Knud Josefsen

Gut Microbiota Composition Is Correlated to Grid Floor Induced Stress and Behavior in the BALB/c Mouse

Stress has profound influence on the gastro-intestinal tract, the immune system and the behavior of the animal. In this study, the correlation between gut microbiota composition determined by Denaturing Grade Gel Electrophoresis (DGGE) and tag-encoded 16S rRNA gene amplicon pyrosequencing (454/FLX) and behavior in the Tripletest (Elevated Plus Maze, Light/Dark Box, and Open Field combined), the Tail Suspension Test, and Burrowing in 28 female BALB/c mice exposed to two weeks of grid floor induced stress was investigated. Cytokine and glucose levels were measured at baseline, during and after exposure to grid floor. Stressing the mice clearly changed the cecal microbiota as determined by both DGGE and pyrosequencing. Odoribacter, Alistipes and an unclassified genus from the Coriobacteriaceae family increased significantly in the grid floor housed mice. Compared to baseline, the mice exposed to grid floor housing changed the amount of time spent in the Elevated Plus Maze, in the Light/Dark Box, and burrowing behavior. The grid floor housed mice had significantly longer immobility duration in the Tail Suspension Test and increased their number of immobility episodes from baseline. Significant correlations were found between GM composition and IL-1α, IFN-γ, closed arm entries of Elevated Plus Maze, total time in Elevated Plus Maze, time spent in Light/Dark Box, and time spent in the inner zone of the Open Field as well as total time in the Open Field. Significant correlations were found to the levels of Firmicutes, e.g. various species of Ruminococccaceae and Lachnospiraceae. No significant difference was found for the evaluated cytokines, except an overall decrease in levels from baseline to end. A significant lower level of blood glucose was found in the grid floor housed mice, whereas the HbA1c level was significantly higher. It is concluded that grid floor housing changes the GM composition, which seems to influence certain anxiety-related parameters.

Dependence of Antigen Expression on Functional State of β-Cells

Antigen expression corresponding to anti-islet cell surface monoclonal antibodies IC2 and A2B5 was studied. IC2 is a rat-rat hybridoma autoantibody produced from the BB rat; among islet cells, IC2 is β-cell specific. A2B5 is an anti-ganglioside antibody described as labeling β-cells. Islets of Langerhans from Lewis rats were isolated and cultured for 18 h in RPMI-1640 with five different glucose concentrations (2.2, 3.3, 5.5, 11.1, and 18.3 mM). In some experiments, islets were precultured for 2 or 3 days. After isolation of islet cells and antibody labeling, the percent of IC2+ β-cells in the different groups increased from 33.3, 34.5, 40.9, and 57.2 to 58.6% (P < 10−6). For A2B5, the percent of labeled islet cells increased from 37.4, 41.8, 46.7, and 53.8 to 56.2% (P < 10−4). Thus, increasing glucose concentration leading to higher β-cell activity implies an increase in antigen expression. Neither A2B5 nor IC2 reacts with insulin, as shown by absorption experiments and immune electron microscopy of binding sites. Electron microscopy of IC2-gold-labeled islet cells substantiated the β-cell specificity of IC2. In conclusion, expression of the corresponding antigens to IC2 and A2B5 depends on the functional state of the β-cells; because this has been shown to be an important factor in the development of insulin-dependent diabetes, our findings may be of potential pathogenetic interest.

A Possible Link between Food and Mood: Dietary Impact on Gut Microbiota and Behavior in BALB/c Mice

Major depressive disorder is a debilitating disease in the Western World. A western diet high in saturated fat and refined sugar seems to play an important part in disease development. Therefore, this study is aimed at investigating whether saturated fat or sucrose predisposes mice to develop behavioral symptoms which can be interpreted as depression-like, and the possible influence of the gut microbiota (GM) in this. Fourty-two mice were randomly assigned to one of three experimental diets, a high-fat, a high-sucrose or a control diet for thirteen weeks. Mice on high-fat diet gained more weight (pu200a=u200a0.00009), displayed significantly less burrowing behavior than the control mice (pu200a=u200a0.034), and showed decreased memory in the Morris water maze test compared to mice on high-sucrose diet (pu200a=u200a0.031). Mice on high-sucrose diet burrowed less goal-oriented, showed greater latency to first bout of immobility in the forced swim test when compared to control mice (pu200a=u200a0.039) and high-fat fed mice (pu200a=u200a0.013), and displayed less anxiety than mice on high-fat diet in the triple test (pu200a=u200a0.009). Behavioral changes were accompanied by a significant change in GM composition of mice fed a high-fat diet, while no difference between diet groups was observed for sucrose preferences, LPS, cholesterol, HbA1c, BDNF and the cytokines IL-1α, IL-1β, IL-6, IL-10, IL-12(p70), IL-17 and TNF-α. A series of correlations was found between GM, behavior, BDNF and inflammatory mediators. In conclusion, the study shows that dietary fat and sucrose affect behavior, sometimes in opposite directions, and suggests a possible association between GM and behavior.

