Dysregulated liver lipid metabolism and innate immunity associated with hepatic steatosis in neonatal BBdp rats and NOD mice.

In a previous study we reported that prediabetic rats have a unique gene signature that was apparent even in neonates. Several of the changes we observed, including enhanced expression of pro-inflammatory genes and dysregulated UPR and metabolism genes were first observed in the liver followed by the pancreas. In the present study we investigated further early changes in hepatic innate immunity and metabolism in two models of type 1 diabetes (T1D), the BBdp rat and NOD mouse. There was a striking increase in lipid deposits in liver, particularly in neonatal BBdp rats, with a less striking but significant increase in neonatal NOD mice in association with dysregulated expression of lipid metabolism genes. This was associated with a decreased number of extramedullary hematopoietic clusters as well as CD68+ macrophages in the liver of both models. In addition, PPARÉ£ and phosphorylated AMPKα protein were decreased in neonatal BBdp rats. BBdp rats displayed decreased expression of antimicrobial genes in neonates and decreased M2 genes at 30 days. This suggests hepatic steatosis could be a common early feature in development of T1D that impacts metabolic homeostasis and tolerogenic phenotype in the prediabetic liver.

Spontaneous obesity-linked type 2 diabetes in the absence of islet amyloid in a cynomolgus monkey infected with bovine spongiform encephalopathy.

A 9-year-old cynomolgus monkey (Macaca fascicularis) infected orally with bovine spongiform encephalopathy (BSE) was presented for necropsy following euthanasia 4 years post infection (p.i.). This macaque R984 was exposed to a BSE dose that causes a simian form of variant Creutzfeldt-Jakob disease (vCJD) within 5 years p.i. in other macaques. All orally BSE-infected macaques developed a significant weight gain within the first 2 years p.i. compared with non-BSE-infected age- and sex-matched control animals, suggesting increased risk of type 2 diabetes (T2D). In contrast, macaque R984 developed rapid weight loss, hyperglycemia, and glucosuria and had to be euthanatized 4 years p.i. before clinical signs of vCJD. Pancreas histopathological evaluation revealed severe islet degeneration but, remarkably, no islet amyloid deposits were present. Immunostaining of pancreas sections for insulin and glucagon confirmed the loss of endocrine cells. In addition, prions were present in the adenohypophysis but not in other areas of the brain, indicating centripetal prion spread from the gut during the preclinical phase of BSE infection. Plasma glucose and insulin concentrations of macaque R984 became abnormal with age and resembled T2D. This unusual case of spontaneous T2D in the absence of islet amyloid deposits could have been due to early prion spread from the periphery to the endocrine system or could have occurred spontaneously.

An exploration of Glo-3A antibody levels in children at increased risk for type 1 diabetes mellitus.

AIMS: To determine whether Glo-3A, (formerly referred to as homologue of Glb1 or Glb1) antibodies are associated with islet autoimmunity (IA) in children at increased risk for type 1 diabetes (T1D) and to investigate their relation with environmental correlates of T1D. METHODS: We selected a sample from the Diabetes Autoimmunity Study in the Young (DAISY), a prospective study of children at increased risk for T1D. Cases were positive for insulin, glutamic acid decarboxylase (GAD), or insulinoma-associated antigen-2 (IA-2) autoantibodies on two consecutive visits and either diagnosed with diabetes mellitus or still autoantibody positive when selected. Controls were from the same increased risk group, of similar age as the cases but negative for autoantibodies. Sera from 91 IA cases and 82 controls were analyzed in a blinded manner for immunoglobulin G (IgG) antibodies to Glo-3A by ELISA. RESULTS: Adjusting for family history of T1D and human leukocyte antigen (HLA)-DR4 positivity, Glo-3A antibodies were not associated with IA case status (OR: 1.01, 95% CI: 0.99-1.03). Adjusting for age, family history of T1D, and HLA-DR4 positivity, Glo-3A antibody levels were inversely associated with breast-feeding duration (beta = -0.08, p = 0.001) and directly associated with current intake of foods containing gluten (beta = 0.24, p = 0.007) in IA cases but not in controls. Zonulin, a biomarker of gut permeability, was directly associated with Glo-3A antibody levels in cases (beta = 0.73, p = 0.003) but not in controls. CONCLUSION: Differing correlates of Glo-3A antibodies in IA cases and controls suggest an underlying difference in mucosal immune response.

