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Changes in Intestinal Morphology and Permeability in the BioBreeding Rat Before the Onset of Type 1 Diabetes

Neu, Josef; Reverte, Christopher M; Mackey, Amy D; Liboni, Kellym; Tuhacek-Tenace, Lauren M; Hatch, Marguerite; Li, Nan; Caicedo, Ricardo A; Schatz, Desmond A; Atkinson, Mark

Journal of Pediatric Gastroenterology and Nutrition: May 2005 - Volume 40 - Issue 5 - p 589-595
doi: 10.1097/01.MPG.0000159636.19346.C1
Original Articles

Objective: Type 1 diabetes is an autoimmune disorder that occurs in genetically susceptible individuals. It has been hypothesized that the disease could be triggered by environmental agents that gain entry into the body through small intestinal absorption. Increased intestinal permeability has been reported both in spontaneous animal models of type 1 diabetes and human type 1 diabetes. In these studies, we examined both the physical and functional permeability characteristics of the small intestine in diabetes-prone and control rats.

Methods: In a series of studies, BioBreeding diabetes-prone(n = 31), BioBreeding diabetes-resistant (n = 20) and control Wistar (n = 25) rats were examined at intervals from 21 to 125 days of age.

Results: The percentage of goblet cells and the mucosal crypt depth were significantly greater in BioBreeding diabetes-prone than BioBreeding diabetes-resistant rats (P < 0.001 and P = 0.01, respectively). BioBreeding diabetes-prone and BioBreeding diabetes-resistant rats expressed less of the tight junction protein claudin (P < 0.05) and exhibited greater intestinal permeability (P < 0.001) than did Wistar rats. Intestinal permeability measured both in vivo and ex vivo decreased in all rat strains as age increased (P < 0.001).

Conclusions: In a genetically susceptible rodent model of diabetes, early increased intestinal permeability might allow unregulated passage of environmental antigens that could potentially trigger the autoimmune response leading to type 1 diabetes.

Department of Pediatrics and Department of Pathology, University of Florida, Gainesville, Florida

Received October 12, 2004; accepted February 7, 2005.

Address correspondence and reprint requests to Josef Neu, College of Medicine, Department of Pediatrics, Division of Neonatology, University of Florida, PO Box 100296, 1600 SW Archer Road, Gainesville, FL 32610. (e-mail:

Supported by grants from the Children's Miracle Network and the National Institutes of Health, Bethesda, Maryland (No. AI42288).

Please note these rats were obtained from Biomedical Research Models; a different nomenclature, BBdp and BBc is used for outbred nondiabetic strains maintained in Canada.

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Type 1 diabetes mellitus is an immunologically mediated disorder that occurs in genetically susceptible individuals (1). Although much progress has occurred in defining the immunologic markers that predict disease and genetic loci that determine susceptibility to the disorder, little progress has been made in defining the triggers and the actual processes that might facilitate triggering the autoimmune destruction of pancreatic islets. Multiple environmental triggers have been proposed, but no single candidate has emerged (2). Recent evidence has suggested that the small intestine might play a role in the events leading to the development of type 1 diabetes (3,4). It is hypothesized that an underlying structural or developmental defect in the intestine may facilitate the entry of multiple agents that have the potential to influence (or trigger) the disease process.

The small intestine is an important immune organ, providing a massive surface area where the external environment and the internal aspects of the body can interact (5,6). In healthy individuals, the surface barrier of the intestine acts as an efficient gatekeeper allowing the absorption of nutrients and preventing the passage of potential dietary, bacterial and viral antigens. In individuals genetically predisposed to diabetes, gut barrier function might be impaired. Indeed, increased permeability, or leakiness, facilitates the entry of potentially antigenic substances that can initiate a host immunologic response that ultimately might lead to islet cell autoimmunity and diabetes. Increased intestinal permeability in type 1 diabetes has been reported in humans (7,8) and in spontaneous models of the disease (9). Other inflammatory or autoimmune conditions such as celiac disease (10,11) and Crohn disease (12) are also associated with compromised gastrointestinal permeability. In addition, there is an increased frequency of celiac disease associated with type 1 diabetes (13-15).

