The BB (BioBreeding) rat is one of the best models of spontaneous autoimmune diabetes and is used to study non-MHC loci contributing to Type 1 diabetes. Type 1 diabetes in the diabetes-prone BB ...(BBDP) rat is polygenic, dependent upon mutations at several loci. Iddm1, on chromosome 4, is responsible for a lymphopenia (lyp) phenotype and is essential to diabetes. In this study, we report the positional cloning of the Iddm1/lyp locus. We show that lymphopenia is due to a frameshift deletion in a novel member (Ian5) of the Immune-Associated Nucleotide (IAN)-related gene family, resulting in truncation of a significant portion of the protein. This mutation was absent in 37 other inbred rat strains that are nonlymphopenic and nondiabetic. The IAN gene family, lying within a tight cluster on rat chromosome 4, mouse chromosome 6, and human chromosome 7, is poorly characterized. Some members of the family have been shown to be expressed in mature T cells and switched on during thymic T-cell development, suggesting that Ian5 may be a key factor in T-cell development. The lymphopenia mutation may thus be useful not only to elucidate Type 1 diabetes, but also in the function of the Ian gene family as a whole.
We have identified for the first time an age-dependent spontaneous loss of tolerance to two self-antigenic epitopes derived from putative diabetes-associated antigens glutamic acid decarboxylase ...(GAD65) and glial fibrillary acidic protein (GFAP) in RIP-B7/DRB1*0404 HLA transgenic mice. Diabetic and older non-diabetic mice exhibited a proliferative response to an immunodominant epitope from GAD65 (555–567) and also from GFAP (240–252) but not from an immunogenic epitope from diabetes-associated islet-specific glucose-6-phosphatase catalytic subunit-related protein. The response to both of these self-antigens is not observed in young mice but is observed in older non-diabetic mice and is accompanied by histological evidence of insulitis in the absence of overt diabetes. Islet infiltrates in older non-diabetic mice and diabetic mice contain CD4
+/FoxP3
+ cells and suggest the presence of a regulatory mechanism prior and during diabetic disease. Diabetes penetrance in RIP-B7/DR0404 mice is 23% with a mean onset age of 40 weeks and is similar to that reported for RIP-B7/DR0401 mice. A gender preference is observed in that 38% of female mice become diabetic compared to 8% of male mice.
The purpose of this descriptive case study is to understand, identify, and describe the perceived barriers some parents of low performing students in two middle schools in a school district in ...suburban northern California have that hinder them from providing academic support for their students. Little information is known from the parent perspective of why they are not able to support their middle school students with academic support. There is little argument that achievement gaps are still prevalent among America’s youth when considering academic performance. Understanding the barriers that some parents of academically struggling middle school students encounter may provide valuable information to school leaders on how best to assist parents with supporting their middle school students with academics. Research findings suggest six major themes that appeared in the data gathered from the one-on-one interviews and the parent focus group. Recommendations for educational practitioners and leaders include improved communication between the teacher and parents that is specific to individual student needs and is explicit on teacher expectations. Consideration of middle school students’ need for developing skills of self-advocacy and confidence is also an area for future focus. Finally, participants in the study indicated a desire for parent support on how best to help their students with academic assistance.
We describe a novel monoclonal antibody raised towards the N-terminus of the 65 kDa isoform of glutamate decarboxylase (GAD65). This N-GAD65 mAb is highly specific for GAD65 and can be used in a wide ...range of applications such as Western blot analysis, immunoprecipitation and immunocytochemistry. Full-length cDNAs coding for N-GAD65 mAb heavy and light chains were cloned and characterized by nucleotide sequence analysis. Both heavy and light chain-specific cDNAs are functional and are members of mouse heavy chain subgroup IgG1 and kappa chain group 2, respectively. Comparing the N-GAD65 mAb gene sequences with GenBank shows that the cDNAs were not reported previously.
