Fraser W. Scott

Effect of dose and modification of viscous properties of oat gum on plasma glucose and insulin following an oral glucose load

An extract from oats known as oat gum (OG) is composed mainly of the polysaccharide (1-->3) (1-->4)-beta-D-glucan, which is highly viscous in aqueous solution. Viscous polysaccharides are known to attenuate postprandial plasma glucose and insulin responses. The purposes of this study were to determine the dose-response to OG and establish quantitatively the effect of viscosity on plasma glucose and insulin levels of healthy humans consuming 50 g glucose. Increasing the dose of OG successively reduced the plasma glucose and insulin responses relative to a control without gum. Reduction of the viscosity of OG by acid hydrolysis reduced or eliminated the capacity to decrease postprandial glucose and insulin levels. The ability of OG to modify glycaemic response was unchanged following agglomeration in the presence of maltodextrin. Agglomerated gum dispersed smoothly in a drink without formation of lumps, and development of maximum viscosity was delayed. These properties improve palatability. There was a highly significant linear relationship between log[viscosity] of the mixtures consumed and the glucose and insulin responses. The relationship shows that 79-96% of the changes in plasma glucose and insulin are attributable to viscosity, and that changes occur at relatively low doses and viscosities.

Potential Mechanisms by Which Certain Foods Promote or Inhibit the Development of Spontaneous Diabetes in BB Rats: Dose, Timing, Early Effect on Islet Area, and Switch in Infiltrate From Th1 to Th2 Cells

Certain diets can have major effects on the development of IDDM in DP-BB rats, but data are scant on the timing, dose, and mechanisms involved. We therefore determined the dose response, timing, and duration of exposure required to induce diabetes, and characterized the effects of nutritionally adequate diets with widely different diabetogenicity on the pancreatic islet area and cytokines. DP-BB rats were fed a diabetogenic, cerealbased, NIH-07 (NIH) diet or a protective, casein or hydrolyzed casein (HC)-based, semipurified diet. Rats were fed from weaning to 50 or 100 days with the HC diet and then switched to the NIH diet, or fed the NIH diet from weaning to 50 days and switched to the HC diet. Pancreas histology and diabetes outcome were determined. Semiquantitative morphometric analyses of hematoxylin and eosin–stained sections of pancreas from 41-day-old rats were also carried out. Diet-induced effects on pancreatic cytokine levels were measured at 70 days using reverse transcriptase–polymerase chain reaction analysis of γ-interferon (IFN-γ), interleukin-10 (IL-10), and transforming growth factor-β (TGF-β). Long-term daily exposure, particularly around the beginning of puberty to late adolescence (50–100 days), was important for development of diabetes. DP-BB rats could be rescued from diabetes development by feeding them a low-diabetogen HC diet as late as 50 days. Diabetes frequency was highest in rats fed 70% and 100% NIH diets. By age 41 days, before classic insulitis, the islet area in HC-fed DP-BB rats was 65% greater than in NIH-fed rats. By 70 days, when mononuclear cells were visible in the islets of most NIH-fed, but not HC-fed rats, the more pronounced inflammatory process in NIH-fed rats was associated with a Thl cytokine pattern (high IFN-γ and low IL-10 and TGF-β), whereas the pancreases of HC-fed rats showed fewer infiltrating cells, low levels of IFN-7, and high levels of TGF-p, typical of a Th2 cytokine pattern. Thus dietary modification can occur as late as puberty. Further, long-term exposure to sufficient amounts of food diabetogens between 50 and 100 days was required for maximum diabetes induction. The islet area was modified by diet before signs of classic insulitis. Pancreatic inflammation in NIH-fed animals is a Thl-dependent phenomenon. The HC diet inhibited insulitis and was associated with a Th2 cytokine pattern in the pancreas, protecting diabetes-prone rats from developing diabetes.