Sulphatide and sulphatide antibodies in insulin-dependent diabetes mellitus

Insulin-dependent diabetes mellitus (IDDM) is associated with neurological disorders. Sulph I, a monoclonal antibody to sulphatide (a neural epitope), stained secretory granules in alpha and beta cells of rat islets of Langerhans, but not exocrine tissue. Sera from 88% of 57 newly diagnosed IDDM patients was anti-sulphatide positive, and 76% were positive 6 months later. All 135 healthy controls were negative. Sulphatide antibody may be an IDDM marker.

Sulphatide in islets of Langerhans and in organs affected in diabetic late complications: a study in human and animal tissue

SummarySulphatide has been found in rat islets of Langerhans and anti-sulphatide antibodies have been demonstrated in patients with insulin-dependent diabetes mellitus. Using a specific monoclonal antibody, Sulph I, directed against sulphatide, we investigated the in situ distribution of this glycolipid immunohistochemically; furthermore, the sulphatide concentration was determined in several organs and cells by thin-layer chromatography. The islets of Langerhans in all species examined, mouse, rat, pig, and monkey were intensively stained but exocrine tissue remained unlabelled. The sulphatide concentration in human islets was 150±46 pmol/100 islets. The only glycolipid-antigen detected was sulphatide. Regarding other tissues, sulphatide was found to be located in distal tubules in the kidney, peripheral nerves, distinct scattered spot-like structures in the choreoid layer of the eye, the ovum, and peripheral granulocytes. Sulph I injection in mice showed homing to kidney tubules. Lung, heart, liver, adrenal, spleen, lymph node and thymus were not stained by Sulph I. Thus, the distribution of sulphatide shows an association with organs known to be affected in diabetes, either initially or in late complications.

Dietary gluten and the development of type 1 diabetes

Gluten proteins differ from other cereal proteins as they are partly resistant to enzymatic processing in the intestine, resulting in a continuous exposure of the proteins to the intestinal immune system. In addition to being a disease-initiating factor in coeliac disease (CD), gluten intake might affect type 1 diabetes development. Studies in animal models of type 1 diabetes have documented that the pathogenesis is influenced by diet. Thus, a gluten-free diet largely prevents diabetes in NOD mice while a cereal-based diet promotes diabetes development. In infants, amount, timing and mode of introduction have been shown to affect the diabetogenic potential of gluten, and some studies now suggest that a gluten-free diet may preserve beta cell function. Other studies have not found this effect. There is evidence that the intestinal immune system plays a primary role in the pathogenesis of type 1 diabetes, as diabetogenic T cells are initially primed in the gut, islet-infiltrating T cells express gut-associated homing receptors, and mesenteric lymphocytes transfer diabetes from NOD mice to NOD/severe combined immunodeficiency (SCID) mice. Thus, gluten may affect diabetes development by influencing proportional changes in immune cell populations or by modifying the cytokine/chemokine pattern towards an inflammatory profile. This supports an important role for gluten intake in the pathogenesis of type 1 diabetes and further studies should be initiated to clarify whether a gluten-free diet could prevent disease in susceptible individuals or be used with newly diagnosed patients to stop disease progression.

Sulphatide antigen in islets of Langerhans and in diabetic glomeruli, and anti-sulphatide antibodies in type 1 diabetes mellitus.