Wheat protein-induced proinflammatory T helper 1 bias in mesenteric lymph nodes of young diabetes-prone rats.

AIMS/HYPOTHESIS: Type 1 diabetes is the result of an inflammatory T helper 1 (Th1) lymphocyte-mediated beta cell destructive process. The majority of diabetes-prone BioBreeding (BBdp) rats fed wheat protein-based diets, such as NTP-2000, develop type 1 diabetes and display a mild coeliac-like enteropathy. Mesenteric lymph nodes (MLNs), which drain the gut, are the major inductive site where dietary antigens are recognised in the gut-associated lymphoid tissue (GALT). We hypothesised that this compartment could be a site of abnormal wheat protein-induced Th1 cell activation. METHODS: MLN cells were isolated from BBdp and BB control (BBc) rats that were fed NTP-2000 or a hydrolysed casein (HC)-based diet at ages that pre-date classic insulitis. The inflammatory status, phenotype and proliferation of these cells in response to wheat protein were determined. RESULTS: The expression ratio of T-bet : Gata3, master transcription factors for Th1 and Th2 cytokines, was increased in the MLN from NTP-2000-fed BBdp rats compared with that from BBc rats, mainly due to decreased Gata3 expression. CD3(+)CD4(+)IFN-gamma(+) T cells were more prevalent in the MLN of wheat-fed BBdp rats, but remained at control levels in BBdp rats fed a diabetes-retardant HC diet. BBdp MLN cells proliferated in response to wheat protein antigens in a specific, dose-dependent manner, and >93% of cells were CD3(+)CD4(+) T cells. This proliferation was associated with a low proportion of CD4(+)CD25(+) T cells and a high proportion of dendritic cells in the MLN of BBdp rats. CONCLUSIONS/INTERPRETATION: Before insulitis is established, the MLNs of wheat-fed BBdp rats contain an unusually high proportion of Th1 cells that proliferate specifically in response to wheat protein antigens.

Insulin-dependent diabetes and gut dysfunction: the BB rat model.

Accumulating data indicate that intestinal dysfunction and dysregulation of the gut immune system may play a role in the development of type 1 diabetes. This review deals with the occurrence of gut damage and dysfunction in BB rats, an animal model of spontaneous immune type 1 diabetes, placing special emphasis on the effect of diet on the incidence of diabetes in BB rats, the identification of a type 1 diabetes-related protein from wheat, and preliminary observations documenting anomalies in the inductive tissues of the gut immune system (Peyer's patch cells and mesenteric lymph node cells) and pancreatic lymph node cells of diabetes-prone BB rats. In addition to histological evidence of gut damage, the review will also draw attention to altered intestinal disaccharidase activity, changes in intestinal peroxidase activity, glucagon-like peptide 1 anomalies, and perturbation of both intestinal permeability and mucin content in BB rats. In all these cases, the findings in rats fed a diabetes-promoting diet are compared to those collected in animals receiving a protective diabetes-retardant diet.

Enteropathy precedes type 1 diabetes in the BB rat.

BACKGROUND AND AIMS: There is increasing evidence implicating intestinal immune responses to dietary proteins in the pathogenesis of type 1 autoimmune diabetes (T1D). Here we investigated the association between intestinal pathology and dietary factors in T1D by examining the mucosal architecture in the BB rat model. METHODS: BB control (BBc) and diabetes prone (BBdp) rats were fed either a diabetes retardant hydrolysed casein based diet or one of two cereal based diets that promote the development of diabetes. Intestinal architecture was assessed in the jejunum by microdissection, histology, and immunohistology, and by measuring peroxidase activity and brush border invertase levels. RESULTS: Enteropathy was present in BBdp rats soon after weaning, as assessed by increases in crypt length and in the proliferative activity of crypt epithelial cells in the jejunum, and this remained constant until 120 days of age. There was also a decrease in invertase activity, as well as increased numbers of intraepithelial lymphocytes, increased levels of mucosal peroxidase activity, and infiltration of the mucosa by CD4(+) T lymphocytes. Equivalent enteropathy was present at all times in BBdp rats and was not influenced by the nature of the diet or by thymectomy at three weeks at age, procedures which prevent the development of diabetes. CONCLUSION: Enteropathy is a consistent feature in the diabetes prone BB rat but it precedes the onset of insulitis and appears to be due to mechanisms distinct from those which cause diabetes. The beneficial effects of the diabetes retardant hydrolysed casein diet on diabetes are not due to an effect on intestinal architecture per se but mucosal damage may be necessary for the development of autoreactivity in the pancreas.