The smaller isoform of glutamic acid decarboxylase, GAD65, is an important autoantigen implicated in the pathogenesis of type 1 diabetes whereas the larger isoform, GAD67 appears to play no major ...role. The primary difference between the two isoforms resides in the N-ter-minal part of the molecule including the GAD65 membrane-anchoring domain. The aim of this study was to generate mutants of the membrane targeting domain spanning amino acids 24 to 31 of GAD65 to determine effects on enzyme activity and antibody recognition. Three GAD65 mutants were generated by substituting two, nine or eleven nucleotides coding for the membrane targeting with the corresponding bases of GAD67. SDS-PAGE and Western blotting wildtype (wt) and mutated GAD65 ascertained that they were of similar size and recognized GAD65-specific antibodies. No difference in enzymatic activity was found between the mutants and wt GAD65. GAD65 antibody positive sera from type 1 diabetes patients immu-noprecipitated mutated GAD65 whether two, nine or eleven nucleotides were replaced. Mono-or polyclonal antibodies to the N-terminal region demonstrated that the mutated GAD65 with two or nine nucleotides replaced was immunoprecipitated markedly better than wt whereas no difference was detected using antibodies specific for the PLP-binding site in the middle part of G AD65 or the C-terminal region. Taken together, these data suggest that no major conformational changes have been introduced by mutating the membrane-anchoring domain of GAD65.
GAD65-specific autoantibodies enhance the presentation of an immunodominant T-cell epitope from GAD65.
H Reijonen ,
T L Daniels ,
A Lernmark and
G T Nepom
Virginia Mason Research Center, Benaroya ...Research Institute, Seattle, Washington 981010, USA. reijonen@vmresearch.org
Abstract
GAD65 autoantibodies (GAD65Ab) are highly prevalent in type 1 diabetes, but their functional role in the pathogenesis of the
disease and their relationship to T-cell reactivity to GAD65 is still unclear. We tested the hypothesis that GAD65Ab modulate
presentation of GAD65 to T-cells. T-cell hybridoma T33.1, which recognizes the GAD65 274-286 epitope in the context of HLA-DRB
1*0401, was incubated with antigen-presenting cells exposed to recombinant human GAD65 alone or complexed with GAD65Ab' or
GAD65Ab- sera. Stimulation of the T33.1 hybridoma was greatly enhanced by multiple GAD65Ab+ sera. The enhancement effect was
most prominent with sera from patients with high GAD65 autoantibody levels. Sera from GAD65Ab- subjects had no effect. The
correlation between T-cell stimulation and GAD65Ab levels was not absolute, suggesting that other variables such as autoantibody
recognition of different regions of GAD65 and variable effects on processing of the 274-286 epitope may contribute. Uptake
of antibody-complexed GAD65 was Fc receptor (FcR)-mediated because the enhancement of presentation was inhibited by monoclonal
antibodies against FcR. Our results support the hypothesis that GAD65Ab modulate presentation of GAD65 to T-cells. Increased
antigen uptake and heterogeneity in the autoantibody specificity may provide a mechanism for antibody-facilitated T-cell response
influencing the progression of type 1 diabetes.
The purpose of this study was to examine the applicability of internal labor market theory to the career advancement of community and state college workforce administrators. The study investigated ...(1) career lines or sequences of positions, (2) career advancement and mobility activities, (3) education level, and (4) demographic characteristics of workforce administrators. Requests for participation were sent to the 88 workforce administrators who are members of the Occupational Education Standing Committee (OESC) for Florida State and Community Colleges. There were 56 usable surveys completed for a 64% response rate. Survey results are presented for all workforce administrators completed the survey. The survey solicited detailed information from participants in regards to educational background, work history, and level of participation in activities that may have influenced career advancement or career mobility. Internal labor market theory was tested to determine the extent of internal hiring thereby examining the extent of boundaries between community college workforce education administrator labor markets and labor markets external to postsecondary education. Findings in this study did not support the findings of Massie's 2003 study in which internal labor market theory was applicable only the community college administrative positions of president and chief academic officer, not workforce education administrator. The difference in findings could possibly be attributed to factors such as year the study was conducted (2003 vs. 2010), sample size (16 vs. 56), or location (Nationwide vs. Florida). We found that internal labor market theory was applicable to community and state college workforce education administrators. Workforce education administrators were more likely to be selected from internal labor markets. In order to advance to the workforce administrative rank at a community or state college, there were certain internal labor market customs and practices related to career path and education level that one must follow. The majority of the participants advanced through the community college labor market through traditional academic pathways.