Paracellular gut permeability is regulated by cell-to-cell contacts known as tight junctions. More than 40 different proteins have been identified to reside in the tight junctions of epithelia (16). There is evidence that the expression and function of these proteins are controlled through a series of signaling molecules (17). Two major transmembrane proteins of the intestinal tight junction, claudin and occludin, participate in the structural integrity of the tight junction and regulate permeability of the intestinal-mucosa surface. Although occludin (molecular weight 62-82 kDa) (18-20) is present in abundance in epithelial tight junctions, its role in regulating permeability is not fully understood. This protein binds to the intracellular tight junction protein zonula occludens 1 (ZO-1), the critical connection that links the intramembrane tight junction components to the intracellular cytoskeletal actin filaments (21). Claudins also are integral protein components of the tight junction complex. These proteins are thought to partner with occludin to seal the intercellular space of epithelia (22). Claudins are reported to have greater adhesion properties than occludin, which makes them well-suited to close the paracellular path (23). A total of 24 different claudins thus far have been identified (molecular weight 20-27 kDa), each expressed differently in various tissues and species, with claudin-1 as a major form in the intestine (24).

Diabetes-prone BioBreeding (BBDP) rats from Biomedical Research Models (Worcester, MA) are a good model of human type I diabetes. A majority of these animals(>90%) spontaneously develop diabetes (i.e., overt glycosuria, hyperglycemia and ketosis) beginning at approximately 70 days of age (25). BioBreeding diabetes-resistant (BBDR) rats are genetically similar to BBDP rats but do not develop spontaneous diabetes as readily as BBDP rats and require an added stimulus such as exposure to a virus to develop diabetes. All BioBreeding rats are descended from a colony of outbred Wistar rats at the BioBreeding Laboratories in which spontaneous hyperglycemia and ketosis appeared 30 years ago (26). Previous studies using a different strain of BioBreeding rat developed in Canada (the BBdp rat) have suggested that diabetes-prone BioBreeding rats fed a standard rodent diet exhibit increased gastrointestinal permeability to the low-molecular-weight sugar markers lactulose and mannitol beginning at day 50 (9). We hypothesized that abnormalities in the tight junction protein complex would increase the likelihood that dietary, bacterial or viral antigens might cross the intestinal barrier before the onset of diabetes. We therefore conducted a series of studies to examine the expression of the tight junction proteins claudin and occludin and to assess permeability and morphologic characteristics in the intestine longitudinally in BBDP and BBDR rats and their progenitor Wistar rats before the onset of diabetes.

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Male BioBreeding diabetes-prone (BBDP) and BioBreeding diabetes-resistant (BBDR) rats 21 to 30 days of age were purchased from Biomedical Research Models, Inc. (Worcester, MA). Male Wistar rats (Hsd:WI) (21 to 30 days of age) were purchased from Harlan (Indianapolis, IN). Rats were kept in conventional housing that was maintained at constant temperature with a 12-h light/dark cycle. A total of 31 BBDP, 20 BBDR and 25 Wistar rats were studied. Animals had free access to a commercially available rodent diet (Harlan Teklad, Indianapolis, IN) and water. The Institutional Animal Care and Use Committee at the University of Florida approved all procedures.

Onset of diabetes was assessed by monitoring rats for polyuria, polydipsia and/or lethargy. Diabetes was diagnosed clinically using urinalysis reagent strips to measure urine concentrations of glucose and/or ketone (Multistix 9 SG; Bayer Corp., Pittsburgh, PA). Rats with glucose concentration >1000 mg/dL and/or ketone concentrations >40 mg/dL were considered diabetic.

Rats were anesthetized with intraperitoneal sodium pentobarbital (50 mg/kg) and exsanguinated by cardiac puncture. For each diabetic rat, BBDR and/or Wistar rats were used as controls. If diabetes developed before the end of the experimental period, the diabetic rat was immediately killed as described above. For anatomic studies, the small intestine was harvested and flushed with phosphate-buffered saline at 4°C to remove intraluminal contents. The small intestine was divided into 3 equal sections to demarcate the duodenum, jejunum and ileum. Small pieces of each section were used for histologic analyses, and the remaining intestine was cut longitudinally to obtain the mucosa by scraping with a glass slide. All procedures were done on ice or at 4°C to minimize proteolysis.

