Explore the vast range of titles available.

Aims/hypothesis An immunohistochemical and genomic analysis of human pancreatic development from 9-23 weeks of fetal age was undertaken to provide a comparative analysis of human and murine islet development. Methods Human fetal pancreases obtained at gestational ages 9-23 weeks were processed in parallel for immunohistochemistry and gene expression profiling by Affymetrix microarrays. Results By 9-11 weeks, the pancreas was made up principally of mesenchymal tissue infiltrated by branched epithelial structures containing scattered hormone-negative neurogenin3-positive endocrine cells. Protoacinar structures emerged by 15-19 weeks, along with clusters of endocrine cells producing either glucagon or insulin. By 20-23 weeks, vascularised islet-like structures appeared. More than 70% of endocrine cells produced a single hormone at any age. Analysis of Ki67 immunoreactivity showed that the replicative rate of endocrine cells was low and suggested that the endocrine expansion was derived from hormone-negative precursors. Insulin, glucagon, somatostatin, ghrelin and pancreatic polypeptide transcripts were present at 9-10 weeks and increased progressively, commensurate with the expansion of endocrine cell volume. The human equivalent of a mouse endocrine secondary transition was not evident, neither in terms of morphology nor in dramatic changes in endocrine-specific transcriptional regulators. By contrast, exocrine genes showed a marked transition at around 11 weeks, associated with a greater than sixfold increase in exocrine gene transcripts. Conclusions/interpretation The observed extension of terminal differentiation of human endocrine tissue into late gestation is in contrast with findings in the mouse. It indicates that the human fetal pancreas could provide an abundant islet precursor cell population that could be expanded ex vivo for therapeutic transplantation.

Aims/hypothesis We investigated whether screening for insulinoma-associated protein (IA-2) beta (IA-2β) autoantibodies (IA-2βA) and zinc transporter-8 (ZnT8) autoantibodies (ZnT8A) improves identification of first-degree relatives of type 1 diabetic patients with a high 5-year disease risk, which to date has been based on assays for insulin autoantibodies (IAA), GAD autoantibodies (GADA) and IA-2 autoantibodies (IA-2A). Methods IA-2βA and ZnT8A (using a ZnT8 carboxy-terminal hybrid construct, CW-CR, carrying 325Arg and 325Trp) were determined by radiobinding assay in 409 IAA + , GADA + and/or IA-2A + siblings or offspring (<40 years) of type 1 diabetic patients consecutively recruited by the Belgian Diabetes Registry. The median (interquartile range) age of the first-degree relatives was 12 (6–19) years. Results Of the first-degree relatives, 24% were IA-2A + ( n  = 97), 14% ( n  = 59) IA-2βA + and 20% ( n  = 80) ZnT8A + . IA-2βA and ZnT8A were significantly ( p  < 0.001) associated with IA-2A and prediabetes ( n  = 86); in IA-2A − first-degree relatives ( n  = 312) the presence of IA-2βA and ZnT8A was associated with an increased progression rate to diabetes ( p  < 0.001). Positivity for IA-2A and/or ZnT8A emerged as the most sensitive combination of two markers to identify first-degree relatives with a 5-year progression rate to diabetes of 45% (survival analysis) and as strongest predictor of diabetes (Cox regression analysis). Omission of first-degree relatives protected by HLA-DQ genotypes or maternal diabetes reduced the group to be followed from n  = 409 to n  = 246 (40%) with minor loss in the number of prediabetic IA-2A + or ZnT8A + first-degree relatives identified ( n  = 3). Conclusions/interpretation IA-2A + and/or ZnT8A + first-degree relatives may be the participants of choice in future secondary prevention trials with immunointervention in relatives of type 1 diabetic patients.

Aims/hypothesis The destruction of pancreatic beta cells leading to type 1 diabetes in humans is thought to occur mainly through apoptosis and necrosis induced by activated macrophages and T cells, and in which secreted cytokines play a significant role. The transcription factor nuclear factor kappa-B (NF-κB) plays an important role in mediating the apoptotic action of cytokines in beta cells. We therefore sought to determine the changes in expression of genes modulated by NF-κB in human islets exposed to a combination of IL1β, TNF-α and IFN-γ. Methods Microarray and gene set enrichment analysis were performed to investigate the global response of gene expression and pathways modulated in cultured human islets exposed to cytokines. Validation of a panel of NF-κB-regulated genes was performed by quantitative RT-PCR. The mechanism of induction of BIRC3 by cytokines was examined by transient transfection of BIRC3 promoter constructs linked to a luciferase gene in MIN6 cells, a mouse beta cell line. Results Enrichment of several metabolic and signalling pathways was observed in cytokine-treated human islets. In addition to the upregulation of known pro-apoptotic genes, a number of anti-apoptotic genes including BIRC3, BCL2A1, TNFAIP3, CFLAR and TRAF1 were induced by cytokines through NF-κB. Significant synergy between the cytokines was observed in NF-κB-mediated induction of the promoter of BIRC3 in MIN6 cells. Conclusions/interpretation These findings suggest that, via NF-κB activation, cytokines induce a concurrent anti-apoptotic pathway that may be critical for preserving islet integrity and viability during the progression of insulitis in type 1 diabetes.