A wheat-based, diabetes-promoting diet induces a Th1-type cytokine bias in the gut of NOD mice.

Dietary antigens are candidate environmental factors in the pathogenesis of type 1 diabetes. In the non-obese diabetic (NOD) mouse, an animal model of type 1 diabetes, cereal-based diets promote disease development, whereas the diets based on hydrolysed proteins or non-diabetogenic proteins are protective. The hypothesis that diabetogenic diets modulate the cytokine balance in the gut was tested. NOD mice were fed with NTP-2000 (mainly a wheat-based milk-free diet) or Prosobee (a semi-purified hypoallergenic diet based on soy protein isolate) or Prosobee plus casein (milk protein fraction). The mRNA levels of IFN-gamma, IL-10, TNF-alpha, TGF-beta, and inducible NO synthase in the small intestine and the Peyer's patches were determined by semi-quantitative RT-PCR. Mice fed on the cereal-based NTP-2000 diet expressed higher levels of the Th1-type and pro-inflammatory markers IFN-gamma, TNF-alpha, and inducible NO synthase mRNA compared to the Prosobee-fed animals. The expression of the counterregulatory cytokines IL-10 and TGF-beta was unaffected. This resulted in a significant bias of the intestinal cytokine balance towards T helper cell type 1 after feeding NTP-2000. The cytokine mRNA levels in the gut-associated Peyer's patches were not affected. Thus, modulation of gut immunoreactivity by diet may contribute to disease development in NOD mice.

A multi-centre, blinded international trial of the effect of A(1) and A(2) beta-casein variants on diabetes incidence in two rodent models of spontaneous Type I diabetes.

AIMS/HYPOTHESIS: The diabetes-inducing potential of cows' milk is still debated and there is no consensus on the diabetogenicity of individual milk proteins. A(1)-beta-casein has been associated with increased diabetes frequency in ecological studies and in NOD mice. Our aim was to ascertain whether A(1)-beta-casein was more diabetogenic than A(2) and to test the diabetogenicity of a milk-free diet in animals representing different forms of spontaneous Type I (insulin-dependent) diabetes mellitus. METHODS: Defined diets were coded and shipped to laboratories in New Zealand (NOD/NZ), Canada (BB) and the UK (NOD/Ba). Base diets were Pregestimil (PG) and ProSobee (PS). Purified fractions of whole casein (WC), A(1)or A(2)-beta-casein were added at 10%. A milk-free, wheat-predominant, NTP-2000 diet was the control. Animals were fed from weaning up to 150 or 250 days, and insulitis, diabetes frequency and expression of pancreatic cytokines were assessed. RESULTS: Diabetes incidence was highest in three locations in animals fed NTP-2000. PG and PS diets were protective except for NOD/Ba mice fed PG+WC where incidence was similar to NTP-2000. A(1) and A(2) diets were protective in both models, but A(1) beta-casein was slightly more diabetogenic in PS-fed BB rats. The New Zealand study was confounded by an infection. CONCLUSION/INTERPRETATION: A milk-free, wheat-predominant diet was highly diabetogenic in three widely separate locations in both animal models. A previous result that A(1) beta-casein was more diabetogenic than A(2) beta-casein in NOD mice was not confirmed; both beta-casein variants were protective in BB rats and NOD mice. Whole Casein promoted diabetes in NOD/Ba but protected BB showing that unique diabetes haplotypes react differently to dietary proteins. A(1)- was more diabetogenic than A(2)-beta-casein only in PS-fed BB rats. Neither the analysis of insulitis nor of pancreatic cytokine gene expression showed a difference between A(1) or A(2) beta-casein fed animals. Milk caseins are unlikely to be exclusive promoters of Type I diabetes, but could enhance the outcome of diabetes in some cases. Other diet components such as wheat could be more important promoters of Type I diabetes.