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Intestinal segments approximately 5 cm long from the duodenum, jejunum and ileum were fixed in 10% (v/v) neutral buffered formalin for 24 hours for light microscopy. The intestine was paraffin embedded, cut into 4-μm sections, mounted on glass slides and stained with hematoxylin and eosin according to standard procedures. Villus length and width and crypt depth were measured using a light microscope. Intestinal sections were also analyzed for goblet cells expressed as goblet cells per total cells within a villus.

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Permeability Experiments

In vivo permeability.

Lactulose and mannitol (Sigma-Aldrich Co.; St. Louis, MO). were used as markers of paracellular and transcellular intestinal permeability, respectively. Rats (aged 30 to 50 days) were placed in metabolic cages (Nalgene; Rochester, NY) and given the sugars in their drinking water to provide 120 mg lactulose and 80 mg mannitol as described by Meddings et al. (9). There were no major differences in the volume of treated drinking water consumed by the animals, and the quantity provided was consumed within 24 hours. Urine was subsequently collected for 24 hours, and no significant differences in urine output among the rats were observed. Each urine sample filtered using a 0.45 micrometer syringe filter and stored at −80°C until analyses. Lactulose and mannitol were quantified by amperometric high performance liquid chromatography (9).

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Ex vivo permeability.

Flux experiments were conducted using proximal and distal mid-ileum (3 sections from each segment) removed from diabetes-prone and matched non-diabetes prone rats at 30 and 60 days of life. Each group consisted of 2 rats. As described previously (27), flat sheets of intestinal tissue were mounted in modified Ussing chambers with an exposed tissue area of 0.64 cm2. The sheets were bathed on both sides by 10 mL of standard saline solution at 37°C that was vigorously circulated by bubbling with a gas mixture of 95% O2/5% CO2. The standard saline contained the following solutes (mmol/L): Na+ 139.4, K+ 5.4, Ca2+ 1.2, Mg2+ 1.2, Cl 123.2, HCO3 21.0, H2PO4 0.6, HPO2− 2.4 and glucose 10. The mucosal to serosal flux of mannitol (nmol·cm−2·h−1) was measured using 14C-mannitol D-[1-14C] mannitol (specific activity of 58 mCi/mmol (2.15 GBq/mmol) from Amersham Pharmacia Biotech) as a marker indicating alterations in paracellular pathway permeability. Each tissue was spiked with 1 microCurie. Fluxes of mannitol were measured in the mucosal to serosal direction and the concentration of mannitol in the solution bathing the tissue was 0.5 mM. The magnitude of the unidirectional flux (J) was measured for a control period of 45 minutes (Per I) at 15-minute intervals, under short-circuit conditions. The electrical parameters of the tissue were also recorded at 15-minute intervals throughout the entire experiment. Tissue conductance (GT, mS·cm−2) was calculated as the ratio of the open-circuit potential (mV) to the short-circuit current (Isc, μA·cm−2).

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Western Blot Analyses

The remaining intestine was cut longitudinally and placed on a glass plate chilled on ice. Mucosa was obtained by scraping luminal surface with a glass slide. Mucosa was weighed and promptly transferred to phosphate-buffered saline containing a general use protease-inhibitor cocktail (Sigma Chemical; St. Louis, MO). Mucosa was homogenized with a Polytron homogenizer and homogenates were stored in aliquots at −80°C until analyses. Gel electrophoresis was performed using the Bio-Rad electrophoresis system (Carlsbad, CA). Intestinal protein samples (30 μg) and pre-stained protein standards (Kaleidoscope; Bio-Rad) were loaded on a 7.5% (w/v) acrylamide Criterion pre-cast gel (Bio-Rad) and electrophoresed at 100 V for ~2 hours. Protein bands were electroblotted onto a polyvinyldifluoride membrane (Millipore Corporation; Bedford, MA) at 100 V for 1.5 hours. Visualization of the protein bands was performed by staining the membrane with amido black to confirm transfer. Polyvinyldifluoride membranes were blocked for 1 hour in 5% (w/v) non-fat dried milk in Tris-buffered saline containing 0.1% Tween-20 (Fisher Scientific; Atlanta, GA). Using fresh non-fat dried milk in Tris-buffered saline containing 0.1% Tween-20, the membranes were incubated with rabbit antioccludin or anticlaudin antibodies obtained from Zymed Laboratories (San Francisco, CA) at 1:1000 dilution overnight at 4°C with gentle rocking. Membranes were washed with multiple changes of Tris-buffered saline containing 0.1% Tween-20 and subsequently incubated with a goat-anti-rabbit HRP-conjugated secondary antibody (Santa Cruz Biotechnology; Santa Cruz, CA). Antibody that did not specifically bind to the membrane was removed by multiple washes with in Tris-buffered saline containing 0.1% Tween-20. A chemiluminescent substrate (ECL-Plus) was then applied to the membrane and incubated for 5 minutes at room temperature. Excess substrate was blotted away and protein bands were visualized by exposure of membrane to X-OMAT scientific imaging film (Eastman-Kodak Corporation; Rochester, NY), followed by development using a Kodak M35A X-OMAT processor (Kodak Diagnostic Imaging Inc.; Rochester, NY). Protein bands were quantified by densitometry using Adobe Photoshop software (Adobe; San Jose, CA).