Aims/hypothesis The appearance of autoantibodies (Abs) before diabetes onset has mainly been studied in young children. However, most patients develop type 1 diabetes after the age of 15 years. In first-degree relatives aged under 40 years, we investigated the frequency of seroconversion to (persistent) Ab positivity, progression to diabetes and baseline characteristics of seroconverters according to age. Methods Abs against insulin (IAA), glutamate decarboxylase (GADA), insulinoma-associated protein 2 (IA-2A) and zinc transporter 8 (ZnT8A) were measured during follow-up of 7,170 first-degree relatives. Results We identified 379 (5.3%) relatives with positivity for IAA, GADA, IA-2A and/or ZnT8A (Ab + ) at first sampling and 224 (3.1%) at a later time point. Most seroconversions occurred after the age of 10 years (63%). During follow-up, Abs persisted more often in relatives initially Ab + (76%) than in seroconverters (53%; p  < 0.001). In both groups diabetes developed at a similar pace and almost exclusively with Ab persistence (136 of 139 prediabetic individuals). For both groups, progression was more rapid if Abs appeared before the age of 10 years. Baseline characteristics at seroconversion did not vary significantly according to age. Conclusions/interpretation Seroconversion to (persistent) Ab + occurs regardless of age. Although the progression rate to diabetes is higher under age 10 years, later seroconverters (up to age 40 years) have similar characteristics when compared with age-matched initially Ab + relatives and generate an important minority of prediabetic relatives, warranting their identification and, eventually, enrolment in prevention trials.

Aims/hypothesis We analysed the association between humoral autoreactivity to zinc transporter-8 (ZnT8) and the SLC30A8 rs13266634 polymorphism (Arg325Trp), which is located at the most distal loop in the ZnT8 protein. Methods Autoantibodies to ZnT8 were determined by RIA in 270 patients with type 1 diabetes using ZnT8 carboxy-terminal constructs (amino acids 268-369) carrying 325Trp(CW) and 325Arg(CR) and a hybrid construct (CW-CR). Forty-four ZnT8 autoantibody-positive sera with genomic DNA were used to examine the association between reactivity to ZnT8 constructs and the rs13266634 genotype. Results Seventy-five patients reacted to the CW-CR hybrid construct, whereas 37 and 36 patients reacted to the CW and CR constructs, respectively. All sera positive for either CW or CR autoantibodies were positive for CW-CR autoantibodies. Among 19 patients with a 325Arg(CC) genotype, 5% had CW-specific autoantibodies, 42% had CR-specific autoantibodies and 32% had dual reactivity. Conversely, 73% of 15 patients with the 325Trp(TT) genotype had CW-specific autoantibodies, no patients had CR-specific autoantibodies and 13% had dual reactivity. Nine of the ten patients (90%) with the CT genotype reacted with either CR or CW constructs. The titre of CR autoantibodies in patients carrying the C allele was significantly higher than that in TT homozygotes (p < 0.0001). In contrast, the titre of CW autoantibodies in patients carrying a T allele was significantly higher than that in CC homozygotes (p < 0.005). No evidence of an association between rs13266634 and type 1 diabetes was observed. Conclusions/interpretation These results indicate that variant residue at amino acid 325 is a key determinant of humoral autoreactivity to ZnT8 and that the SLC30A8 genotype is an important determinant of autoantibody specificity.

Aims/hypothesis Secondary type 1 diabetes prevention trials require selection of participants with impending diabetes. HLA-A and - B alleles have been reported to promote disease progression. We investigated whether typing for HLA-B*18 and -B*39 may complement screening for HLA-DQ8 , -DQ2 and -A*24 and autoantibodies (Abs) against islet antigen-2 (IA-2) and zinc transporter 8 (ZnT8) for predicting rapid progression to hyperglycaemia. Methods A registry-based group of 288 persistently autoantibody-positive (Ab + ) offspring/siblings (aged 0–39 years) of known patients (Ab + against insulin, GAD, IA-2 and/or ZnT8) were typed for HLA-DQ , - A and - B and monitored from the first Ab + sample for development of diabetes within 5 years. Results Unlike HLA-B*39 , HLA-B*18 was associated with accelerated disease progression, but only in HLA-DQ2 carriers ( p  < 0.006). In contrast, HLA-A*24 promoted progression preferentially in the presence of HLA-DQ8 ( p  < 0.002). In HLA-DQ2 - and/or HLA-DQ8 -positive relatives ( n  = 246), HLA-B*18 predicted impending diabetes ( p  = 0.015) in addition to HLA-A*24 , HLA-DQ2/DQ8 and positivity for IA-2A or ZnT8A ( p  ≤ 0.004). HLA-B*18 interacted significantly with HLA-DQ2/DQ8 and HLA-A*24 in the presence of IA-2 and/or ZnT8 autoantibodies ( p  ≤ 0.009). Additional testing for HLA-B*18 and -A*24 significantly improved screening sensitivity for rapid progressors, from 38% to 53%, among relatives at high Ab-inferred risk carrying at least one genetic risk factor. Screening for HLA-B*18 increased sensitivity for progressors, from 17% to 28%, among individuals carrying ≥3 risk markers conferring >85% 5 year risk. Conclusions/interpretation These results reinforce the importance of HLA class I alleles in disease progression and quantify their added value for preparing prevention trials.

