Incomplete Freund's adjuvant reduces diabetes in the non-obese diabetic mouse.

As the study of type 1 diabetes moves towards preventive therapy, the role of adjuvants needs to be addressed. Incomplete Freund's adjuvant (IFA) is thought of as "immunologically inert" as, unlike complete FA (CFA), it has no components designed to provoke an immune response. We investigated the effect of IFA as an immunomodulator on the disease process leading to type 1 diabetes in the non-obese diabetic (NOD) mouse. 24 NOD mice were injected intradermally (i.d.) at 8 and 12 weeks of age with a 1:1 mixture of IFA and saline; 24 controls received saline alone. Splenocytes were tested against antigens thought to be involved in the disease process, namely insulin, a GAD peptide, a beta-casein peptide, a Glut-2 peptide and concanavalin A (ConA) as a non-specific antigen. In the IFA experiment diabetes incidence was 13% compared to 38% in the controls (p < 0.05). In vitro, splenocytes from IFA treated animals showed non-specific immunosuppression with ConA (p < 0.01), whereas the response to 1-casein and Glut-2 was raised in IFA treated animals with respect to controls. ELISA using supernatants from IFA treated animals, showed a typical Th2 cytokine pattern, whereas controls showed a Th1 pattern. In conclusion, IFA alone can reduce diabetes incidence in the NOD mouse apparently by modulating the immune response towards beta-cell related specific antigens. As IFA has been adopted as an adjuvant in preventive trials in the NOD mouse, this might have implications for the interpretation of previous and future results.     

Increased incidence of true type I diabetes acquired during pregnancy.

A longitudinal study was carried out of all patients with newly acquired insulin dependent diabetes during pregnancy (as distinct from non-insulin-dependent gestational diabetes) seen at the Copenhagen Centre for Diabetes and Pregnancy during 1966 to 1980. The series comprised 63 patients with a mean age of 27 (SEM 1) years. At diagnosis the mean fasting blood glucose concentration was 15.6 (1.3) mmol/l and mean maximal insulin dose 49 (3) IU/day. At a prospective follow up examination a mean of 8 (SEM 1) years after diagnosis 46 of 60 patients (77%) were being treated with insulin (35 (2) IU/day) and had a very low mean stimulated plasma C peptide value (0.12 (0.02) nmol/l) suggesting absent or nearly absent beta cell function. The remaining 14 patients (23%), not currently receiving insulin, appeared to be severely glucose intolerant, having a mean fasting blood glucose concentration of 13.4 (1.2) mmol/l. Thus most of these patients developing insulin dependent diabetes during pregnancy had true type I disease. Compared with the age specific incidence of type I diabetes in the background population of women the incidence was at least 70% higher in pregnant than non-pregnant women (p less than 0.001; chi 2 = 11.6; f = 1). This increased incidence occurred in the third trimester when the risk of developing type I diabetes was 3.8 times that of non-pregnant women (p less than 0.000001; chi 2 = 35.6; f = 1). Finally, the risk of developing insulin dependent diabetes during pregnancy was lower when conception occurred in the winter (p less than 0.05; chi 2 = 4.18; f = 1).     

Extracellular matrix distribution and islet morphology in the early postnatal pancreas: anomalies in the non-obese diabetic mouse