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

Summary 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 (IFNγ) 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. [Diabetologia (1998) 41: 844–847]

Cytokine gene expression in the BB rat pancreas: natural course and impact of bacterial vaccines

Summary In diabetes prone BB rat pancreas the Th1/Th2 cytokine balance and the expression of inducible nitric oxide synthase (iNOS) was determined by mRNA analysis before and after the onset of insulitis. Specific mRNA was amplified by reverse transcriptase polymerase chain reaction, quantitated with radiolabelled probes by phosphoimaging and calibrated with the amount of co-amplified β -actin mRNA. At 50 days of age, prior to recognizeable insulitis, there was already significantly enhanced expression of both, Th1 and Th2 cytokines, and of iNOS mRNA, when compared to Wistar rat pancreas (p < 0.001). This supports the concept of an inconspicuous early phase of islet infiltration by single immunocytes, called single cell insulitis. At 70 days of age mononuclear infiltration of islets had begun and was associated with upregulation of interferon γ (IFNγ ) and iNOS, but downregulation of interleukin-10 and transforming growth factor β mRNA (p < 0.001). These findings correlate the onset of insulitis with a shift of the Th1/Th2 cytokine balance towards Th1 cell reactivity. Indeed there was a close correlation of the Th1/Th2 cytokine ratio but not of absolute IFNγ mRNA levels with the insulitis score. Vaccination at day 50 with tetanus toxoid did not affect cytokine gene expression while diphtheria toxoid and even more strongly BCG administration induced a shift towards Th2 reactivity (p < 0.001) while iNOS mRNA was decreased (p < 0.01). Oral dosing with immunostimulatory components of Escherichia coli also changed the quality of inflammation. Oral lipopolysaccharide (LPS) from E. coli and OM-89, an endotoxin free extract containing immunostimulatory glycolipopeptides and heat shock protein (hsp) 65, both downregulated IFNγ mRNA while only OM-89 in addition suppressed iNOS mRNA and enhanced Th2 cytokine gene expression (p < 0.001). We conclude that the onset of insulitis is associated with a shift towards Th1 cytokine and iNOS gene expression. Diphtheria toxoid and BCG vaccination stimulates Th2 reactivity but does not downregulate Th1. The latter can be achieved through oral administration of LPS or a glycopeptide fraction (OM-89) from E. coli. [Diabetologia (1996) 39: 1448–1454]

Flow cytometric analysis of intracellular IFN-γ, 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.

Intervention in autoimmune diabetes by targeting the gut immune system

BB rats and nonobese diabetic (NOD) mice spontaneously develop autoimmune insulin dependent diabetes and serve as models for human type I diabetes. During progression of the disease the cytokine pattern elaborated by islet infiltrating immune cells shifts from a Th2 or Th0 toward Th1 type. Only the latter is associated with destructive insulitis. We discuss here attempts to modulate disease progression by targeting the gut immune system with bacterial immunostimulants. Oral dosing of diabetes prone BB rats with lipopolysaccharide (LPS) or the Escherichia coli extract OM-89 lead to a Th2-shift of pancreatic mRNA expression. In vitro studies showed that repeated exposure toward LPS or OM-89 lead to downregulation of proinflammatory macrophage responses. In the NOD mouse, repeated oral dosing of OM-89 caused a Th2 shift in the gut cytokine gene expression, probably because of desensitization of macrophages and other antigen presenting cells. Concomitantly, diabetes prevention by oral insulin was improved. In conclusion, oral dosing with bacterial immunostimulants dampens Th1 type immune reactivities of the gut immune system and thereby promotes oral tolerance mechanisms. Downregulation of proinflammatory immune reactivities by repeated exposure to bacterial stimulants requires intact desensitization mechanisms in macrophages or other antigen presenting cells.

Insulin Therapy of Prediabetes Suppresses TH1 Associated Gene Expression in BB Rat Pancreas

Subcutaneous insulin treatment of young diabetes prone BB rats has been shown previously to suppress the development of autoimmune diabetes. In this study the hypothesis was tested that exogenous insulin may deviate the autoimmune process by acting on the Th1/Th2 cytokine balance in the pancreas. BB rats were implanted with pellets which continuously released insulin, at 50 d of age. Three weeks later cytokine mRNA expression in the pancreas and insulitis score were determined. While in control BB rats high levels of IFNgamma mRNA were detectable by RT-PCR, insulin treatment almost completely suppressed IFNgamma mRNA levels without concomitant upregulation of counterregulatory IL-10 and TGFbeta gene expression. Insulin also suppressed gene expression of inducible nitric oxide synthase. Mean insulitis scores were decreased after insulin treatment. We conclude that the protective effects of insulin treatment may not be due to the induction of protective Th2 immune reactivity but to general downregulation of immune activation in the pancreas, and hence also of Th1 autoimmunity.

Food and the development of autoimmune disease

Abstract Autoimmune diseases, where the immune system inappropriately reacts against the bodys own components, may be affected by foods. The identification and characterization of the food factors involved is advancing and this information may provide practical solutions to understanding the pathogenesis and nutritional epidemiology of autoimmune diseases.