Clinical coincidence between diabetes and neurological disorders, and sharing of antigen determinants between islets of Langerhans and neural tissue, has been suggested. Sulphatide is a neural epitope which can be visualized with a monoclonal antibody Sulph I. Different tissues were examined by immunohistological methods. Sulphatide and anti‐sulphatide antibodies were determined by thin‐layer chromatographic techniques. IgG was isolated using protein A columns. A specific staining by Sulph I was found of rat islets, assigned to the secretory granules of both alpha and beta cells. No labelling of the exocrine tissue or other body tissues was seen, except for nerve and kidney structures. The latter showed staining of the distal tubules and, in addition, but only in the diabetic kidney, of glomeruli located in the subendothelial area in the capillary loops and the mesangial space. Sera from 38% of 40 spontaneously diabetic BB rats displayed anti‐sulphatide antibodies, mainly IgG, whereas all 30 control Lewis rats were negative. Most recently we have demonstrated anti‐sulphatide antibodies in 88% of 57 patients with newly diagnosed Type 1 diabetes (titres of > 1:400); all 135 healthy control persons were negative. The sulphatide antibody reactivity was present in the IgG fractions of the patients sera. Thus, sulphatide is demonstrated in islets of Langerhans and in kidney related to the diabetic lesion, and, furthermore, anti‐sulphatide antibodies exist in Type 1 diabetes mellitus.

Prevention of diabetes mellitus in BB rats by neonatal stimulation of β cells.

Abstract Antigen expression in type 1 (insulin-dependent) diabetes may depend on the functional state of β cells. At birth, β cells are immature, produce only a basal amount of insulin, and are unresponsive to glucose—but are sensitive to glucagon and arginine. β cells of spontaneously diabetic BB rats were stimulated for the first 6 days after birth by glucose with glucagon or arginine to accelerate β cell maturation, and possibly to induce antigen expression and tolerance. Over the first 200 days of life, only 23% of glucose and glucagon-treated BB rats, and 20% of glucose and arginine-treated BB rats developed diabetes, compared with 65% of untreated controls. This finding may explain the observation that children of mothers who have type 1 diabetes are three times less likely to develop the disease than children of fathers with type 1 diabetes. Earlier maturation of β cells during the diabetic pregnancy may protect against diabetes in later life.

Reduction of diabetes incidence in NOD mice by neonatal glucose treatment

The aim of this study was to investigate whether neonatal glucose treatment influences the incidence of diabetes in NOD mice. Thirty‐nine NOD mice (19 males, 20 females) were treated with 8 g glucose/ kg BW/day administered by subcutaneous injections twice a day for the first six days of life. Thirty‐six untreated NOD mice (20 males, 16 females) served as a control group. In the glucose‐treated group, 33% became diabetic compared with 58% in the control group (X2= 5.3, p = 0.021). Among the glucose‐treated males, 16% became diabetic compared with 50% of the untreated males (X2= 5.5, p = 0.019), whereas 50% of the glucose‐treated females became diabetic compared with 69% of the untreated females (X2= 1.1, NS). We conclude that neonatal glucose treatment can reduce the diabetes incidence in NOD mice. These results could have implications for the prevention of type 1 diabetes mellitus in humans.

Presence of sulphatide (3′-sulphogalactosylceramide) in pericytes in the choroid layer of the eye: sharing of this glycolipid autoantigen with islets of Langerhans

SummaryThe aim of the study was to investigate the distribution in the eye of sulphatide, an acid glycolipid which has previously been demonstrated in islets of Langerhans, nervous tissue and in kidney glomeruli of diabetic patients, and against which antibodies have been found in patients with newly diagnosed insulin-dependent diabetes mellitus. A specific monoclonal antibody, Sulph I, was used for detection of sulphatide by thin-layer chromatography, and light and electron microscope immunohistochemistry. A distinct, patchy staining was found in the choroid layer and the ciliary processes. The antigen was confirmed to be sulphatide and its concentration in human eyes was 30 nmol sulphatide/g wet tissue. By electron microscopy, anti-sulphatide choroid labelling was demonstrated in pericytes and in smooth muscle cells surrounding vessels. No Sulph I-negative pericytes were seen. Double labelling with Sulph I and anti-smooth muscle actin revealed that only pericytes in the eye contained sulphatide and not those in heart, lung, liver, adrenal, spleen, lymph node, thymus, or pancreatic tissue. Thus, sharing of the autoantigen sulphatide has been demonstrated between islets of Langerhans and pericytes in the choroid layer of the eye.