Previously, we reported elevated numbers of macrophages in the pancreas of NOD mice, a spontaneous animal model for T1D, during the early postnatal period. Extracellular matrix plays an important role in the tissue trafficking and retention of macrophages as well as in postnatal pancreas development. Therefore, we have examined the expression and distribution of laminin and fibronectin, two major extracellular matrix proteins and their corresponding integrin receptors, in the pre-weaning pancreases of NOD mice and control mouse strains. In addition, we have characterized the pancreas morphology during this period, since the morphology of the pre-weaning pancreas before the onset of lymphocytic peri-insulitis, when the pancreas is still subject to developmental changes, has been poorly documented. We show that laminin labeling is mainly associated with exocrine tissue, whereas fibronectin labeling was mostly localized at the islet-ductal pole, islet periphery and in intralobular septa. Moreover, the protein expression level of fibronectin was increased in NOD pancreases at the early stage of postnatal development, as compared to pancreases of C57BL/6 and BALB/c mouse strains. Interestingly, pancreatic macrophages were essentially found at sites of intense fibronectin labeling. The increased fibronectin content in NOD neonatal pancreas coincided with altered islet morphology, histologically reflected by enlarged and irregular shaped islets and increased percentages of total endocrine area as compared to that of control strains. In conclusion, increased levels of the extracellular matrix protein fibronectin were found in the early postnatal NOD pancreas, and this is associated with an enhanced accumulation of macrophages and altered islet morphology.This work was supported by the Centre Nationale de la Recherche Scientifique, Université Paris V and grants from the 5th PCRD MONODIAB.     

Effect of indomethacin on plasma levels of vitamin D metabolites, oestradiol and progesterone in rabbits during early pregnancy

1. Inhibition of prostaglandin (PG) synthesis by indomethacin (Id) during early pregnancy in rabbits apparently disrupts the process of sex steroid production by the ovaries. 2. The role of PGs as mediators in steroidogenesis was tested by investigating the effect of Id alone or in combination with progesterone, with oestradiol and progesterone, or with a mixture of PGs, on plasma levels of 25-hydroxyvitamin D (25(OH)D) and 1,25-dihydroxyvitamin D (1,25(OH)2D) in rabbits at an early stage of pregnancy. 3. Id alone significantly reduced plasma levels of both 25(OH) D and 1,25(OH)2D. Treatment with Id in combination with either oestradiol and progesterone, or with a mixture of PGs, resulted in the restoration of plasma levels of both vitamin D metabolites as well as the restoration of plasma levels of progesterone, to their control values.     

Overexpression of the reg gene in non-obese diabetic mouse pancreas during active diabetogenesis is restricted to exocrine tissue.

We demonstrated pancreatic reg gene overexpression in non-obese diabetic (NOD) mice during active diabetogenesis. The aim of this study was to determine in which part of the pancreas (endocrine and/or exocrine) the gene(s) and the protein(s) were expressed and if their localization changed with progression of the disease. In situ hybridization analysis and immunocytochemical studies were carried out on pancreas of female and male NOD mice. Both develop insulitis but diabetes develops only in females and in males only when treated by cyclophosphamide. Our results show that whatever the age, sex, and presence of insulitis and/or diabetes, the expression of reg mRNAs and of the corresponding protein(s) was restricted to exocrine tissue. Moreover, reg remains localized in acinar cells in the two opposite situations of (a) cyclophosphamide-treated males in a prediabetic stage presenting a high level of both insulin and reg mRNAs, and (b) the overtly diabetic females with no insulin but a high level of reg mRNA. These findings suggest that overexpression of the reg gene(s) might represent a defense of the acinar cell against pancreatic aggression.     

Uterine and oviducal protein secretion during early pregnancy in the mouse

Changes in the protein composition of the embryo's environment during early development were studied by analysis of proteins synthesized and secreted by oviducal and uterine explants on Days 1-6 of pregnancy. Although secretions from ampullar and isthmic oviduct and uterus contained many proteins in common, each area also produced its own characteristic proteins. In the uterus, changes in the secretion pattern were found during the peri-implantation period, including both increases and decreases in particular proteins which appear to be dependent on the presence of embryos. Embryo-induced effects on uterine secretion began between 09:00 h of Day 4 and 09:00 h of Day 5. Oviducal secretions exhibited many of the embryo-dependent proteins found in the uterus, but the expression of these proteins did not appear to be influenced by the presence of embryos on Day 1 or Day 3. The characteristic pattern of secreted protein expression by each portion of the reproductive tract may reflect the specialization of each area for certain developmental events.     

