Faculty Bibliography

Introduction: Gut fermentation or auto-brewery syndrome is a relatively uncommon medical condition that presents a diagnostic and therapeutic challenge. Case Report: Here we present a unique case of a 45-yearold obese, male, diabetic patient treated with two courses of antibiotics for deviated nasal septum and dental procedure who reported episodes of diarrhea, vomiting, edema, seizures, hallucinations, intermittent fevers, chills, slurred speech, and loss of consciousness precipitated after meals for a duration of 14 months. The patient denied alcohol consumption at any time. His blood ethanol levels, measured on multiple occasions, were elevated and corroborated by a 24-hour meal test administered in the hospital. Small bowel and fecal contents collected during the endoscopic procedures were remarkable for the growth of Saccharomyces cerevisiae from both samples. Gram stain was negative for Salmonella, Shigella, or Campylobacter. A rapid membrane enzyme immunoassay for C difficile antigen and toxins A and B was also negative. The patient responded well to oral fluconazole but relapsed after a month and needed further assessment. Microbiological studies undertaken on gastric and small bowel contents collected during the follow-up upper and lower endoscopic procedures were positive for Candida intermedia, sensitive to fluconazole (0.250 mcg/ mL) and amphotericin B (0.250 mcg/mL). Bacterial cultures showed rare, positive growth for Klebsiella pneumoniae and Enterococcus faecium. The patient responded dramatically to a no-carbohydrate diet and intravenous administration of an antifungal agent through a peripherally inserted central catheter line. The patient has been asymptomatic ever since.
Summary: Auto-brewery syndrome in a setting of diabetes, obesity, and high carbohydrate intake presented with signs and symptoms of elevated serum ethanol levels. The importance of microbiological studies on carefully collected intestinal secretions is emphasized in this report, in the absence of validated diagnostic and treatment protocols in the literature

OBJECTIVE: This study investigated the utility of advanced computational techniques to large-scale genome-based data to identify novel genes that govern murine pancreatic development. METHODS: An expression data set for mouse pancreatic development was complemented with high-throughput data analyzer to identify and prioritize novel genes. Quantitative real-time polymerase chain reaction, in situ hybridization, and immunohistochemistry were used to validate selected genes. RESULTS: Four new genes whose roles in the development of murine pancreas have not previously been established were identified: cystathionine beta-synthase (Cbs), Meis homeobox 1, growth factor independent 1, and aldehyde dehydrogenase 18 family, member A1. Their temporal expression during development was documented. Cbs was localized in the cytoplasm of the tip cells of the epithelial chords of the undifferentiated progenitor cells at E12.5 and was coexpressed with the pancreatic and duodenal homeobox 1 and pancreas-specific transcription factor, 1a-positive cells. In the adult pancreas, Cbs was localized primarily within the acinar compartment. CONCLUSIONS: In silico analysis of high-throughput microarray data in combination with background knowledge about genes provides an additional reliable method of identifying novel genes. To our knowledge, the expression and localization of Cbs have not been previously documented during mouse pancreatic development.

More than one-half of the ~50 human chemokines have been associated with or implicated in the pathogenesis of type 1 diabetes, yet their actual expression patterns in the islet environment of type 1 diabetic patients remain, at present, poorly defined. Here, we have integrated a human islet culture system, murine models of virus-induced and spontaneous type 1 diabetes, and the histopathological examination of pancreata from diabetic organ donors with the goal of providing a foundation for the informed selection of potential therapeutic targets within the chemokine/receptor family. Chemokine (C-C motif) ligand (CCL) 5 (CCL5), CCL8, CCL22, chemokine (C-X-C motif) ligand (CXCL) 9 (CXCL9), CXCL10, and chemokine (C-X3-C motif) ligand (CX3CL) 1 (CX3CL1) were the major chemokines transcribed (in an inducible nitric oxide synthase-dependent but not nuclear factor-kappaB-dependent fashion) and translated by human islet cells in response to in vitro inflammatory stimuli. CXCL10 was identified as the dominant chemokine expressed in vivo in the islet environment of prediabetic animals and type 1 diabetic patients, whereas CCL5, CCL8, CXCL9, and CX3CL1 proteins were present at lower levels in the islets of both species. Of importance, additional expression of the same chemokines in human acinar tissues emphasizes an underappreciated involvement of the exocrine pancreas in the natural course of type 1 diabetes that will require consideration for additional type 1 diabetes pathogenesis and immune intervention studies.

OBJECTIVE: The SLC30A8 gene encodes the islet-specific transporter ZnT-8, which is hypothesized to provide zinc for insulin-crystal formation. A polymorphic variant in SLC30A8 is associated with altered susceptibility to type 2 diabetes. Several groups have examined the effect of global Slc30a8 gene deletion but the results have been highly variable, perhaps due to the mixed 129SvEv/C57BL/6J genetic background of the mice studied. We therefore sought to remove the conflicting effect of 129SvEv-specific modifier genes. METHODS: The impact of Slc30a8 deletion was examined in the context of the pure C57BL/6J genetic background. RESULTS: Male C57BL/6J Slc30a8 knockout (KO) mice had normal fasting insulin levels and no change in glucose-stimulated insulin secretion (GSIS) from isolated islets in marked contrast to the approximately 50% and approximately 35% decrease, respectively, in both parameters observed in male mixed genetic background Slc30a8 KO mice. This observation suggests that 129SvEv-specific modifier genes modulate the impact of Slc30a8 deletion. In contrast, female C57BL/6J Slc30a8 KO mice had reduced ( approximately 20%) fasting insulin levels, though this was not associated with a change in fasting blood glucose (FBG), or GSIS from isolated islets. This observation indicates that gender also modulates the impact of Slc30a8 deletion, though the physiological explanation as to why impaired insulin secretion is not accompanied by elevated FBG is unclear. Neither male nor female C57BL/6J Slc30a8 KO mice showed impaired glucose tolerance. CONCLUSIONS: Our data suggest that, despite a marked reduction in islet zinc content, the absence of ZnT-8 does not have a substantial impact on mouse physiology.

