The Relationship between insulin variable number of tandem repeats (INS-VNTR) -23 A/T and cytotoxic Tlymphocyte associated protein-4 (CTLA-4) +49 A/G polymorphisms with islet autoantibodies in persons with diabetes.

Background: Both genetic and environmental factors are important in pathogenesis of diabetes. Non HLA (Human Leukocyte Antigen) genes such as INS-VNTR and CTLA-4 in addition of HLA genes have influence on genetic susceptibility for diabetes mellitus. In this study the association of +49 A/G CTLA-4 and -23 A/T INS-VNTR polymorphisms with diabetes and their association with islet autoantibodies were investigated. Methods: Thirty four autoantibody positive adult persons with diabetes mellitus and 39 persons with Type 1diabetes mellitus (T1DM), 40 autoantibody negative Type 2 diabetes mellitus (T2DM) patients and 40 healthy controls were studied using Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) technique. Results: The frequencies of -23 A/T INS-VNTR genotypes were not significantly different among study groups. It was shown that the distribution of the +49A/G CTLA-4 allele and genotype frequencies did not differ between T1DM patients, autoantibody positive adult patients and controls. With increasing CTLA-4 G allele and GG/AG genotypes, the frequency of Glutamic Acid Decarboxylase Autoantibody (GADA), Islet Cell Autoantibody (ICA) and Islet Antigen 2 Antibody (IA2A) positive patients were increased. Conclusion: Our results suggest that susceptibility allele A of -23A/T INS-VNTR does not have any role in the pathogenesis of diabetes in our patients and susceptibility allele G of +49 A/G CTLA-4 if not, has a small role in pathogenesis of diabetes in T1DM and autoantibody positive adult patients and in spite of significant increase in autoantibody negative T2DM group it does not have any role in disease pathogenesis.

Development of an immune function assay by measuring intracellular adenosine triphosphate (iATP) levels in mitogen-stimulated CD4+ T lymphocytes.

We developed an immune function assay for monitoring CD4+ T cells activity based on changes in intracellular adenosine triphosphate (iATP) levels after phytohemagglutinin (PHA) stimulation. Blood samples were obtained from 40 healthy subjects and 30 RTRs and incubated with 5 µg/mL of PHA for 15-18 hr at 37°C and 5% CO2. Afterward, the CD4+ T cells were separated by antibody-coated magnetic beads and lysed. Then, iATP content in unstimulated and stimulated conditions was measured by luciferin-luciferase reaction using a log-log standard curve. The iATP levels showed significant increase in CD4+ T cells in both healthy persons (mean: 550 ± 142 ng/mL vs. 109 ± 54 ng/mL) and RTRs (mean: 394 ± 160 ng/mL vs. 52 ± 37 ng/mL) after PHA stimulation (P < 0.001). However, the iATP production in RTRs was significantly lower than that in healthy individuals; both prior to and after stimulation with PHA (P < 0.001). No gender-specific difference in iATP production was observed between women and men subjects. This rapid and low-cost assay reflects the degree of immune cell function through assessment of CD4+ T cells activation. Thus, it can be used for evaluation of immune system status in immunodeficient individuals as well as in immunosuppressed transplant recipients who needs drug adjustment.

A simple graphene-based pipette tip solid-phase extraction of malondialdehyde from human plasma and its determination by spectrofluorometry.

Determination of malondialdehyde (MDA) in human blood plasma is important because of its role as a biomarker of lipid peroxidation in biological and medical sciences. In this work, a miniaturized graphene-based pipette tip solid-phase extraction technique was developed for very efficient extraction of MDA as its dithiobarbituric acid (TBA) adduct from human plasma. Two milligrams of graphene as sorbent were placed into a pipette tip and MDA-TBA compound was extracted and preconcentrated by it, after 4 repeated aspirating/dispensing cycles, then the column was eluted with 80 µL of dimethyl sulfoxide by 4 repeated aspirating/dispensing cycles and elusion was measured spectrofluorimetrically. Various effective parameters such as type and volume of eluent solvent, temperature, sample volume, number of cycles of extraction and desorption, derivatization reaction time, and pH of the sample solution were investigated and optimized. Under optimum conditions, a linear calibration curve was obtained in the range of 0.5-90 µg L(-1) (r (2) = 0.991) with a detection limit of 0.3 µg L(-1). The relative standard deviations for 8 replicate measurements of 10 and 40 µg L(-1) of MDA were found to be 4.51 and 3.78 % respectively. The developed protocol was successfully applied to the determination of MDA in a human blood plasma sample. Graphical Abstract A simple graphene-based pipette tip solid-phase extraction of malondialdehyde from human plasma and its determination by spectrofluorometry.