Aims/hypothesis Islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP, now known as glucose-6-phosphatase, catalytic, 2 [G6PC2]) has recently been identified as a major autoantigen in mouse and human type 1 diabetes. Strategies designed to suppress expression of the gene encoding G6PC2 might therefore be useful in delaying or preventing the onset of this disease. However, since the function of G6PC2 is unclear, the concern with such an approach is that a change in G6PC2 expression might itself have deleterious consequences. Methods To address this concern and assess the physiological function of G6PC2, we generated G6pc2-null mice and performed a phenotypic analysis focusing principally on energy metabolism. Results No differences in body weight were observed and no gross anatomical or behavioural changes were evident. In 16-week-old animals, following a 6-h fast, a small but significant decrease in blood glucose was observed in both male (-14%) and female (-11%) G6pc2 -/- mice, while female G6pc2 -/- mice also exhibited a 12% decrease in plasma triacylglycerol. Plasma cholesterol, glycerol, insulin and glucagon concentrations were unchanged. Conclusions/interpretation These results argue against the possibility of G6PC2 playing a major role in pancreatic islet stimulus secretion coupling or energy homeostasis under physiological conditions imposed by conventional animal housing. This indicates that manipulating the expression of G6PC2 for therapeutic ends may be feasible.

Mice transgenic for an expanded CAG repeat in the Huntington's disease gene develop diabetes. M S Hurlbert , W Zhou , C Wasmeier , F G Kaddis , J C Hutton and C R Freed Department of Medicine, University of Colorado Health Sciences Center, Denver, USA. Abstract The autosomal dominant neurological syndrome of Huntington's disease has been modeled in transgenic mice by the expression of a portion of the human huntingtin gene together with 140 CAG repeats (the R6/2 strain). The mice develop progressive chorea with onset at approximately 9 weeks of age and with death at approximately 13 weeks. Associated symptoms include weight loss and polyuria in the absence of eating or drinking deficits. We have found that these mice have insulin-responsive diabetes. Fasting glucose was 211 + 19 mg/dl in R6/2 mice compared with 93 + 5 mg/dl in C57/B6 controls (n = 12, both groups; P < 0.01). Administration of insulin intraperitoneally led to a reduction in blood glucose. At 12.5 weeks, animals were killed and pancreas weighed and analyzed for insulin and glucagon. Pancreatic mass in R6/2 mice was the same as controls, and islets appeared normal in morphology without lymphocytic infiltration. Immunohistochemical staining showed dramatic reductions in glucagon in the alpha-cells and in insulin in the beta-cells. Direct tissue assays showed glucagon and insulin content were reduced to only 10 and 15% of controls, respectively. Diabetes has been reported as being more common in Huntington's disease and other triplet repeat disorders. The R6/2 mouse should prove useful for elucidating the mechanism of diabetes in these genetic diseases.

Aims/hypothesis Transplantation of islets is a viable option for the treatment of diabetes. A significant proportion of islets is lost during isolation, storage and after transplantation as a result of apoptosis. cAMP response element binding protein (CREB) is an important cell survival factor. The aim of the present study was to determine whether preservation of CREB function is needed for survival of human islets. Materials and methods To determine the effects of downregulation of CREB activity on beta cell apoptosis in a transplantation setting, adenoviral vectors were used to express two dominant negative mutant forms of CREB in human islets isolated from cadaveric donors. Markers of apoptosis were determined in these transduced islets under basal conditions and following treatment with growth factor. Results Expression of CREB mutants in human islets resulted in significant (p < 0.001) activation of caspase-9, a key regulatory enzyme in the mitochondrial pathway of apoptosis, when compared with islets transduced with adenoviral beta galactosidase. Immunocytochemical analysis showed the activation of caspase-9 to be predominantly in beta cells. Other definitive markers of apoptosis such as parallel activation of caspase-3, accumulation of cleaved poly-(ADP-ribose) polymerase and nuclear condensation were also observed. Furthermore, the anti-apoptotic action of growth factors exendin-4 and betacellulin in human islets exposed to cytokines was partially lost when CREB function was impaired. Conclusions/interpretation Our findings suggest that impairment of CREB-mediated transcription could lead to loss of islets by apoptosis with potential implications in islet transplantation as well as in the mechanism of beta cell loss leading to diabetes.