Establishment and Characterization of a New Muscle Cell Line of Zebrafish (Danio rerio) as an In Vitro Model for Gene Expression Studies.

A new continuous fibroblast cell line was established from the muscle tissue of healthy juvenile Danio rerio (Zebrafish) through explant method. Fish cell lines serve as useful tool for investigating basic fish biology, as a model for bioassay of environmental toxicant, toxicity ranking, and for developing molecular biomarkers. The cell line was continuously subcultured for a period of 12 months (61 passages) and maintained at 28 °C in L-15 medium supplemented with 10% FBS and 10 ng/mL of basic fibroblastic growth factor (bFGF) without use of antibiotics. Its growth rate was proportional to the FBS concentration, with optimum growth at 15% FBS. DNA barcoding (16SrRNA and COX1) was used to authenticate the cell line. Cells were incubated with propidium iodide and sorted via flow cytometry to calculate the DNA content to confirm the genetic stability. Significant green fluorescent protein (GFP) signals confirmed the utility of cell line in transgenic and genetic manipulation studies. In vitro assay was performed with MTT to examine the growth potential of the cell line. The muscle cell line would provide a novel invaluable in vitro model to identify important genes to understand regulatory mechanisms that govern the molecular regulation of myogenesis and should be useful in biomedical research.

Experimental determination of carbon dioxide evolution during aerobic composting of agro-wastes.

This work aims at optimal composting of agro-wastes like sugarcane bagasse, wood straw and soya husk. A mixture of these substances along with small quantity of food waste as the seed was composted aerobically and carbon dioxide evolved was determined experimentally using a composting system comprising aerobic digester, operating in near-optimal conditions with regard to adequacy of oxygen and temperature in the system. During aerobic composting of agro-waste carbon dioxide is produced due to degradation of different carbon fractions in the substrate. Carbon dioxide production rate, which is a measure of bacterial/fungal activity in composting systems, can be related to various process parameters like different carbon fractions present in the substrate and their reaction rates, progress and termination of compost phenomenon and stabilization of organic matter. This gives a balanced compromise between complexity of mathematical model and extensive experimentation, and can be used for determining optimum conditions for composting.

Isolation of integral membrane proteins of Leishmania promastigotes and evaluation of their prophylactic potential in hamsters against experimental visceral leishmaniasis.

The integral membrane proteins (IMP's) of promastigotes of two virulent strain of Leishmania (L.) donovani Dd8 and Leishmania (L.) infantum LV9 and one avirulent viscerotropic strain Leishmania tropica UR6 were extracted by phase separation technique using a non-ionic detergent "Triton X-114". This detergent is homogeneous at 0 degrees C but divides in an aqueous phase and a detergent phase at above 20 degrees C. The phase partition resulted in solubilisation of hydrophilic proteins in aqueous phase, and IMP's with an amphiphilic nature were recovered in the detergent phase. The strain wise quantitative recovery rates of IMP's were estimated. These proteins were purified by chill methanol centrifugation and used as vaccinogen, alone or in combination with adjuvant against L. donovani challenge in hamster model. Among all the combinations, hamsters immunised with IMP of L. donovani (Dd8) in combination with CFA resulted in 75% parasite inhibition in spleen (P <0.001), however, 61.14% (P <0.001) and 77.60% (P <0.001) parasitic inhibition was achieved in liver and bone marrow respectively as compared to their unvaccinated counter part. Similar combinations with UR6 and LV9 strain inhibited the parasite establishment up to 65.12% (P <0.001) and 66.87% (P <0.001), respectively on splenic site. The specific IgG level against (Dd8 strain) soluble leishmania promastigote antigen was monitored at different stages by enzyme linked immunosorbent assay (ELISA) corresponds to the level of parasitic establishment. Similar observations were made in cases of LV9 and UR6 strains. However, significant lymphoproliferative response to IMPs of Dd8 strain (SI 3.5-4.9, P <0.001) was noticed in all IMP + CFA vaccinated animals. Thus, this study will provide a lead for more manipulative trials to develop a subunit vaccine against the fatal disease.

Metabolic and histological analysis of mesenchymal stem cells grown in 3-D hyaluronan-based scaffolds.