Sex-specific effect of insulin-dependent diabetes 4 on regulation of diabetes pathogenesis in the nonobese diabetic mouse

Many human autoimmune diseases are more frequent in females than males, and their clinical severity is affected by sex hormone levels. A strong female bias is also observed in the NOD mouse model of type I diabetes (T1D). In both NOD mice and humans, T1D displays complex polygenic inheritance and T cell-mediated autoimmune pathogenesis. The identities of many of the insulin-dependent diabetes (Idd) loci, their influence on specific stages of autoimmune pathogenesis, and sex-specific effects of Idd loci in the NOD model are not well understood. To address these questions, we analyzed cyclophosphamide-accelerated T1D (CY-T1D) that causes disease with high and similar frequencies in male and female NOD mice, but not in diabetes-resistant animals, including the nonobese diabetes-resistant (NOR) strain. In this study we show by genetic linkage analysis of (NOD x NOR) x NOD backcross mice that progression to severe islet inflammation after CY treatment was controlled by the Idd4 and Idd9 loci. Congenic strains on both the NOD and NOR backgrounds confirmed the roles of Idd4 and Idd9 in CY-T1D susceptibility and revealed the contribution of a third locus, Idd5. Importantly, we show that the three loci acted at distinct stages of islet inflammation and disease progression. Among these three loci, Idd4 alleles alone displayed striking sex-specific behavior in CY-accelerated disease. Additional studies will be required to address the question of whether a sex-specific effect of Idd4, observed in this study, is also present in the spontaneous model of the disease with striking female bias.     

Effect of different insulin administration modalities on vitamin D metabolism of insulin-dependent diabetic patients

The vitamin D status of IDDs was studied in 3 groups of patients who were treated for several months with (i) conventional insulin therapy (group I, n = 17, HbA1 = 10.1 +/- 0.5%); (ii) continuous subcutaneous insulin infusion (CSII, group II, n = 11, HbA1 = 8.9 +/- 0.6%); and (iii) continuous intraperitoneal insulin infusion (CPII, group III, n = 13, HbA1 = 8.0 +/- 0.4%). In all patient groups the plasma concentration of vitamin D metabolites were within normal range. However plasma 25 OH D (ng/ml) was significantly lower in groups I (13.0 +/- 0.8, P less than 0.01) and II (12.5 +/- 1.5, P less than 0.02) than in group III: 22.1 +/- 2.3 (normal range 7-27). Plasma 24,25-(OH)2D (ng/ml) was positively correlated to plasma 25 OH D and was significantly decreased in groups I (1.5 +/- 0.2, P less than 0.05) and II (1.4 +/- 0.2, P less than 0.05) compared with group III: 2.3 +/- 0.3. No significant differences were found in plasma 1,25-(OH)2D between the three groups of diabetics. Plasma PTH was similar in the three groups. The same differences in plasma 25 OH D were observed between the patients treated with CPII and 15 subcutaneously treated patients matched for diabetic control (HbA1 less than 10 per cent). The present results seem to indicate that insulin might have a stimulatory effect on the hepatic 25 hydroxylase activity.     

The pro-apoptotic BH3-only protein Bid is dispensable for development of insulitis and diabetes in the non-obese diabetic mouse

Type 1 diabetes is caused by death of insulin-producing pancreatic beta cells. Beta-cell apoptosis induced by FasL may be important in type 1 diabetes in humans and in the non-obese diabetic (NOD) mouse model. Deficiency of the pro-apoptotic BH3-only molecule Bid protects beta cells from FasL-induced apoptosis in vitro. We aimed to test the requirement for Bid, and the significance of Bid-dependent FasL-induced beta-cell apoptosis in type 1 diabetes. We backcrossed Bid-deficient mice, produced by homologous recombination and thus without transgene overexpression, onto a NOD genetic background. Genome-wide single nucleotide polymorphism analysis demonstrated that diabetes-related genetic regions were NOD genotype. Transferred beta cell antigen-specific CD8+ T cells proliferated normally in the pancreatic lymph nodes of Bid-deficient mice. Moreover, Bid-deficient NOD mice developed type 1 diabetes and insulitis similarly to wild-type NOD mice. Our data indicate that beta-cell apoptosis in type 1 diabetes can proceed without Fas-induced killing mediated by the BH3-only protein Bid.     