One-way analysis of variance was used to determine statistical differences among intestinal permeability and intestinal morphologic measurements. Multiple comparisons were performed using Bonferroni's test. Two-way analysis of variance was used to determine statistical differences in intestinal morphology among the groups using region of intestine and diabetic status as factors. Two-way analysis of variance was also used to detect statistical differences in measures of intestinal permeability and protein expression among the groups using age and diabetic status as factors. All statistical analyses were done using SigmaStat software (Jandel Scientific; San Rafael, CA).

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Morphological and functional characteristics of BBDP, BBDR and Wistar rat small intestine were determined in separate studies to identify biologic markers that occur before the development of diabetes.

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Intestinal Morphology of BBDP Rats and Age-matched BBDR Rats

Five rats in each group were studied and followed from 21 days of age. BBDP rats were killed at the time they developed diabetes and BBDR control rats were killed at the same time on days 63 (n = 2), 77 (n = 1), 82 (n = 1) and 93 (n = 1). Hematoxylin and eosin stained slides of proximal, middle and distal small intestine were examined for morphology. There was a greater percentage of goblet cells in BBDP compared with BBDR rats (12% versus 9%, P < 0.001) (Fig. 1). BBDP rats also exhibited significantly greater crypt depth in the proximal small intestine compared with the BBDR rats (137 ± 2 versus 126 ± 3 mm, P = 0.01). No differences between the BBDP and BBDR rats were found for villus height or width. Adequate comparison of goblet cells between BB and Wistar rats could not be made because the Wistar rats were larger and had longer villi at the same age as the BB rats.

FIG. 1

FIG. 1

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Intestinal Permeability in BBDP and Control Wistar Rats Measured Ex Vivo

Ex vivo intestinal permeability was measured in BBDP (n = 6) and Wistar (n = 6) rats at 45, 67 and 80 days of age. Two rats were examined at each time point and 2 sections of the middle small intestine were used. As shown in Table 1, there was a significant decline (P < 0.01) in 14C-mannitol permeability in the proximal portion of the middle ileum with increasing age regardless of metabolic status. A similar result was obtained for the distal portion of the middle ileum (data not shown). There were no differences in the flux (Jms) of 14C-mannitol between the BBDP and Wistar rats at 45 or 67 days; however, the BBDP rats exhibited a 27%-lower mannitol permeability (P < 0.05) than did Wistar rats at 80 days (Table 1). Using the assumption that mannitol moves exclusively via the paracellular pathway, the flux results indicate that permeability of this pathway is decreased during development. The fact there were no changes in GT, however, would also suggest that transcellular conductance increases during development, as GT is the sum of cell and paracellular conductances.



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In Vivo Intestinal Permeability

In vivo intestinal permeability (urinary excretion of lactulose and mannitol) was measured in BBDP (n = 7), BBDR (n = 7) and Wistar (n = 6) rats at mean ages 42, 49 and 53 days. Consistent with the ex vivo permeability data, both lactulose and mannitol excretion significantly decreased (P < 0.001) with increasing age, suggesting a developmental maturation in intestinal permeability (Fig. 2). However, BBDP and BBDR rats excreted a significantly greater amount of lactulose than did Wistar rats at 42, 49 and 53 days, implying greater intestinal permeability (P < 0.001). Although urinary mannitol excretion was not significantly different among the rats at 42 (P = 0.155), 49 (P = 0.06) or 53 days (P = 0.141), the urinary lactulose:mannitol excretion ratio was significantly greater in the BBDP and BBDR rats compared with Wistar rats (P = 0.006). Interestingly, there were no differences detected between the BBDP and BBDR rats.