Sheep mesenchymal stem cells (MSCs) were isolated and expanded using the principle of plastic adherence. Their identity as progenitor cells was confirmed by induction along the osteoblastic lineage using osteogenic supplements and observation of calcific deposits by von Kossa staining. MSCs were seeded onto two types of hyaluronan-based cylindrical scaffolds in high concentrations and cultured for varying time points up to three weeks. Culture medium was supplied using the following conditions: statically, on a shaker, by stirring with a magnetic stirrer or by perfusion in a tubular flow circuit. Total cell metabolism was assessed by MTT assay and the quality of cell coverage and matrix formation observed by SEM and histological analysis of thin sections of the constructs. Perfusion culture was established as the most appropriate culturing conditions, with cell metabolism increasing by approximately 300% over three weeks. The coverage of the scaffold surface was very good and the deposition of collagenous matrix was superior in these conditions compared to the, static and other dynamic culture conditions.

Heterogeneity in proliferative potential of ovine mesenchymal stem cell colonies.

Bone marrow biopsies were taken from the iliac crest of 28 individual sheep from three different breeds, ranging in age from 4 months to 8 years and mesenchymal stem cells (MSCs) isolated using selection due to plastic adherence. Cells were cultured in medium that had been selected for its effect on observed MSC proliferation, until populations of greater than 50 million had been obtained from each biopsy. The identity of the isolated cell populations as progenitors of the mesenchymal lineage was verified by deriving both osteoblastic and chondrocytic phenotypes when cultured in osteogenic and chondrogenic medium supplements, respectively. The rate of cell proliferation for each marrow biopsy was measured at each passage and the number of initial stem cells in each sample estimated. There was no statistically significant correlation between the age of the sheep and MSC proliferative potential, or age and estimated initial MSC number. There was no apparent significant difference between proliferation rate and sheep breed and colonies established from frozen cells grew at similar rates to pre-frozen cells. Counter intuitively, there appeared to be a negatively correlated trend between proliferation rate and MSC concentration in the samples. It is concluded that no initial descriptive statistics of the marrow biopsies can assist in estimating the proliferative potential, and therefore the timing of future surgeries, of MSCs sampled for the purposes of tissue engineering.

Prophylactic potential of autoclaved Leishmania donovani with BCG against experimental visceral leishmaniasis.

The prophylactic efficacy of autoclaved Leishmania donovani (ALD) and autoclaved L. major (ALM)--a heterologous vaccine developed against cutaneous leishmaniasis (used as a reference vaccine), along with BCG--was evaluated against L. donovani in hamsters (Mesocricetus auratus). Animals were immunized with triple doses (21 days apart) of either ALD or ALM (1.0 mg) with or without BCG (0.1 mg) and challenged 21 days later with 1 x 10(6) L. donovani amastigotes intracardially. Animals immunized with ALM + BCG and ALD + BCG yielded 94.3% and 86.1% parasite inhibition respectively in comparison to the BCG only and unvaccinated controls. Fifty and 33.3% of the vaccinated animals (ALM + BCG and ALD + BCG respectively) were completely devoid of parasites when tested on day 45 post-challenge (p.c.) and survived till the experiment was terminated. The mean survival of ALM + BCG and ALD + BCG groups (animals harbouring parasites) was longest (168 and 139 days respectively). No significant increase in anti-leishmanial antibody level (ELISA) was noticed in ALD + BCG and ALM + BCG groups whereas it increased progressively in the rest of the experimental groups. The lymphoproliferative responses to PHA and Con A, of the 2 vaccinated groups were comparable to that of normal controls on day 45 p.c. The study suggests that ALD along with BCG can offer substantial protection against visceral leishmaniasis in hamsters.

Establishment of asymptomatic Leishmania donovani infection in Indian langurs (Presbytis entellus) through intradermal route.

Indian langurs, which were previously reported to be highly susceptible, were infected intradermally using variable numbers of promastigotes along with different doses, 1/2 pair, 5 pairs and 10 pairs respectively of salivary gland lysate (SGL). Although, all the monkeys developed mild infection and remained subclinically infected throughout the observation period, which later resolved, none of them could develop the classical disease. No marked antigen specific antibody or lymphoproliferative response was noticed throughout the experimental period. However, a late IFN-gamma response (by day 90 pi.) was demonstrated in monkeys infected with 2 x 10(6) promastigotes +10 pairs SGL. It seems that a single intradermal dose of promastigotes with or without SGLs had a vaccines like effect. Perhaps, multiple frequent inoculations, as happens in the natural situation, may be necessary for the development of full-blown disease.