Altered islet homeostasis before classic insulitis in BB rats.

Young diabetes-prone BioBreeding (BBdp) rats fed a diabetes-promoting, cereal-based, NIH-07 (NIH) diet have decreased islet area compared with rats fed a diabetes-retardant diet at a time when classic insulitis is minimal. This finding raised the possibility that islet homeostasis in BBdp rats may be abnormal. To investigate this possibility further, comparisons were made between BBdp and BB control (BBc) rats fed a diabetes-promoting NIH diet for 22 days after weaning. Pancreatic sections were fixed in Bouin's solution and evaluated using immunohistochemistry and image analysis by staining with antibodies for islet hormones: insulin, glucagon; cell proliferation markers: PCNA, BrdU; markers of islet neogenesis: PDX-1, cytokeratin 20; apoptosis was assessed by morphological changes and TUNEL staining. Body weight of BBdp rats was significantly smaller than BBc rats. Although the total number of islets was higher in BBdp compared with BBc, both islet and beta-cell fraction were similar. BBdp rats had a lower beta-cell mass than BBc rats, although this was not statistically significant. Alpha-cell fraction and beta-cell size were similar. Apoptotic bodies were rare in beta-cells but more frequent in acinar tissue of BBdp rats. When the day-night cycle was reversed to synchronize the apoptotic process, the number of apoptotic bodies in islets and in acinar cells was increased. Apoptotic bodies and BrdU+ or PCNA+ beta-cells were more frequently encountered in islets of BBdp rats. Although the frequency of CK20+ islets in BBdp rats was not different, CK20+ area fraction was smaller in BBdp. The number of extra-islet insulin+ and glucagon+ clusters (<4 cells) was significantly greater in BBdp rats. These data are consistent with an enhanced compensatory or "repair" process in the pancreas of BBdp rats that attempts to maintain islet cell mass by altering homeostasis through increased islet neogenesis.

Hydrolysed casein diet protects BB rats from developing diabetes by promoting islet neogenesis.

Feeding diabetes-prone BioBreeding (BBdp) rats a hydrolysed-casein (HC)-based semi-purified diet results in two-to-three-fold fewer diabetes cases compared with feeding cereal-based diets such as NIH-07 (NIH). We showed previously that young NIH-fed BBdp rats had decreased islet area at a time when classic insulitis was minimal. Rats fed an HC diet maintained near normal islet area followed 3-4 weeks later by a deviation of the pancreas cytokine pattern from Th1 to Th2/Th3. This finding raised the possibility that BBdp rats were more susceptible to diet-induced changes in islet homeostasis. To investigate this possibility further, BBdp rats were fed an NIH or HC diet from days 23 to 45. Bouin's fixed sections of pancreas were stained with H & E or antibodies for insulin and glucagon. Cell proliferation nuclear antigen (PCNA) was used as a marker of cell proliferation and cells were stained for putative markers of islet neogenesis, cytokeratin 20 (CK20) and Bcl-2. Apoptotic bodies were recognized by morphological features and by TUNEL-positive staining. BBdp rats fed an HC diet had a significantly higher beta-cell fraction than rats fed NIH, whereas alpha-cell fraction and beta-cell size were not affected by diet or rat type. Apoptotic bodies of beta-cells were rare and unaffected by diet. The number of PCNA(+)beta-cells was not affected by diet. CK20 expression was localized in the ductular system and at the periphery of islets in rats aged 7 and 45 days. There were more CK20(+)islets in BBdp rats fed NIH than in those fed HC but the CK20 area fraction was unaffected by diet. Bcl-2 expression was scattered among ducts and central acinar cells. The number of extra-islet insulin(+)and glucagon(+)clusters (

Flow cytometric analysis of intracellular IFN-gamma, IL-4 and IL-10 in CD3(+)4(+) T-cells from rat spleen.