Genome-wide association studies have shown that a polymorphic variant in SLC30A8, which encodes zinc transporter-8, is associated with altered susceptibility to type 2 diabetes (T2D). This association is consistent with the observation that glucose-stimulated insulin secretion is decreased in islets isolated from Slc30a8 knockout mice. In this study, immunohistochemical staining was first used to show that SLC30A8 is expressed specifically in pancreatic islets. Fusion gene studies were then used to examine the molecular basis for the islet-specific expression of SLC30A8. The analysis of SLC30A8-luciferase expression in betaTC-3 cells revealed that the proximal promoter region, located between -6154 and -1, relative to the translation start site, was only active in stable but not transient transfections. VISTA analyses identified three regions in the SLC30A8 promoter and a region in SLC30A8 intron 2 that are conserved in the mouse Slc30a8 gene. Additional fusion gene experiments demonstrated that none of these Slc30a8 promoter regions exhibited enhancer activity when ligated to a heterologous promoter whereas the conserved region in SLC30A8 intron 2 conferred elevated reporter gene expression selectively in betaTC-3 but not in alphaTC-6 cells. Finally, the functional effects of a single nucleotide polymorphism (SNP), rs62510556, in this conserved intron 2 enhancer were investigated. Gel retardation studies showed that rs62510556 affects the binding of an unknown transcription factor and fusion gene analyses showed that it modulates enhancer activity. However, genetic analyses suggest that this SNP is not a causal variant that contributes to the association between SLC30A8 and T2D, at least in Europeans.

OBJECTIVE: Islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), now known as G6PC2, is a major target of autoreactive T cells implicated in the pathogenesis of type 1 diabetes in both mice and humans. This study aimed to determine whether suppression of G6p2 gene expression might therefore prevent or delay disease progression. RESEARCH DESIGN AND METHODS: G6pc2(-/-) mice were generated on the NOD/ShiLtJ genetic background, and glycemia was monitored weekly up to 35 weeks of age to determine the onset and incidence of diabetes. The antigen specificity of CD8(+) T cells infiltrating islets from NOD/ShiLtJ G6pc2(+/+) and G6pc2(-/-) mice at 12 weeks was determined in parallel. RESULTS: The absence of G6pc2 did not affect the time of onset, incidence, or sex bias of type 1 diabetes in NOD/ShiLtJ mice. Insulitis was prominent in both groups, but whereas NOD/ShiLtJ G6pc2(+/+) islets contained CD8(+) T cells reactive to the G6pc2 NRP peptide, G6pc2 NRP-reactive T cells were absent in NOD/ShiLtJ G6pc2(-/-) islets. CONCLUSIONS: These results demonstrate that G6pc2 is an important driver for the selection and expansion of islet-reactive CD8(+) T cells infiltrating NOD/ShiLtJ islets. However, autoreactivity to G6pc2 is not essential for the emergence of autoimmune diabetes. The results remain consistent with previous studies indicating that insulin may be the primary autoimmune target, at least in NOD/ShiLtJ mice.

BACKGROUND: We recently reported an association between Type 1 diabetes and the telomeric major histocompatibility complex (MHC) single nucleotide polymorphism (SNP) rs1233478. As further families have been analyzed in the Type 1 Diabetes Genetics Consortium (T1DGC), we tested replication of the association and, with more data, analyzed haplotypic associations. METHODS: An additional 2717 case and 1315 control chromosomes have been analyzed from the T1DGC, with human leukocyte antigen (HLA) typing and data for 2837 SNPs across the MHC region. RESULTS: We confirmed the association of rs1233478 (new data only: P=2.2E-5, OR=1.4). We also found two additional SNPs nearby that were significantly associated with Type 1 diabetes (new data only rs3131020: P=8.3E-9, OR=0.65; rs1592410: P=2.2E-8, OR=1.5). For studies of Type 1 diabetes in the MHC region, it is critical to account for linkage disequilibrium with the HLA genes. Logistic regression analysis of these new data indicated that the effects of rs3131020 and rs1592410 on Type 1 diabetes risk are independent of HLA alleles (rs3131020: P=2.3E-3, OR=0.73; rs1592410: P=2.1E-3, OR=1.4). Haplotypes of 12 SNPs (including the three highly significant SNPs) stratify diabetes risk (high risk, protective, and neutral), with high-risk haplotypes limited to approximately 20,000 bp in length. The 20,000-bp region is telomeric of the UBD gene and contains LOC729653, a hypothetical gene. CONCLUSIONS: We believe that polymorphisms of the telomeric MHC locus LOC729653 may confer risk for Type 1 diabetes.