Immunological studies in multiple low dose streptozotocin induced diabetes in mice

1. Multiple low doses of streptozotocin (MSZ) treatment successfully induced diabetes in male TO, MFI and HO lean mice. In contrast however, BALB/c mice failed to develop persistent hyperglycaemia. Single streptozotocin (SSZ) treatment also produced diabetes in TO mice. SSZ treatment however, produced severe weight loss and atrophy of the lymphoid organs. MSZ treatment on the other hand, was not cytotoxic towards lymphoid organs and, whilst there was no loss of body weight, growth rates were reduced in MSZ treated mice. 2. Following sheep red blood cell (SRBC) immunisation of MSZ-treated mice, haemagglutination titres, and numbers of antigen reactive cells and plaque forming cells were all significantly lower than control values. 3. In vitro proliferation of spleen cells in response to phytohaemagglutinin (PHA) and conconavalin A (ConA) was found to be significantly depressed in MSZ treated mice. However, T-lymphocyte responses were intact when the mice were not overtly hyperglycaemic. In contrast, however, T cell independent responses to lipopolysaccharide (LPS) were generally intact throughout the study period. 4. Cell mediated immunity, as assessed by measurements of delayed (Type IV) hypersensitivity, was also depressed in MSZ treated mice. This suppression could be reversed by insulin therapy. 5. Both natural killer cell activity and antibody dependent cell mediated cytotoxicity were found to be significantly increased in MSZ treated mice. 6. Histological examination of the pancreas showed the presence of insulitis, in MSZ treated mice, and cytotoxic effector cells against obese mice islet cells (as assessed by 51Cr release) and HIT-T15 cells (as assessed by insulin secretion) were found to be significantly increased. Furthermore, these effector cells were also found to show increased proliferation in the presence of homogenates prepared from HIT-T15 cells. Examination of the Sera from MSZ treated mice showed that islet cell surface antibodies were present.

The kallikrein-kinin system in relation to retinal vessel tone in the streptozotocin-diabetic rat model

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

The kallikrein-kinin system in relation to retinal vessel tone in the streptozotocin-diabetic rat model

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Nephrin and the Pathogenesis of Nephropathy - Emphasis on Type I Diabetes

End-stage renal disease is an increasingly common pathologic condition, with a current incidence of 87 per million inhabitants in Finland. It is the end point of various nephropathies, most common of which is the diabetic nephropathy. This thesis focuses on exploring the role of nephrin in the pathogenesis of diabetic nephropathy. Nephrin is a protein of the glomerular epithelial cell, or podocyte, and it appears to have a crucial function as a component of the filtration slit diaphragm in the kidney glomeruli. Mutations in the nephrin gene NPHS1 lead to massive proteinuria. Along with the originally described location in the podocyte, nephrin has now been found to be expressed in the brain, testis, placenta and pancreatic beta cells. In type 1 diabetes, the fundamental pathologic event is the autoimmune destruction of the beta cells. Autoantibodies against various beta cell antigens are generated during this process. Due to the location of nephrin in the beta cell, we hypothesized that patients with type 1 diabetes may present with nephrin autoantibodies. We also wanted to test whether such autoantibodies could be involved in the pathogenesis of diabetic nephropathy. The puromycin aminonucleoside nephrosis model in the rat, the streptozotocin model in the rat, and the non-obese diabetic mice were studied by immunochemical techniques, in situ -hybridization and the polymerase chain reaction -based methods to resolve the expression of nephrin mRNA and protein in experimental nephropathies. To test the effect of antiproteinuric therapies, streptozotocin-treated rats were also treated with aminoguanidine or perindopril. To detect nephrin antibodies we developed a radioimmunoprecipitation assay and analyzed follow-up material of 66 patients with type 1 diabetes. In the puromycin aminonucleoside nephrosis model, the nephrin expression level was uniformly decreased together with the appearance of proteinuria. In the streptozotocin-treated rats and in non-obese diabetic mice, the nephrin mRNA and protein expression levels were seen to increase in the early stages of nephropathy. However, as observed in the streptozotocin rats, in prolonged diabetic nephropathy the expression level decreased. We also found out that treatment with perindopril could not only prevent proteinuria but also a decrease in nephrin expression in streptozotocin-treated rats. Aminoguanidine did not have an effect on nephrin expression, although it could attenuate the proteinuria. Circulating antibodies to nephrin in patients with type 1 diabetes were found, although there was no correlation with the development of diabetic nephropathy. At diagnosis, 24% of the patients had these antibodies, while at 2, 5 and 10 years of disease duration the respective proportions were 23%, 14% and 18%. During the total follow-up of 16 to 19 years after diagnosis of diabetes, 14 patients had signs of nephropathy and 29% of them tested positive for nephrin autoantibodies in at least one sample. In conclusion, this thesis work could show changes of nephrin expression along with the development of proteinuria. The autoantibodies against nephrin are likely generated in the autoimmune process leading to type 1 diabetes. However, according to the present work it is unlikely that these autoantibodies are contributing significantly to the development of diabetic nephropathy.