Successful vaccination against Leishmania donovani infection in Indian langur using alum-precipitated autoclaved Leishmania major with BCG.

Autoclaved Leishmania major (ALM) along with BCG, presently undergoing phase II clinical trial by WHO for its vaccine potential against cutaneous leishmaniasis, has been successfully evaluated in single and triple dose schedules against L. donovani in Indian langurs (Presbytis entellus). Encouraged with the results, another formulation alum-precipitated ALM (provided by WHO) along with BCG has been evaluated in this system. Eight monkeys were vaccinated with alum-precipitated ALM + BCG (1 mg of each per animal) while four were kept as unvaccinated controls. All were challenged with 100 x 10(6) amastigotes i.v. on day 60 post vaccination. Parasitic assessment in splenic tissue was performed on day 45, 90 and 180 p.c. Initially, seven of the eight vaccinated monkeys developed infection (two to six amastigotes per 1000 cell nuclei), which resolved by day 180 p.c., while the eighth monkey had a parasite burden of 14 amastigotes per 1000 cell nuclei on day 45 p.c. and died on day 130 p.c. On the other hand, there was progressive infection in unvaccinated control animals and three out of four died between days 110 and 120 p.c., and one monkey, which had low parasite burden, died on day 178 p.c. Prior to challenge, there was an initial rise in antileishmanaial antibodies in the vaccinated group compared to the unvaccinated control group, which later came down to normal level, while it remained higher in the unvaccinated control group. An increasing pattern of antigen-specific proliferative responses and interferon-gamma level to the two antigens--autoclaved L. donovani (ALD) and ALM--was observed in vaccinated monkeys throughout the experiment. There was a good correlation between parasite burden and IFN-gamma level on days 90 and 180 p.c., indicating IFN-gamma response as a sensitive parameter of immune status. The findings suggest alum-precipitated ALM+BCG as a potential vaccine against visceral leishmaniasis and warrants clinical trials.

Leishmania donovani: cellular and humoral immune responses in Indian langur monkeys, Presbytis entellus.

We have previously reported that disease mimicking human visceral leishmaniasis can be established in Presbytis entellus, the Indian langur monkey, following a single intravenous challenge of 10(8) Leishmania donovani amastigotes. In the present report, infection was assessed in monkeys infected intravenously with a single dose of 10(8) amastigotes (HDA group), three weekly doses of 10(7) amastigotes (LDA group) and three weekly doses of 5 x 10(7) promastigotes (HDP group). Typical clinical infection was established in all three groups with significant parasite load. There was a gradual and sustained rise in anti-leishmania specific immunoglobulin G response, and a severe fall in the lymphoproliferative response to the T cell mitogens PHA and Con A by day 80 post infection (p.i.). The antibody level remained elevated until death in monkeys of the HDA and HDP groups; the T-cell responses showed a recovery prior to death. T-cell responses to leishmania antigen, however, could not be demonstrated in any of these monkeys prior to death. One monkey of the LDA group survived for 155 days and two monkeys spontaneously eradicated the infection. Surprisingly, one monkey of the HDA group also achieved spontaneous cure. In the three monkeys which eradicated infection spontaneously, the antibody level declined to baseline levels on day 180 p.i. with a well demonstrable antigen specific lymphoproliferative response; no parasites could be demonstrated in splenic aspirates by direct examination of culture. These data demonstrate that disease severity may be the function of the total inoculum dose rather than the stage of the parasite and that the immunological responses in the Indian langur model parallel the reported changes in human visceral leishmaniasis. This makes the langur a potentially useful model for the evaluation of candidate anti-leishmanial drugs and vaccines.

Vaccination of langur monkeys (Presbytis entellus) against Leishmania donovani with autoclaved L. major plus BCG.

The protective potential of killed Leishmania major (ALM) along with BCG was evaluated against L. donovani in Indian langur monkeys in single and triple dose schedules. A delayed protection was observed in monkeys after a single dose schedule of ALM (3 mg)+BCG (3 mg) given intradermally 2 months before intravenous challenge with L. donovani. Triple dose schedule each of 1 mg ALM + 1 mg BCG was more effective. The status remained unchanged until the end of the experiment (approximately 8 months). The study indicates that a combination of ALM + BCG may be a good candidate vaccine for exploiting against human Kala-azar.

Metabolic disposition of methyl [5-[[4-(2-pyridinyl)-1-piperazinyl] carbonyl]-1-H-benzimidazol-2-yl] carbamate in hamsters: a study to understand chemoprophylactic action against experimental Ancylostomiasis.