FIG. 2

FIG. 2

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Expression of Tight Junction Proteins

In separate studies, claudin and occludin expression was assessed by immunoblotting in age-matched BBDP (n = 8), BBDR (n = 8) and Wistar (n = 8) rats at different time points. Three rats from each group were examined at 34 days and 4 rats at 41 days of age. Densitometry measurements revealed significantly less claudin-1 protein expression in the BBDP and BBDR rats compared with Wistar rats (P = 0.01) (Fig. 3). There also was a significantly greater abundance of both claudin and occludin in all rats at 34 days than at 41 days of age (data not shown, P = 0.04). No differences in occludin expression were detected among the Wistar, BBDP and BBDR rats (data not shown). No significant differences were seen in actin staining of the same blots between the three groups.

FIG. 3

FIG. 3

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In these studies, we examined the developing small intestines of BBDP, BBDR and Wistar rats to identify potential differences or abnormalities that might occur before the onset of diabetes. We tested the hypothesis that tight junction complexes in the small intestine of diabetes-prone BBDP rats were abnormal and might be associated with increased intestinal permeability. Our reasoning was that increased permeability might allow the entry of dietary, viral or bacterial antigens that could trigger the induction of autoimmunity, leading to beta-cell destruction.

Long-term studies by a number of investigators have established that approximately 90% of BBDP rats will develop type 1 diabetes, a finding consistent with our studies. We compared this genetically susceptible strain with another BioBreeding strain of rats that were not as likely to develop diabetes (BBDR) as the BBDP internal control. Because both the BBDP and BBDR stains originate from a common progenitor colony of Wistar rats, we used Wistar rats as controls for both BBDP and BBDR rats (28). In our study, none of the BBDR rats developed diabetes.

Small intestinal histology of these rats revealed a greater percentage of goblet cells in the BBDP rats than in BBDR rats, suggesting the presence of an inflammatory response before the onset of diabetes. Increased mucus secretion from goblet cells results from the inflammatory response in the small intestine. This finding has previously been demonstrated in streptozotocin-induced diabetic rats (29). The increase in goblet cells in the intestine of BBDP rats in our studies is consistent with previous reports of increased number of goblet cells in the intestine of the spontaneously diabetic Chinese hamster (29) and the streptozotocin-induced diabetic rat (29). More recently, Westerholm-Ormio et al. (30) reported that IL-1, IL-4 and IL-4 mRNA were increased in the cells of the lamina propria in children with type 1 diabetes, also suggesting an inflammatory response in the small intestine. The BBdp rat, a strain different from the BBDP strain used in this study, has been reported to have elevated myeloperoxidase activity and infiltrating neutrophils in the distal small intestine before the development of diabetes (at 45 days) compared with control rats (31). Data from our laboratory (not shown) confirm higher myeloperoxidase in BBDP than in BBDR and Wistar rat intestine at approximately 50 days of age. In BBdp rats fed a diabetogenic diet, there was an age-related increase in peroxidase activity (32), as well as impaired disaccharidase activities compared with controls (33). Collectively, these data demonstrate the presence of inflammation in the small intestine before the onset of diabetes. This is supported by the goblet cell data. However, the percentage of goblet cells in the villi of the Wistar animals was not different from that of the BBDP rats. Whether this was a function of the size of the Wistar rats (the Wistar rats were significantly larger, as were their intestinal villi) compared with the BB rats is speculative. The BBDP and BBDR rats were similar in size at similar ages. To address this question, future studies may include measurement of the levels of cyclooxygenase-2, an important modulator of epithelial permeability and immune responses, in the goblet cells (34).