Supramolecular insulin assembly II for a sustained treatment of type 1 diabetes mellitus

Diabetes is a chronic disease requiring continuous medical supervision and patient education to prevent acute secondary complications. In this study, we have harnessed the inherent property of insulin to aggregate into an oligomeric intermediate on the pathway to amyloid formation, to generate a form that exhibits controlled and sustained release for extended periods. Administration of a single dose of the insulin oligomer, defined here as the supramolecular insulin assembly II (SIA-II), to experimental animals rendered diabetic by streptozotocin or alloxan, released the hormone capable of maintaining physiologic glucose levels for > 120 days for bovine and > 140 days for recombinant human insulin without fasting hypoglycemia. Moreover, the novel SIA-II described here not only improved the glycemic control, but also reduced the extent of secondary diabetic complications.

LIBS Based Screening of Glycemic Elements of Ficus religiosa

The effectiveness of various trace element concentrations in medicinal plants in the cure of various diseases can be determined by their quantitative estimation. Elemental concentrations of aqueous extract of F. religiosa leaves were measured by Laser-induced breakdown spectroscopy (LIBS). LIBS is a very powerful and efficient analytical tool for determining elemental constitution. The present study deals with the LIBS-based validation of elements responsible for the glycemic potential of aqueous extract of F. religiosa leaves in streptozotocin-induced diabetic models. The significant decrease in blood glucose level and marked improvement in glucose tolerance test of diabetic models is correlated to the concentration of elements present in the extract as revealed by LIBS spectra. Elements such as Mg and Ca have been observed in the LIBS spectra of F. religiosa.

Mechanisms Responsible for the Trophic Effect of Beta-Adrenoceptors on the I-to Current Density in Type 1 Diabetic Rat Cardiomyocytes

Background/Aims: In diabetic ventricular myocytes, transient outward potassium current (I-to) amplitude is severely reduced because of the impaired catecholamine release that characterizes diabetic autonomic neuropathy. Sympathetic nervous system exhibits a trophic effect on I-to since incubation of myocytes with noradrenaline restores current amplitude via beta-adrenoceptor (beta AR) stimulation. Here, we investigate the intracellular signalling pathway though which incubation of diabetic cardiomyocytes with the beta AR agonist isoproterenol recovers I-to amplitude to normal values. Methods: Experiments were performed in ventricular myocytes isolated from streptozotocin-diabetic rats. I-to current was recorded by using the patch-clamp technique. Kv4 channel expression was determined by immunofluorescence. Protein-protein interaction was determined by coimmunoprecipitation. Results: Stimulation of beta AR activates first a G alpha s protein, adenylyl cyclase and Protein Kinase A. PKA-phosphorylated receptor then switches to the G alpha i protein. This leads to the activation of the beta AR-Kinase-1 and further receptor phosphorylation and arrestin dependent internalization. The internalized receptor-arrestin complex recruits and activates cSrc and the MAPK cascade, where Ras, c-Raf1 and finally ERK1/2 mediate the increase in Kv4.2 and Kv4.3 protein abundance in the plasma membrane. Conclusion: beta(2)AR stimulation activates a G alpha s and G alpha i protein dependent pathway where the ERK1/2 modulates the Ito current amplitude and the density of the Kv4.2 and Kv4.2 channels in the plasma membrane upon sympathetic stimulation in diabetic heart.