Methyl [5-[[4-(2-pyridinyl-1-piperazinyl] carbonyl]-1H- benzimidazol-2-yl] carbamate (CDRI Compound 81-470) exhibits a long prophylactic action against experimental ancylostomiasis, when given parenterally but not orally. To find out an explanation for such a behaviour, metabolic disposition studies were performed in hamsters using [3H] compound 81-470. Following intramuscular administration, the compound was found to form a depot at the site of injection and to remain there in substantial amount for more than 7 weeks. The compound was fairly distributed in all the organs studied and the presence of radioactivity could be easily detected up to 7 weeks of observation period. The compound was very slowly eliminated from the body and only 38% of the radioactivity could be recovered in the urine and faeces during 14 days. The oral dose, to the contrary, was poorly absorbed and more than 62.8% was excreted in the faeces within 48 hr. Consequently, this dose yielded lesser area under plasma curve. More than 95% of the oral dose was eliminated within a week and hardly and radioactivity could be detected in the tissues after day 14. In accord with this pattern, in blood also the im dose was detected up to 7 weeks while the orally given compound reached undetectable level within 6 days only. The lower clearance and prolonged stay in the body of the im dose compared to quick elimination of the oral dose may be responsible for the long chemoprophylactic action of compound 81-470 when given through im route.

Role of reactive oxygen species in the chemoprophylactic action of methyl [5-[[4-(2-pyridinyl)-1-piperazinyl]-carbonyl]-1H-benzimidazol-2 yl] carbamate in hamster against Ancylostoma ceylanicum.

To delineate mechanisms involved in the prophylactic action of methyl [5-[[4-(2-pyridinyl)-1-piperazinyl]-carbonyl]-1H-benzimidazol-2 yl] carbamate (compound 81/470) in hamster against Ancylostoma ceylanicum infection, plasma level of the compound and status of reactive oxygen metabolites in jejunum at different periods of the drug treatment were examined. The compound was found to enhance the generation of both O2- and H2O2 by the jejunum possibly by activating xanthine oxidase. This stimulation was found to be both time and dose dependent. At 100 mg/kg dose the increase in O2- production could be recorded at least upto 50 days, whereas at 25 mg/kg the stimulation remained effective upto 20 days only, and at 5 mg/kg there was no change in the activity. This correlated well with the reported prophylactic pattern of the compound i.e. upto 45 and 7 days by 100 and 25 mg/kg doses, respectively. Plasma level of the compound also exhibited dose dependent variation. The compound given at 100 mg/kg dose could be detected in significant concentration upto at least 42 days while that given in 25 and 5 mg/kg doses was present in equivalent concentration upto 14 days and 1 day, respectively. It is concluded that the activation of respiratory burst in the jejunum induced by the persistent presence of compound 81/470 may represent one of the important mechanisms for the chemoprophylactic activity of this anthelmintic.

Evaluation of enzyme-linked immunosorbent assay in the diagnosis of kala-azar in Malda district (West Bengal).

An immunological test based on indirect (plate) ELISA has been successfully standardized and modified using promastigote soluble antigen. The test carried out on 813 subjects from a kala-azar endemic area (including parasitologically confirmed patients, subjects presenting with clinical symptoms of visceral leishmaniasis and endemic controls) and a non-endemic area (with diseases other than kala-azar and apparently normal subjects) was found to detect, specifically, antileishmanial antibodies. The plate ELISA has been simplified to a more sensitive dot-ELISA where the results are read within 2-3 h. The antigen requirement is 250 ng per test. No cross-reactivity with sera from patients of malaria, tuberculosis, leprosy, amoebiasis and filariasis was observed. The follow up monitoring of antibodies in successfully treated kala-azar patients showed a decline of antibodies. A drop of blood taken on filter paper is sufficient to conduct the test. Dot ELISA therefore is a simple, inexpensive and stable test in serodiagnosis of visceral leishmaniasis.

Role of reactive oxygen species in expulsion of Nippostrongylus brasiliensis from rats.