The application of multi-parameter flow cytometry for the assessment of T-cell and cytokine functioning has been used by several groups for studying human and mouse samples, although little has been reported for the rat. Here we report the optimisation of immunofluorescent staining for cell surface and intracellular antigens using three-colour flow cytometric analysis to measure the frequency of rat CD3(+)4(+) T-cells that produce IFN-gamma, IL-4 and IL-10. In vitro stimulation of IFN-gamma production required incubation of splenocytes with PMA and ionomycin in the presence of the protein transport inhibitor brefeldin A for 6 h. Three stimulation protocols for IL-4 and IL-10 production were evaluated. In vitro priming of splenic T-cells with antibodies against CD3 and CD28 and recombinant cytokines (IL-2 and IL-4) for 5 days followed by restimulation with PMA and ionomycin was required to stimulate cells to produce either IL-4 or IL-10. Brefeldin A was found to be a more suitable protein transport inhibitor than monensin. This method will be useful for analysing the nature of individual rat cytokine-producing cells in a variety of experimental model systems.

Dietary effects on insulin and nutrient metabolism in mesenteric lymph node cells, splenocytes, and pancreatic islets of BB rats.

The present studies were performed to determine if a protective diet has different effects on the metabolic activity or function of islet cells, as well as the metabolic activity of mesenteric lymph node (MLN) cells and spleen cells, from BioBreeding (BB) rats. Diabetes-prone BB (BBdp) rats and control non-diabetes-prone BB (BBc) rats were fed for about 20 days either a mainly plant-based diabetogenic diet, NIH-07 (NIH), or a protective semipurified diet with hydrolyzed casein (HC) as the amino acid source. At 6 to 8 weeks of age, BBdp rats had high plasma D-glucose and low insulin concentrations, low insulin content, and low metabolic and secretory responses to D-glucose in isolated pancreatic islets. Islet metabolism, as measured by accumulation of 14C-acidic metabolites, amino acids, and the ratio of D-[U-14C]glucose oxidation and D-[5-3H]glucose utilization was increased in control rats fed HC (P < .05); a similar trend in BBdp rats was not significant. Feeding the HC diet increased islet insulin content (P < .01) by 13% in BBdp and 23% in BBc rats; other metabolic and hormonal variables were unaffected. Compared with BBc rats, BBdp rats displayed higher rates of L-[U-14C]glutamine oxidation, D-[5-3H]glucose utilization, and D-[U-14C]glucose oxidation in MLN cells, but not in splenocytes. There was a dramatic decrease of L-[U-14C]glutamine oxidation in MLN cells from BBc and BBdp rats fed HC. Glycolysis was decreased in control rats. We conclude that the protection afforded by feeding BBdp rats a HC diet is associated with increased insulin in target beta cells and downregulation of metabolic activity in gut-associated MLN cells. Metabolic activity in splenocytes, cells representative of the systemic immune system, was less affected. These data suggest that diet-induced metabolic changes occur in the islets and nearby cells of the gut immune system in the period before classic insulitis. Changes in the islets were smaller in comparison to the dramatic remodeling of nutrient catabolism in MLN cells. MLN downregulation may reflect baseline metabolic activity in the absence of diabetogenic (or other) food antigens and further highlights an important interaction between diabetogenic food antigens and the gut immune tissues.

Feeding a protective hydrolysed casein diet to young diabetic-prone BB rats affects oxidation of L[U-14C]glutamine in islets and Peyer's patches, reduces abnormally high mitotic activity in mesenteric lymph nodes, enhances islet insulin and tends to normalize NO production.

The present studies were undertaken to examine concomitant diet-induced changes in pancreatic islets and cells of the gut immune system of diabetes-prone BB rats in the period before classic insulitis. Diabetes-prone (BBdp) and control nondiabetes prone (BBc) BB rats were fed for approximately 17 days either a mainly plant-based standard laboratory rodent diet associated with high diabetes frequency, NIH-07 (NIH) or a protective semipurified diet with hydrolyzed casein (HC) as the amino acid source. By about 7 weeks of age, NIH-fed BBdp rats had lower plasma insulin and insulin/glucose ratio, lower insulin content of isolated islets, lower basal levels of NO but higher responsiveness of NO production to IL-1beta in cultured islets, and higher Con A response and biosynthetic activities in mesenteric lymphocytes than control rats fed the same diet. In control rats, the HC diet caused only minor changes in most variables, except for a decrease in oxidation of L-[U-14C]glutamine in Peyer's patch (PP) cells and an increase in protein biosynthesis in mesenteric lymphocytes. In BBdp rats, however, the HC diet increased plasma insulin concentration, islet insulin/protein ratio, and tended to normalize the basal and IL-1beta-stimulated NO production by cultured islets. The HC diet decreased oxidation of L[U-14C]glutamine in BBdp pancreatic islets, whereas oxidation of L-[U-14C]glutamine in PP cells was increased, and the basal [Methyl-3H]thymidine incorporation in mesenteric lymphocytes was decreased. These findings are compatible with the view that alteration of nutrient catabolism in islet cells as well as key cells of the gut immune system, particularly changes in mitotic and biosynthetic activities in mesenteric lymphocytes, as well as basal and IL-1beta stimulated NO production, participate in the sequence of events leading to autoimmune diabetes in BB rats. Thus, the protection afforded by feeding a hydrolysed casein-based diet derives from alterations in both the target islet tissue and key cells of the gut immune system in this animal model of type 1 diabetes.

