Isolated islets in diabetes research.

This review highlights some recent developments and diversified applications of islets in diabetes research as they are rapidly emerging as a model system in biomedical and biotechnological research. Isolated islets have formed an effective in vitro model in antidiabetic drug development programme, screening of potential hypoglycaemic agents and for investigating their mechanisms of action. Yet another application of isolated islets could be to understand the mechanisms of beta cell death in vitro and to identify the sites of intervention for possible cytoprotection. Advances in immunoisolation and immunomodulation protocols have made xeno-transplantation feasible without immunosuppression thus increasing the availability of islets. Research in the areas of pancreatic and non pancreatic stem cells has given new hope to diabetic subjects to renew their islet cell mass for the possible cure of diabetes. Investigations of the factors leading to differentiation of pancreatic stem/progenitor cells would be of interest as they are likely to induce pancreatic regeneration in diabetics. Similarly search for the beta cell protective agents has a great future in preservation of residual beta cell mass left after diabetogenic insults. We have detailed various applications of islets in diabetes research in context of their current status, progress and future challenges and long term prospects for a cure.

Pancreatic islet-cell viability, functionality and oxidative status remain unaffected at pharmacological concentrations of commonly used antibiotics in vitro.

Environmental factors such as diet, physical activity, drugs, pollution and life style play an important role in the progression and/or precipitation of diseases like diabetes, hypertension, obesity and cardiovascular disorders. Indiscriminate use of antibiotics to combat infectious diseases is one of the commonest forms of misuse of drugs. Antibiotics seem to have a correlation with diabetes and pancreatic function. There are controversial reports about the effect of antibiotics on the pancreatic islets; some suggesting their harmless action, some depicting a beneficial role and others indicating deleterious effect. Moreover, use of antibiotics is mandatory during islet isolation and cultivation to reduce incidences of microbial contamination. It is likely that antibiotic treatment may adversely affect islet viability and its functioning leading to failure of islet transplantation. The present in vitro study was undertaken to examine the effect of commonly used antibiotics such as gentamycin, penicillin, streptomycin, tetracycline, neomycin, erythromycin and chloramphenicol on islet viability, its functioning and induction of oxidative stress if any. The viability and insulin production data showed that none of the antibiotics used in the present study affect the viability and the functioning of the islets at their pharmacological concentrations. Free radical levels measured in terms of melonyldialdehyde (MDA), nitric oxide (NO) and reduced glutathione (GSH) reveal that except for a marginal increase in lipid peroxidation with tetracycline and slight increase in NO levels with streptomycin, none of these antibiotics affect the oxidative status of the cells. Antioxidant enzymes such as superoxide dismutase and catalase remain unaffected after this treatment. Our results reveal the innocuous nature of the antibiotics used at pharmacological concentrations, suggesting their safety whenever prescribed to combat infections and also during islet isolation procedures.

Growth modulation of fibroblasts by chitosan-polyvinyl pyrrolidone hydrogel: implications for wound management?

Wounds in adults and fetuses differ in their healing ability with respect to scar formation. In adults, wounds lacking the epidermis exhibit excess collagen production and scar formation. Fibroblasts synthesize and deposit a collagen rich extracellular matrix. The early migration and proliferation of fibroblasts in the wound area is implicated in wound scarring. We have synthesized a hydrogel from chitosan-polyvinyl pyrrolidone (PVP) and examined its effect on fibroblast growth modulation in vitro. The hydrogel was found to be hydrophilic as seen from its octane contact angle (141.2+/-0.37 degrees). The hydrogel was non-toxic and biocompatible with fibroblasts and epithelial cells as confirmed by the 3(4,5-dimethylthiazolyl-2)-2, 5-diphenyl tetrazolium bromide (MTT) as-say. It showed dual properties by supporting growth of epithelial cells (SiHa) and selectively inhibiting fibro-blast (NIH3T3) growth. Growth inhibition of fibroblasts resulted from their inability to attach on to the hydrogel. These findings are supported by image analysis, which revealed a significant difference (P<0.05) between the number of fibroblasts attached to the hydrogel in tissue culture as compared to tissue culture treated polystyrene (TCPS) controls. However, no significant difference was observed (P>0.05) in the number of epithelial (SiHa) cells attached on to the hydrogel as compared to the TCPS control. Although in vivo experiments are awaited, these findings point to the possible use of chitosan-PVP hydrogels in wound-management.