ABSTRACT
Objectives:
The conformation of tissue transglutaminase might influence the performance of immunoassays to detect autoantibodies from patients with celiac disease. The present study ...investigated how the exposure of tissue transglutaminase kept in a liquid phase and fixed to a solid support affected the binding of immunoglobulin (Ig)A and IgG autoantibodies in children with untreated and treated celiac disease.
Methods:
Included were 73 untreated celiac disease children, 50 controls and 80 children with treated celiac disease. IgA and IgG antitissue transglutaminase were measured with solid phase enzyme‐linked immunoassay (ELISA) and liquid phase radioligand binding assays. For IgG antitissue transglutaminase detection with radioligand binding assays antihuman IgG and protein A were used. IgA endomysial autoantibodies were measured by indirect immunofluorescence.
Results:
Both ELISA and radioligand binding assays detected IgA antitissue transglutaminase in 65 of 73 untreated celiac disease children and in 2 of 50 controls. One additional control child was detected with radioligand binding assays. Endomysial autoantibodies were present in 62 of 73 celiac disease children and in 2 of 50 controls. IgG antitissue transglutaminase was detected with both ELISA and radioligand binding assays in 40 of 73 untreated celiac disease children and in 2 of 50 controls. Radioligand binding assays using protein A detected 20 of 73 additional untreated celiac disease children and one control child with increased IgG antitissue transglutaminase. In treated celiac disease children, 21 of 80 were IgA antitissue transglutaminase positive with radioligand binding assays, 3 of 80 with ELISA, whereas none had endomysial autoantibodies.
Conclusions:
No qualitative differences between radioligand binding assays and ELISA in IgA or IgG antitissue transglutaminase binding from untreated celiac disease children was demonstrated. However, discrepancies in the binding of IgA antitissue transglutaminase from a subgroup of treated celiac disease children indicated that alterations of tissue transglutaminase might occur on fixation of the antigen. Protein A used for radioligand binding assays seemed not to assess IgG autoantibodies exclusively. IgA antitissue transglutaminase detection in screening of childhood celiac disease can be performed either by ELISA or radioligand binding assays because the two assays are interchangeable.
Wild bank voles (Clethrionomys glareolus) may develop diabetes in laboratory captivity. The aim of this study was to test whether bank voles develop type 1 diabetes in association with Ljungan virus. ...Two groups of bank voles were analyzed for diabetes, pancreas histology, autoantibodies to glutamic acid decarboxylase (GAD65), IA-2, and insulin by standardized radioligand-binding assays as well as antibodies to in vitro transcribed and translated Ljungan virus antigens. Group A represented 101 trapped bank voles, which were screened for diabetes when euthanized within 24 hours of capture. Group B represented 67 bank voles, which were trapped and kept in the laboratory for 1 month before being euthanized. Group A bank voles did not have diabetes. Bank voles in group B (22/67; 33%) developed diabetes due to specific lysis of pancreatic islet beta cells. Compared to nondiabetic group B bank voles, diabetic animals had increased levels of GAD65 (P < .0001), IA-2 (P < .0001), and insulin (P = .03) autoantibodies. Affected islets stained positive for Ljungan virus, a novel picorna virus isolated from bank voles. Ljungan virus inoculation of nondiabetic wild bank voles induced beta-cell lysis. Compared to group A bank voles, Ljungan virus antibodies were increased in both nondiabetic (P < .0001) and diabetic (P = .0015) group B bank voles. Levels of Ljungan virus antibodies were also increased in young age at onset of newly diagnosed type 1 diabetes in children (P < .01). These findings support the hypothesis that the development of type 1 diabetes in captured wild bank voles is associated with Ljungan virus. It is speculated that bank voles may have a possible zoonotic role as a reservoir and vector for virus that may contribute to the incidence of type 1 diabetes in humans.
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