Effects of a protective hydrolyzed casein diet upon the metabolic and secretory responses of pancreatic islets to IL-1beta, cytokine production by mesenteric lymph node cells, mitogenic and biosynthetic activities in Peyer's patch cells, and mitogenic activity in pancreatic lymph node cells from control and diabetes-prone BB rats.

The effects of substituting a plant-based control diabetogenic diet (NIH diet) by a protective hydrolyzed casein diet (HC diet) upon selected metabolic and functional variables were recently investigated in Peyer's patch cells, splenocytes, mesenteric lymph node cells, and pancreatic islets from either control (BBc) or diabetes-prone (BBdp) BB rats. In the present work, the plasma d-glucose and insulin concentrations, the protein and insulin content of pancreatic islets, the metabolism of d-glucose, and its insulinotropic action in islets first cultured for 24 h in the absence or presence of IL-1beta, the production of IFN-gamma and IL-10 by mesenteric lymph node cells cultured for 48 h in the absence or presence of concanavalin A, the mitogenic activity of Peyer's patch cells and pancreatic lymph node cells in the absence or presence of the same lectin, and the biosynthetic activity of Peyer's patch cells were measured in the BBc and BBdp rats fed either the NIH or the HC diet. Two major novel findings emerged from this study. First, in immune cells, diet HC increased to a greater extent the responsiveness to concanavalin A of certain metabolic and functional variables in BBdp rats than in BBc rats. Second, pancreatic islet cells of BBdp rats were less sensitive to IL-1beta than those of BBc rats and this difference was further accentuated when the animals were fed the HC rather than the NIH diet. These findings afford further support to the view that, in BB rats, changes in the biological behavior of Peyer's patch cells, mesenteric and pancreatic lymph node cells, and pancreatic islet cells participate in the pathogenesis of insulin-dependent diabetes mellitus and its prevention by a suitable dietary manipulation.

Long-term immunity in cats vaccinated with an inactivated trivalent vaccine.

OBJECTIVE: To evaluate duration of immunity in cats vaccinated with an inactivated vaccine of feline panleukopenia virus (FPV), feline herpesvirus (FHV), and feline calicivirus (FCV). ANIMALS: 17 cats. PROCEDURE: Immunity of 9 vaccinated and 8 unvaccinated cats (of an original 15 vaccinated and 17 unvaccinated cats) was challenged 7.5 years after vaccination. Specific-pathogen-free (SPF) cats were vaccinated at 8 and 12 weeks old and housed in isolation facilities. Offspring of vaccinated cats served as unvaccinated contact control cats. Virus neutralization tests were used to determine antibody titers yearly. Clinical responses were recorded, and titers were determined weekly after viral challenge. RESULTS: Control cats remained free of antibodies against FPV, FHV, and FCV and did not have infection before viral challenge. Vaccinated cats had high FPV titers throughout the study and solid protection against virulent FPV 7.5 years after vaccination. Vaccinated cats were seropositive against FHV and FCV for 3 to 4 years after vaccination, with gradually declining titers. Vaccinated cats were protected partially against viral challenge with virulent FHV. Relative efficacy of the vaccine, on the basis of reduction of clinical signs of disease, was 52%. Results were similar after FCV challenge, with relative efficacy of 63%. Vaccination did not prevent local mild infection or shedding of FHV or FCV. CONCLUSIONS: Duration of immunity after vaccination with an inactivated, adjuvanted vaccine was > 7 years. Protection against FPV was better than for FHV and FCV. CLINICAL IMPLICATIONS: Persistence of antibody titers against all 3 viruses for > 3 years supports recommendations that cats may be revaccinated against FPV-FHV-FCV at 3-year intervals.