Remodeling of the uterine artery during and early after pregnancy in the mouse

Biomechanics and Modeling in Mechanobiology - Pregnancy associates with dramatic changes in maternal cardiovascular physiology that ensure that the utero-placental circulation can support the...     

Granzyme B is dispensable in the development of diabetes in non-obese diabetic mice

Pancreatic beta cell destruction in type 1 diabetes is mediated by cytotoxic CD8(+) T lymphoctyes (CTL). Granzyme B is an effector molecule used by CTL to kill target cells. We previously showed that granzyme B-deficient allogeneic CTL inefficiently killed pancreatic islets in vitro. We generated granzyme B-deficient non-obese diabetic (NOD) mice to test whether granzyme B is an important effector molecule in spontaneous type 1 diabetes. Granzyme B-deficient islet antigen-specific CD8(+) T cells had impaired homing into islets of young mice. Insulitis was reduced in granzyme B-deficient mice at 70 days of age (insulitis score 0.043±0.019 in granzyme B-deficient versus 0.139±0.034 in wild-type NOD mice p<0.05), but was similar to wild-type at 100 and 150 days of age. We observed a reduced frequency of CD3(+)CD8(+) T cells in the islets and peripheral lymphoid tissues of granzyme B-deficient mice (p<0.005 and p<0.0001 respectively), but there was no difference in cell proportions in the thymus. Antigen-specific CTL developed normally in granzyme B-deficient mice, and were able to kill NOD islet target cells as efficiently as wild-type CTL in vitro. The incidence of spontaneous diabetes in granzyme B-deficient mice was the same as wild-type NOD mice. We observed a delayed onset of diabetes in granzyme B-deficient CD8-dependent NOD8.3 mice (median onset 102.5 days in granzyme B-deficient versus 57.50 days in wild-type NOD8.3 mice), which may be due to the delayed onset of insulitis or inefficient priming at an earlier age in this accelerated model of diabetes. Our data indicate that granzyme B is dispensable for beta cell destruction in type 1 diabetes, but is required for efficient early activation of CTL.     

Immunohistochemical study of caspase-3-expressing cells within the pancreas of non-obese diabetic mice during cyclophosphamide-accelerated diabetes

During insulin-dependent diabetes mellitus, immune cells infiltrate pancreatic islets progressively and mediate beta cell destruction over a prolonged asymptomatic prediabetic period. Apoptosis may be a major mechanism of beta cell loss during the disease. This process involves a proteolytic cascade in which upstream procaspases are activated which themselves activate downstream caspases, including caspase-3, a key enzyme involved in the terminal apoptotic cascade. Here dual-label immunohistochemistry was employed to examine the intra-islet expression, distribution and cellular sources of active caspase-3 in the non-obese diabetic (NOD) mouse given cyclophosphamide to accelerate diabetes. NOD mice were treated at day 95 and caspase-3 expression was studied at days 0, 4, 7, 11 and 14. Its expression was also correlated with advancing disease and compared with age-matched NOD mice treated with diluent alone. At day 0 (=day 95), caspase-3 immunolabelling was observed in several peri-islet and intra-islet macrophages, but not in CD4 and CD8 cells and only extremely rarely in beta cells. At day 4, only a few beta cells weakly expressed the enzyme, in the absence of significant insulitis. At day 7, caspase-3 expression was observed in a small proportion of intra-islet macrophages. At day 11, there was a marked increase in the number of intra-islet macrophages positive for caspase-3 while only a few CD4 cells expressed the enzyme. At day 14, caspase-3 labelling became prominent in a significant proportion of macrophages. Only a few CD4 and CD8 cells expressed the enzyme. Capase-3 labelling was also present in a proportion of macrophages in perivascular and exocrine regions. Surprisingly, beta cell labelling of caspase-3 at days 11 and 14 was rare. At this stage of heightened beta cell loss, a proportion of intra-islet interleukin-1-positive cells coexpressed the enzyme. Caspase-3 was also observed in numerous Fas-positive cells in heavily infiltrated islets. During this late stage, only a proportion of caspase-3-positive cells contained apoptotic nuclei, as judged by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL). We conclude that during cyclophosphamide-accelerated diabetes in the NOD mouse, the predominant immunolabelling of caspase-3 in intra-islet macrophages suggests that apoptosis of macrophages may be an important mechanism for its elimination. The virtual absence of caspase-3 immunolabelling in most beta cells even during heightened beta cell loss supports their rapid clearance following their death during insulin-dependent diabetes mellitus.     

