Construction of a mutation due to a fourteen base-pair insertion in HNF-1α gene causing maturity-onset diabetes of the young (MODY) in Thai patients.

Objective: The aim of this study is to generate a mutation causing maturity-onset diabetes of the young (MODY) in Thai patients by insertion of a fourteen base-pair (bp) into a HNF-1a gene using a modified site-directed ligase-independent mutagenesis (SLIM) method. Methods: Two pairs of long- and short-tailed primers were designed to amplify a plasmid construct containing a HNF-1a and to insert a 14-bp at a desired position. Long-tailed primers contained the overhanging 14-nucleotide (nt) insert at their termini which were complementary to each other. Polymerase chain reactions (PCR) were performed in two separated tubes using different pairs of primers. After amplifications, PCR products from both tubes were pooled together, denatured and then re-annealed to allow formation of double stranded DNA molecules containing the 14-bp insert within HNF-1a. The pooled and reannealed PCR products without ligation were transformed into competent E.coli cells to generate a ligated recombinant plasmid with a 14-bp insertion. Results: Five of 14 bacterial colonies contained the desired recombinant plasmid with a 14-bp insertion within HNF-1a The efficiency of the method for generation of recombinant plasmid was about 36 percent. Conclusion: This method is simple and rapid to insert a long stretch of nucleotides into a plasmid construct containing a gene of interest at a desired position. A recombinant plasmid containing an insertion mutation in a HNF-1a gene was successfully generated, allowing an opportunity to perform functional study of the mutated gene.

Reduced interleukin-17 expression of Burkholderia pseudomallei-infected peripheral blood mononuclear cells of diabetic patients.

Burkholderia pseudomallei is the causative agent of melioidosis. One of the main risk factors for B. pseudomallei infection in endemic areas is diabetes mellitus. The present study investigated IL-17 mRNA and protein expression by peripheral blood mononuclear cells in response to B. pseudomallei infection in 10 diabetic patients in comparison to 10 healthy blood donors. The IL-17 expression in diabetic patients was significantly lower (p < 0.05) than in the controls. However, IL-23 mRNA expression of the 2 groups was comparable. The present findings suggest that melioidosis affects T cell IL-17 production and that patients with diabetes mellitus have a defective IL-17 production in response to this type of infection.

Analysis of the reg1alpha and reg1beta gene transcripts in patients with fibrocalculous pancreatopathy.

Fibrocalculous pancreatopathy is a form of diabetes, associated with tropical chronic calcific pancreatitis, in which islet beta-cell loss and pancreatic stone formation are found. It is likely to be a multifactorial disease with both genetic and environmental components. Regenerating (reg) gene encodes protein that has been involved in pancreatic lithogenesis and the regeneration of islet cells and therefore the abnormality of reg genes could be associated with fibrocalculous pancreatopathy. In this study, regla and reg1beta mRNAs were isolated from peripheral blood lymphocytes obtained from 16 patients with fibrocalculous pancreatopathy, 42 patients with type 1 diabetes, 37 patients with type 2 diabetes, and 22 normal controls. mRNAs were amplified by reverse-transcription polymerase chain reaction (RT-PCR) and analysed by a single strand conformation polymorphism (SSCP) technique. The reg1alpha and reg1beta mRNAs were isolated, indicating the ectopic expression of these genes in peripheral blood lymphocytes; however, variation among mobility patterns was not observed in the SSCP analysis of the RT-PCR products. The results indicated that there was no abnormality of the regla and reg1beta mRNAs obtained from the study groups.

Flow cytometry for the analysis of T cells expressing CD69 after stimulation with glutamic acid decarboxylase.

Type 1 diabetes mellitus is a T-cell mediated autoimmune disease in which the insulin-producing pancreatic beta cells are selectively destroyed. We recently found that the detection of cell-mediated immune response to glutamic acid decarboxylase (GAD) was more useful than the detection of specific autoantibodies for the diagnosis of type 1 diabetes mellitus. In this study, we established a flow cytometric analysis for the detection of activated T cells in whole venous blood, obtained from diabetic patients and normal controls after stimulation by GAD. Two millitiers of peripheral venous blood and 6 hours incubation time were used for performing the test. It was found that 33% (3/9) type 1 diabetic patients, 7.7% (1/13) type 2 diabetic patients and neither patients with fibrocalculous pancreatopathy nor normal controls had > or = 20% CD8+ T cells expressing CD69. The results suggest that flow cytometry may be a useful tool for the detection of surrogate markers of type 1 diabetes mellitus.