Bio-artificial liver from cultured human foetal hepatocytes: feasibility and prospects.

BACKGROUND: Conventional therapy for acute liver failure has not been able to improve survival beyond 40%. Apart from liver transplantation, the most promising development in this field is the utilization of cultured hepatocytes to make 'bio-artificial liver support systems' as a 'bridge to transplantation' or ideally as a 'bridge to total recovery'. This study examines the feasibility of culturing foetal hepatocytes without the use of growth factors and formulating a bio-artificial liver support device in our set-up. METHODS: Foetal hepatocytes were harvested from the liver obtained from mid trimester abortions at Armed Forces Medical College and Command Hospital (SC), Pune. The liver was perfused with Phosphate Buffered Saline (PBS) and collagenase type IV and was cut with a pair of sterile scissors into tiny pieces. Cells so separated, were washed with PBS plus foetal calf serum and stirred to disperse the cell aggregates. Filtered cell suspensions were inoculated in polystyrene flasks containing hepatocyte culture medium (MEM E: 75%, M199: 25%, BSA: 0.1%, Bovine Insulin 5 micrograms/ml, FCS: 10%, Penicillin: 10 i.u., Streptomycin 50 micrograms/ml, Hydrocortisone 5 micrograms/ml and incubated at 37 degrees C. The functional capabilities of the cultured hepatocytes were analyzed by studying production of albumin and a foetoprotein. Structural integrity of hepatocytes was assessed by light and electron microscopy. RESULTS: The hepatocyte yield varied from 2 to 60 x 10(6) cells/L with an average of 38 x 10(6) cells/L in the eight consecutive experiments. Initial hepatocyte viability varied from 25% to 90% with an average of 61%. The yield and the viability of hepatocytes were adversely affected by the condition of foetus at birth and use of intra-amniotic injections for inducing abortions. Hepatocyte monolayers and colonies formed in 75% experiments. The cultures could be maintained in incubation without the use of epidermal or hepatocyte growth factors for 2-25 days with a mean survival of 8.9 days. The cells in culture were found to be structurally normal and functionally active and could be cryo-preserved. These hepatocytes were inoculated into a hollow fiber module to formulate bio-artificial liver support device. The cultures ultimately developed either cellular disintegration or bacterial infections despite use of antibiotics in the culture medium. CONCLUSIONS: We conclude that it is feasible to maintain foetal hepatocyte cultures without the use of expensive growth factors for over 8 days. Bio-artificial liver formulated with cultured foetal hepatocytes is now a step closer to clinical trials.

Factors influencing induction and reversal of virus induced diabetes.

Injection of encephalomyocarditis virus -D strain in SJL/J mice leads to development of diabetes. In order to ascertain various factors involved in this process, effect of age of the host, dose of virus and glucose pretreatment on incidence of diabetes and its possible reversal were studied. Blood and urine glucose levels of control and experimental mice were followed for 6-8 weeks to reveal diabetic and reversal from diabetic state. It is observed that incidence of diabetes is directly proportional to the age of the host and dose of the virus, leading to maximum destruction of beta cells and minimum chances of recovery from the diabetic state. Glucose injection prior to low dose virus inoculation reduced the incidence of diabetes and enhanced the process of reversal of diabetes. The data reveal the importance of age of host, dose of virus, metabolic state of beta cells and residual beta cell mass in recovery and reversal of virus induced.