To understand the mechanism for the expulsion of Nippostrongylus brasiliensis from rats, age-dependent variations in the metabolism of reactive oxygen species in the parasite and the host intestines were examined. N. brasiliensis showed an age-dependent increase in its susceptibility to xanthine-xanthine oxidase and t-butyl hydroperoxide generated oxidants as well as to H2O2. Protection obtained with several scavengers suggested that the worms were damaged by the combined action of oxidants generated by the in vitro systems employed. The level of superoxide dismutase in the nematode and its release into the surroundings exhibited a marked depression with advancement of age. No such alteration was, however, recorded for catalase and glutathione peroxidase. An appreciable decrease in the level of reduced glutathione in older N. brasiliensis appears to render them prone to oxidant attack. The rat intestines, on the other hand, exhibited an appreciable depression in catalase and a reduced glutathione content with progress of the infection. Vitamin E levels were elevated. The release of O2-. and H2O2 by the intestines was also found to be greater during later stages of the infection. The combined effect of the changes observed in N. brasiliensis and in the rat intestines may be at least partly responsible for expulsion of the nematode from the rats after day 10.

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.

Effect of anthelmintics on the antioxidant system of Nippostrongylus brasiliensis.

To understand the mode of anthelmintic action of thiabendazole and methyl-[5-[[4-(2-pyridinyl)-l-piperazinyl]carbonyl]-1H-benzimidazole- 2-yl] carbamate (C.D.R.I. compound 81/470) against Nippostrongylus brasiliensis, their effect on the metabolism of reactive oxygen species in the parasite as well as in rat intestine was examined. Both drugs produced a significant depression in the levels of superoxide dismutase (SOD) and reduced glutathione (GSH) of the parasite. Release of antioxidant enzymes by the drug-treated worms was also found to be appreciably lowered. Both thiabendazole and compound 81/470 induced a depression in the levels of all five constituents of the antioxidant system of rat intestine but significant alterations were detected only in the GSH content of infected and the SOD activity of normal intestine. The production of O2- by treated intestine was, on the other hand, markedly enhanced. Increased formation of O2- by the host intestine accompanied with the reduced level of SOD and GSH in N. brasiliensis appear to have a deleterious effect on the parasite. Consequently, the drug-treated worms are unable to retain themselves in situ and are ultimately expelled. The greater effect produced on these parameters by thiabendazole compared to compound 81/470 is consistent with the relative efficacy of these anthelmintics.

Efficacy of methyl 5(6)-(alpha-hydroxyphenylmethyl) benzimidazole-2-carbamate, a metabolite of mebendazole, against developing forms of experimental helminth parasites.

Methyl 5(6)-(alpha-hydroxyphenylmethyl) benzimidazole-2- carbamate, a metabolite of mebendazole, was evaluated against metamorphic forms of Ancylostoma ceylanicum in hamsters, Nippostrongylus brasiliensis in rats and cysticercoids of Hymenolepis nana in grain beetles. The test compound offered better action than mebendazole except against H. nana cysticercoids where the activity of the compound and mebendazole was comparable, but was inferior to the standard cestodicidal drug, praziquantel. The results suggest that the action was better by ip route compared to per os route of drug administration.

Effects of methyl [5 [[4-(2-pyridinyl)-1-piperazinyl]carbonyl]-1H-benzimidazol-2-yl] carbamate on energy metabolism of Ancylostoma ceylanicum and Nippostrongylus brasiliensis.

Effects of methyl [5[[4-(2-pyridinyl)-1-piperazinyl] carbonyl] 1H-benzimidazol-2-yl] carbamate (CDRI Comp. 81-470) and mebendazole on the energy metabolism of A. ceylanicum and N. brasiliensis were compared. At 10 and 50 microM concentration both compounds inhibited glucose uptake and its conversion into metabolic endproducts. The shift towards the increased production of lactic acid appeared to be the result of inhibition of PEP carboxykinase and increase in LDH activity. The compounds also caused significant inhibition of ATP production in mitochondria.

Chemoprophylactic action of a substituted methyl benzimidazole carbamate against experimental nematode infections.

The prophylactic action of a substituted methylbenzimidazole carbamate was investigated against experimental nematode infections. The compound at the dose of 100 mg/kg x 1, i.m., protected hamsters against Ancylostoma ceylanicum for about 45 days and at 500 mg/kg x 1 protected rats against Nippostrongylus brasiliensis for 28 days. The uteri of female worms recovered from treated animals were devoid of ova so long the compound had considerable phophylactic action. In case of A. ceylanicum the compound exerted lethal action on L4 stage and in N. brasiliensis, mild action was noticed on L4 which became more pronounced on L5. The reference drugs, mebendazole and thiabendazole, had weaker prophylactic action.