaked on day 40. Delayed type of hypersensitivity could not be demonstrated during the course of infection. All the drugs including Comp. 81-470 were effective in removing the parasites. Precipitin antibodies were only demonstrable till day 60 post treatment. ELISA depicted gradual depletion of antibody titre following treatment with mebendazole, albendazole and pyrantel pamoate. In levamisole treated hamsters the initial fall in serum antibody was restored by day 20 post treatment. With Compound 81-470, immediately after the treatment there was sharp rise in antibodies concentration followed by gradual fall and on day 60 post treatment the titre was still higher than the pretreated titre. Thus the study denotes that effective therapy will bring down immune responses of the host if the drug possess no immunopotentiating action. Therefore the immune parameters may be used as supportive indicator to successful therapy particularly in systemic parasites where parasitic forms are nondemonstrable in excreta or blood.

Multilayer-enzyme linked immunosorbent assay (ML-ELISA) for detection of Entamoeba histolytica trophozoite coproantigen.

Multilayer ELISA (ML-ELISA) method for Entamoeba histolytica trophozoite coproantigen was developed for diagnosis of amoebiasis and its usefulness vis-a-vis microscopic examination analysed for paired specimens from 87 patients from Lucknow and Hyderabad, India. All the stool specimens positive for trophozoites were also found to be positive in ML-ELISA. Out of a total 32 samples positive for cysts in microscopy 21 (65.62%) gave positive reactions for coproantigen. Results on 26 samples drawn from otherwise healthy subjects with negative microscopical picture, 4 were positive for coproantigen. The results of the present study indicate that ML-ELISA test is suitable for early diagnosis of acute amoebic patients for timely treatment.

Effect of long-term centchroman treatment on plasma steroid and peptide hormones in female rhesus monkeys (Macaca mulatta).

Effect of weekly oral administration of Centchroman (3,4-trans-2, 2-dimethyl-3-phenyl-4-(p-(beta-pyrrolidinoethoxy)-phenyl)-7-met hoxychroman) at 1 and 2.5 mg/kg for 12 months on plasma estradiol, progesterone, luteinizing hormone and follicle stimulating hormone of female rhesus monkeys was studied. Centchroman administration at both doses did not disturb the menstrual pattern of rhesus monkeys except for a prolongation of the first treatment cycle. The subsequent cycles were of normal duration. The general pattern of plasma estradiol, progesterone, luteinizing hormone and follicle stimulating hormone was not affected by Centchroman treatment. The basal and peak levels were similar in pretreatment, control and treatment cycles. The results clearly indicate that Centchroman treatment up to 1 year does not affect the hypothalamo-pituitary-ovarian axis in female rhesus monkeys.

Enzyme linked immuno-sorbent assay for detection of antibody activity in female genital tract of immunized rabbits.

A method to assay anti-HCG activity was developed. Polystyrene micro-ELISA plates were coated with HCG and uterine and tubal washings were incubated in the wells. Sera were diluted to equate the protein content with that in the washings and were included in the assay to serve as internal reference standards. Anti-rabbit-IgG (sheep) conjugated to horse-radish peroxidase was added to each well and enzyme activity was monitored using ortho-phenyl-diamine as chromogen agent. Enzyme activity was a direct measure of anti-HCG activity. This method was used to compare anti-HCG activity in the genital tract fluid of rabbits immunized with HCG with that of serum from the same animal. The results of the present study show that at equal protein concentrations, the anti-HCG activity was only 2.64% to 18.73% of the activity present in serum. Thus, it seems that antibody activity in the genital tract of the female rabbits immunized systemically may not be sufficient enough to neutralize the biological function of the antigen needs for contraceptive protection.

Toxic effects of quinacrine hydrochloride in rhesus monkeys.

The toxic effects of intraperitoneal, intrauterine and intravenous administration of quinacrine hydrochloride solution were evaluated in female adult rhesus monkeys (Macaca mulatta). A single intraperitoneal injection of 400 mg and above resulted in the development of toxic manifestations leading to death of monkeys. Intrauterine instillation of 500 mg of quinacrine was well tolerated and did not produce any toxic effects. However, intravenous injection of 100 and 75 mg was lethal to the animals.

Effect of chronic local administration of norethisterone enanthate on the testes of adult rat.

Silastic implants containing crystalline Norethisterone Enanthate (NE) inserted into the epididymal fat-pads unilaterally were found to bring about a drastic reduction (P smaller than 0.01) in the size and weight of the testis six weeks after implant insertion on the side bearing the progestin implant. The ipsilateral (treated side) testis, caput and cauda epididymis and the vas deferens showed either a complete absence of spermatozoa or occasional few immotile and decapitated sperms. In contrast, the smears obtained from the contralateral testis and the corresponding epididymal segments and the vas showed a population of entirely normal and motile spermatozoa. There was apparently no effect on the epididymis as the ipsilateral epididymis was almost similar (weight-wise) to that of the contralateral side. The results suggested an apparently local effect of NE implants inserted into the epididymal fat pads. The feasibility of the approach for long-term fertility regulation in males is discussed.

Chemical occlusion of rhesus monkey oviducts with chloroquine.

Fertility control through chemical occlusion of the oviducts with chloroquine was investigated in rhesus monkeys (M.m.). Chloroquine was instilled retrograde through the tubal ostium after laparotomising the monkeys as a 10, 50 and 100% suspension (w/v) in aqueous carboxymethyl cellulose (CMC) solution which was used as a carrier vehicle. Complete occlusion of the Fallopian tubes at the uterotubal junction and also the intramural portions resulted in 15 days after a single instillation.