Potential risk of oral insulin with adjuvant for the prevention of Type I diabetes: a protocol effective in NOD mice may exacerbate disease in BB rats.

The impact of oral treatment with insulin on disease development was studied in diabetes prone BB rats. Because of the positive outcome of a prior study in non obese diabetic (NOD) mice, BB rats received insulin in combination with a bacterial adjuvant. Porcine insulin was given orally twice weekly from 35-100 days of age, the E. coli preparation OM-89 was fed on alternate days. Other groups received vehicle, the bacterial adjuvant, or insulin alone. Both insulin containing oral dosing regimens induced a transient non significant delay in diabetes onset. Insulin alone, however did not decrease the final diabetes incidence. Oral dosing with insulin plus adjuvant caused exacerbation of disease development as judged from the decreased survival rate in comparison with the insulin treated group (p < 0.05). Intra-islet infiltration also increased (p < 0.005) compared with the insulin or vehicle treated groups. The effect correlated with enhanced interferon gamma (IFNgamma) and decreased interleukin 10 (IL-10) gene expression in the gut suggesting a shift towards proinflammatory T helper 1 (Th1) reactivity (p < 0.01). Although treatment with adjuvant alone also increased the degree of insulitis, an enhanced incidence of diabetes and a shift in cytokine expression was only seen in the group receiving insulin plus adjuvant. Taken together, the data suggest that treatment with a bacterial adjuvant and oral insulin may alter the gut immunoregulatory state such that disease promoting rather than protective immune responses are induced.

Impact of dietary fat on Th1/Th2 cytokine gene expression in the pancreas and gut of diabetes-prone BB rats.

The effect of dietary n-3 or n-6 polyunsaturated fatty acids on the development of autoimmune insulitis was analysed in diabetes-prone BB rats. Litter-matched groups of rats received a standard open formula NIH-07 (National Institutes of Health, NIH) diet enriched with 10% fish oil, 10% flaxseed oil or with 10% palm oil plus 2% cholesterol during the period of insulitis onset (50-70 days of age). Analysis of cytokine gene expression in pancreatic RNA revealed an increase of IFN-gamma and a decrease of IL-10 mRNA with onset of insulitis. When compared to unsupplemented NIH, none of the three fat-enriched diets depressed the rise of IFN-gamma gene expression or the influx of leukocytes into islets. However, all of the fat-enriched diets led to significantly higher IL-10 mRNA levels. Although a specific anti-inflammatory effect of fish oil was not seen in the pancreas, a clear shift of the Th1/Th2 cytokine mRNA ratio towards Th2 was seen in the gut-associated immune system. We conclude that diets high in fat support IL-10 without suppressing IFN-gamma gene expression in islet inflammation. A special anti-inflammatory effect of fish oil was not seen in pancreatic lesions of BB rats, although there was strong modulation of the IFN-gamma/IL-10 mRNA ratio in the gut associated immune system.

Raccoon poxvirus live recombinant feline panleukopenia virus VP2 and rabies virus glycoprotein bivalent vaccine.

A raccoon poxvirus (RCNV) recombinant for immunizing against feline panleukopenia and rabies was developed by homologous recombination with a chimeric plasmid for insertional inactivation of the RCNV thymidine kinase gene. The recombinant, RCN-FPV/VP2-rabG, coexpressed the feline panleukopenia virus (FPV) VP2 protein and the rabies virus spike glycoprotein (rabG) under oppositely oriented vaccinia virus P11 promoters. Cats vaccinated subcutaneously with the recombinant showed relatively high neutralizing antibody responses against rabies virus and FPV, and protection against an otherwise virulent FPV challenge with no drop in white blood cell count. Because of containment constraints, no rabies virus challenges were done, but the high concentrations (> 8 IU) of rabies neutralizing antibodies were consistent with levels that usually indicate an ability to counter the infection.