There are data to support both a structural and functional role of occludin in the tight junction, but its role is not independent of other tight junction proteins comprising the tight junction complex. Barrier function of occludin has been observed in studies involving overexpression of occludin in epithelial cells, which increased transepithelial resistance but did not decrease the permeability of a small molecular weight marker, mannitol (35). In our study, differences in occludin abundance in the Western blot analyses were not detected. Occludin is present in the intestine in many forms (membrane bound, cytosolic, phosphorylated and unphosphorylated) and the antibody used in this study might have detected only one form (36). Further investigation of the abundance and form of occludin present in these rats is warranted.

There was a significantly greater abundance of claudin-1 expressed in the Wistar rats than in either of the BioBreeding strains. The tight junction proteins of the claudin family are emerging as the primary regulators of paracellular permeability in epithelial cells (22). Our claudin-1 results suggest that the intestinal barrier in BBDP and BBDR rats may be compromised in comparison with Wistar rats. Deficiency of this major tight junction structural protein in the BBDP and BBDR small intestines corresponds in time to the change in permeability we observed. There was a developmental decline in both claudin-1 and occludin expression, regardless of strain (data not shown). Because these were derived from mucosal scrapings it is possible that several cell types (e.g., epithelium, endothelium) contributed to the changes, but the specific site was not evaluated in this study. We observed an increase in the lactulose:mannitol urinary excretion ratio at day 49 in BB rats. A decline in the urinary excretion of the lactulose and mannitol occurred as the rats matured. The ex vivo mannitol data also suggest that a developmental change in the small intestine occurs to improve transcellular barrier function in both Wistar and BBDP rats. The lack of a major difference between these two strains suggests that the permeability defect evidenced in the in vivo studies is primarily paracellular. Thus there appears to be a period of time in which compromised gut barrier function may facilitate the triggering of the disease process.

Increased intestinal permeability to sugar markers in diabetes has been reported in both humans (7,8) and rats (9). Although it is difficult to ascertain if the reduced barrier function preceded the disease or if the disease was the underlying cause of intestinal barrier dysfunction, Meddings et al. (9) reported that BBdp rats (the Canadian strain) exhibited increased intestinal permeability at 50 days, before the spontaneous onset of diabetes. Despite the persistence of increased permeability in the BBdp rats, Meddings et al. (9) also observed a decline in the lactulose:mannitol urinary excretion from 60 to 110 days of age, suggesting a developmental sealing of the small intestine. Our results are similar in that they show increased permeability before the onset of diabetes in the genetically susceptible BB rat. We did not set out to demonstrate a correlation between the intestinal permeability defect and onset or incidence of diabetes; this will be an aim of future studies.

In our study, both BB strains had greater permeability and lower claudin-1 protein despite the fact that only the BBDP rat develops spontaneous diabetes. It is interesting to speculate therefore, that there might be a susceptible time period postnatally when intestinal tight junctions do not efficiently regulate the passage of dietary, viral or bacterial antigens in subjects genetically predisposed to type 1 diabetes. This impaired gate-keeping could increase the risk of the autoimmune response that ultimately leads to islet cell destruction and diabetes. We speculate that in addition to the tight junction defect seen in the BB rats, there is likely to be an additional as yet undefined defect. A drawback of our study is that rats less than 21 days of age who are fed by their mothers or with formula were not compared. This would be important to consider in future studies because it is possible that the permeability defect could be manifest very early in infancy and this would correspond better to the time during which infant feeding may play a role in the subsequent prevention of type 1 diabetes.

The National Institute of Diabetes and Digestive and Kidney Diseases estimates that 1 in every 400 to 500 children in the United States is affected by type 1 diabetes (37). Global data on the incidence of this form of diabetes forecast that the diagnosis of type 1 diabetes is markedly on the rise (38). It is important to understand the environmental factors that possibly contribute to the triggering of the immunologic response precipitating the development of diabetes. Early intervention to minimize the exposure of the vulnerable permeable intestine to environmental antigens, a facet suggested by our studies, might reduce the risk of diabetes in genetically predisposed individuals.

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The authors would like to thank Mr. Scott McMillen and the Interdisciplinary Center for Biotechnology Research (ICBR) Protein/Glycobiology Core Laboratory and Dr. Jill Reed-Verlander and the Electron Microscopy Core Laboratory at the University of Florida.

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BB rats; Inflammation; Intestinal permeability; Tight junctions; Type 1 diabetes

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