Iranian journal of immunology
Volume:10 Issue: 3, Summer 2013

The antibody response to hepatitis B surface antigen (HBsAg) controls hepatitis B virus infection. The "a" determinant of HBsAg is the most important target for protective antibody response, diagnosis and immunoprophylaxis. Mutations in this area may induce immune escape mutants and affect the performance of HBsAg assays. To construct clinically relevant recombinant mutant forms of HBsAg and assessment of their reactivity with anti-HBs monoclonal antibodies (MAbs). Wild type (wt) and mutant (mt) HBsAg genes were constructed by site directed mutagenesis and SEOing PCR. The amplified genes were inserted into pCMV6-neo plasmid and transfected in CHO cell line. The expression of wt- and mtHBsAg was assessed by commercial ELISA assays and stable cells were established and cloned by limiting dilution. The recombinant mutants were further characterized using a panel of anti-HBs monoclonal antibodies (MAbs) and the pattern of their reactivity was assessed by ELISA. Ten HBsAg mutants having single mutation within the "a" determinant including P120E, T123N, Q129H, M133L, K141E, P142S, D144A, G145R, N146S and C147S together with a wt form were successfully constructed and expressed in CHO cells. Reactivity of anti-HBs MAbs with mtHBsAgs displayed different patterns. The effect of mutations on antibody binding differed depending on the amino acid involved and its location within the ‘‘a’’ determinant. Mutation at amino acids 123 and 145 resulted in either complete loss or significant reduction of binding to all anti-HBs MAbs. Our panel of mtHBsAgs is a valuable tool for assessment of the antibody response to HBV escape mutants and may have substantial implications in HBV immunological diagnostics.

Chemo-immunotherapy is one of the new achievements for treatment of cancer, by which the success of anti-cancer therapy can be increased. In vitro studies have been shown that Arteether (ARE) induces apoptosis in tumor cells, but not in normal cells. To investigate the cytotoxic and immunomodulatory properties of Arteether invivo and in-vitro. In this study, we used MTT assay for evaluation of cytotoxicity of Arteether on tumor cell line and Peripheral Blood Mononuclear Cells (PBMCs) from healthy individuals. Balb/c mice were subcutaneously transplanted with tumor tissue taken from Spontaneous Mouse Mammary Tumor (SMMT) bearing female mice. Arteether was administered to breast tumor-bearing Balb/c mice at a dose of 6mg/kg/day intraperitoneally. Tumor sizes, lymphocyte proliferation, cytokines production, and the percentage of splenic Treg cells were measured. We observed that ARE could reduce the cell growth of 4T1 cell line in a dose-dependent manner but it had no cytotoxic effect on the growth of peripheral blood lymphocytes. ARE administered intraperitoneally to tumor-bearing Balb/c mice could reduce the tumor growth rate and splenic T-reg cells. No difference in the IFN-γ, IL-10 and IL-4 production was observed between tumor antigen-stimulated splenocytes of mice treated with ARE and control mice. These results underscore antitumor properties of Arteether that may aid in development of more effective antitumor agents.

Killer cell immunoglobulin-like receptors (KIR) are expressed on NK cells and a subset of T cells. The variable KIR receptors along with their ligands, HLA class I, influence risk for autoimmune and malignant diseases. To investigate the KIR gene profiles in relation to susceptibility to Graves’ disease in patients with ophthalmopathy. KIR genes profiles were analyzed in 90 patients presenting Graves’ disease with ophthalmopathy representing upper eyelid retraction, swelling, redness, conjunctivitis, and bulging eyes and were compared with the KIR gene profiles of 112 healthy controls. The presence and absence of 11 variable KIR genes were characterized using a gene-specific PCR typing system. There was no significant difference in the distribution of KIR gene profiles between patients and controls. Our data show that none of the KIR genotypes contribute in susceptibility to Graves’ disease; although the role of HLA ligand remains to be characterized.

Increased levels of interleukin-8 (IL-8) and interleukin-6 (IL-6) in acute human brucellosis have been reported. Previous studies have shown that the production and level of IL-6 and IL-8 cytokines are associated with the polymorphism of the encoding genes. To investigate the probable association between IL-6 (-174 C/G) and IL-8 (-251 A/T) gene polymorphisms and susceptibility/resistance to brucellosis. The patient group included 196 patients suffering from Brucella infection and the control group consisted of 82 healthy animal husbandmen from the same geographical area. IL-8 (-251 A/C) and IL-6 (-174 C/G) gene polymorphisms were analyzed by PCR-RFLP and Allele Specific PCR (AS-PCR) respectively. The frequency of -251 IL-8 AA genotype was significantly lower in the controls compared with that of the patients (p=0.0051), while the frequencies of other genotypes (AT and TT) and alleles (A and T) were not significantly different among the participants. No association was found between IL-6 (-174 C/G) polymorphism and brucellosis. This study indicates that the IL-8 -251 AA genotype may be considered as a genetic susceptibility factor for brucellosis.

Chronic low-grade systemic inflammation presented in Type 2 diabetes mellitus plays a major role in disease progression as well as development of micro- and macro-vascular complications of diabetes. Therefore, reducing inflammation can be beneficial in prevention of diabetes complications. To investigate the association between insulin resistance and inflammatory markers, and assessing the effects of oral Calcitriol on inflammatory cytokines in type 2 diabetic patients. In this doubleblind randomized placebo-controlled trial, 70 participants with type-2 diabetes were randomly divided to two groups. One group received two capsules of Calcitriol (0.25 μg 1,25-dihydroxy cholecalciferol per each capsule) per day. The second group received placebo tablets. At the beginning of the study, we assessed insulin resistance and its relation to inflammatory profile. Serum high sensitive C-reactive protein (hsCRP), interleukin-6 and interleukin-18 were also measured at the beginning and the end of the 12-week supplementation trial. Mean calcium, phosphorus and vitamin D concentrations in the study participants were 8.98 ± 0.79 mg/dL, 3.86 ± 0.50 mg/dL and 40.91 ± 30.9 ng/mL, respectively. IL-18 and hsCRP had significant positive associations with insulin resistance markers and negative associations with insulin sensitivity markers. At the end of the 12-week supplementation trial, no significant difference was seen in serum levels of hsCRP, IL-6 and IL-18 between the two groups, while these values were adjusted for baseline values. Although there was an association between insulin resistance and inflammation, Calcitriol had no effect in decreasing hsCRP, IL-6 and IL-18 in diabetic patients.

IL-17 is a major cytokine player in T cell mediated leukocyte associated inflammation. IL-17 is also recognized to participate in the pathophysiology of asthma. To determine the role of IL-17 in predicting severe asthma. We obtained serum samples from asthmatic children under the age of 5-year in three different groups of mild (n=33), moderate (n=28) and severe (n=32) persistent asthma. IL-17 serum concentrations and mRNA expression were determined by ELISA and real time PCR assays, respectively. Serum IL-17 concentrations were significantly higher in patients with severe asthma than the other two groups of children with mild and moderate disease (p=0.00). Mean serum IL-17 values were 142.04 pg/ml in mild group, 180.4 pg/ml in moderate group and 251.25 pg/ml in severe group. IL-17 mRNA levels were also significantly elevated in severe asthmatic patients compared to mild and moderate asthmatic children (p=0.00). Serum IL-17 concentrations and IL-17 mRNA expressions were increased in children with severe asthma compared to those with mild and moderate forms of the disease.