Effect of vitamin D deficiency on skeletal development during early growth in the rat

To define the role of vitamin D in early development, female weanling rats were grown to maturity on a vitamin D-deficient diet and mated with normal males. At Day 20 of pregnancy the weight and total body calcium of fetuses were determined. At various times after parturition, pups were sacrificed. Plasma samples were analyzed for calcium and phorphorus, and femurs were characterized as to volume, dry weight, ash weight, and total calcium. The results indicate that vitamin D deficiency with its accompanying hypocalcemia does not impair placental transfer of calcium nor weight gain of the fetus. Vitamin D deficiency does appear to increase calcium accumulation in the fetus. After parturition vitamin D is functional in maintaining a normocalcemia as early as 3 days postpartum and its importance increases with age of the neonate. Bone mineralization is clearly disrupted by Day 14 as judged by calcium content per unit bone volume and the severity of the defect increases with age. Both vitamin D and normal concentrations of calcium and phosphorus appear to be essential for proper skeletal development during early growth postpartum.     

Insolation effect on vitamin D supply in children during sanatorium treatment

Serum 25/OH/D3 levels were determined in 58 children aged 5-7 years staying from April 1 to September 30 1981 in Ko?obrzeg in a sanatorium. A comparison of isolation conditions in that time period with the serum values of 25/OH/D3 showed that the supply of vitamin D was sufficient only in the period from June to September. In the remaining months no correlation was observed between the values of solar radiation and vitamin D levels. During the study and for three months before it exogenous vitamin D administration was stopped.     

Protein metabolism in the mouse during pregnancy and lactation.

Protein synthesis was measured in vivo in the whole body and in a number of individual tissues in mice at various stages of pregnancy and lactation. The absolute rate of protein synthesis in the whole body increased from 640 mg/day in virgin mice to 1590 mg/day by day 18 of pregnancy, and to 2100 mg/day by day 15 of lactation. Large proportions of these increments were contributed by the rapidly growing foetuses and placentae in the pregnant animals and by protein synthesis in the mammary glands during lactation. In addition, a substantial stimulation of growth and protein synthesis was also observed in the liver and the gastrointestinal tract. Gastrocnemius muscle showed no changes in protein metabolism, indicating that in the well-fed mouse this tissue is not required to play a role as a protein reserve during pregnancy and lactation.     

Lack of effect of vitamin C on the incidence of chromosome anomalies induced by influenza virus in germ cells of mice

Summary Administration of vitamin C did not have any protective effect on the incidence of chromosomal anomalies produced by influenza virus nor did this vitamin itself produce chromosome aberrations in mice spermatocytes.This report is part of the Ph.D. thesis of K.Pichan, whose present address is National Institute of Nutrition, Hyderabad, India