Interleukin-13 gene polymorphisms in patients with Graves' disease

OBJECTIVE: In patients with Graves' disease (GD), an elevation of serum immunoglobulin E (IgE) has been recently reported to be associated with the severity of hyperthyroidism and ophthalmopathy. Interleukin 13 (IL-13) is a major cytokine involved in IgE synthesis and therefore may be a potential candidate gene contributing to the development of GD or influencing the clinical course of the disease. DESIGN: In a case-control study, we examined IL-13 gene single-nucleotide polymorphisms in the 5' promoter region at position -1112 (C to T change, termed as C-1112T) and in exon 4 at position 2044 (G to A change, G2044A, which results in an amino acid exchange Arg130Gln) in 261 patients with GD. The control groups consisted of healthy young subjects (n=168) and subjects over 100 years old with no history of autoimmune or allergic diseases recruited from the Polish Centenarians Project (n=50). MEASUREMENTS: C-1112T and G2044A polymorphisms were defined by fluorescent single-strand conformational polymorphism and by restriction fragment length polymorphism analysis, respectively. RESULTS: In patients with GD, the distribution of IL-13 alleles (-1112T 31%; 2044A 25%) and genotypes (-1112T/T 10%; 2044A/A 7%) did not differ significantly compared to control groups. Subdividing GD patients according to clinically evident ophthalmopathy (NOSPECS class III or higher, n=93) revealed no significant differences in the frequencies of -1112T allele (33%vs. 29%; P=0.4), -1112T/T genotype (13%vs. 8%; P=0.3), 2044A allele (27%vs. 24%; P=0.5) and 2044A/A genotype (9%vs. 7%; P=0.7) between GD patients with and without eye involvement. In order to analyse the association with the severity of hyperthyroidism, we examined patients with a first onset of GD treated with antithyroid drugs (n=32). IL-13 genotypes were not associated with the laboratory findings at diagnosis (thyroid volume, serum levels of FT4, TRAb, TPOAb, TGAb) and with the outcome of antithyroid drug treatment. CONCLUSIONS: Our results suggest that IL-13 gene polymorphisms at positions -1112 (C-->T) and 2044 (G-->A): (1) do not confer genetic susceptibility to Graves' disease; (2) do not contribute to the development of clinically evident ophthalmopathy; (3) are not associated with severity of hyperthyroidism.     

Identification of AP80978, a Novel Small-Molecule Inhibitor of Hepatitis C Virus Replication That Targets NS4B

A small-molecule inhibitor of hepatitis C virus (HCV) designated AP89652 was identified by screening a compound library with an HCV genotype 1b subgenomic replicon assay. AP89652 contains two chiral centers, and testing of two syn enantiomers revealed that activity in the replicon assay resided with only one, AP80978, whose 50% effective concentration (EC₅₀) (the concentration at which a 50% reduction in Renilla luciferase levels was observed relative to an untreated control) was 630 nM. AP80978 was inhibitory against HCV genotypes 1a and 1b but not genotype 2a. In a replicon clearance assay, the potency and clearance rate of AP80978 were similar to those of telaprevir (VX950) and cyclosporine (CsA). AP80978 was nontoxic when tested against a panel of human cell lines, and inhibitory activity was HCV specific in that there was limited activity against negative-strand viruses, an alphavirus, and flaviviruses. By selection of resistant replicons and assessment of activity in genotype 1b/2a intergenotypic replicons, the viral protein target of this compound was identified as NS4B. NS4B F98V/L substitutions were confirmed by site-directed mutagenesis as AP80978 resistance-associated mutations. When tested against HCV produced in cell culture, the compound was significantly more potent than other HCV inhibitors, including VX950, CsA, and 2′-C-methyladenosine (2′C-meA). In addition, AP80977, the enantiomer that was inactive in the replicon assay, had activity against the virus, although it was lower than the activity of AP80978. These results suggest that AP80978 has the potential to be optimized into an effective antiviral drug and is a useful tool to further study the role of NS4B in HCV replication.     

Liver sinusoidal lymphocytes: their immune functions

Abstract  Recent studies strongly suggest that the liver plays an im portant immunoregulatory role. Ev idence of its role in general immune responsiveness originates from ob servation that, in recipients of liver grafts, the survival of other allo-grafts is significantly prolonged. The question arises as to which blood lymphocyte subsets, most likely to be responsible for this phenomenon, marginate in liver sinusoids. To study this problem, a liver ex vivo perfusion model was designed for rats. In situ W/WAG livers were wa shed clear of sinusoidal marginating cells prior to and after 1 h perfusion with syngeneic blood. The number of blood cells retained in liver sinu soids, their phenotypes, the respon siveness to mitogen (PHA, 90 μ g/ ml) and cytotoxicity against YAC-1 tumour cells were examined. Our studies showed that rat liver retains in the sinusoids a population of blood cells, enriched in NK, CD8' and MHC class II+ cells, displaying a high cytotoxic activity and low re sponsiveness to mitogen stimula tion, with a capacity of about 10⁶ cells/g of tissue.     

Post-exercise oxidative stress and obesity in postmenopausal women: the role of beta3-adrenergic receptor polymorphism.

AIM: Some studies indicate that the Trp64Arg polymorphism in the gene encoding the beta3-adrenergic receptor (ADRB3) is associated with obesity, insulin resistance and earlier onset of type 2 diabetes mellitus. The aim of the present study was to evaluate the frequency of this polymorphism and its relationship with obesity and oxidative stress in postmenopausal women. MATERIAL AND METHODS: We performed the study on 200 women, aged 50-60 years. Estimation of anthropometric parameters and total body fat, android and gynoid fat deposits was carried out using dual-energy X-ray absorptiometry. Oxidative stress was estimated by measurement of thiobarbituric acid-reactive substances (TBARS) in serum. Blood for analysis was collected before, directly after and 6 h after a 30-min physical test on a cycle ergometer. ADRB3 genotyping was performed by polymerase chain reaction. RESULTS: The frequency of Trp64/Arg64 genotype in the investigated population was 12%, and of Trp64/Trp64 was 87%. The Arg64/Arg64 genotype was present in only 1% of women. Women bearing the Trp64/Arg64 genotype did not differ in any measured anthropometric parameters from women bearing the Trp64/Trp64 genotype. Moreover, genotype had no influence on oxidative stress parameters. Likewise, in both groups, mean plasma level of TBARS was increased significantly (p < 0.05) directly after the endurance test and remained elevated 6 h after the test. CONCLUSIONS: The Trp64Arg polymorphism of ADRB3 seems to not be related to obesity in postmenopausal women. Moreover, the Trp64Arg polymorphism has no influence on oxidative stress intensification after standardized physical effort in postmenopausal women.     

Signaling of programmed cell death induction in WEHI-231 B lymphoma cells

The murine WEHI-231 B lymphoma is highly sensitive to membrane immunoglobulin ligation which leads to programmed cell death (PCD) in this cell line. To study the molecular pathways involved in PCD induction in these cells, we derived two variants of WEHI-231 resistant to anti-Ig treatment. The level of bcl-2 mRNA was identical in the wild type and the variants, either untreated or anti-Ig treated, suggesting that PCD is not under the control of bcl-2 in WEHI-231 cells. In contrast, c-myc gene expression was markedly different in the wild type and the variants, both in the unstimulated and anti-Ig-stimulated state. Our findings are interpreted in the context of the dual capacity of c-myc to promote cell growth or cell death, in conjunction with other growth regulatory signals.     

Analysis of CD27 surface expression on T cell subsets in MS patients and control individuals

Within the peripheral blood, CD4⁺CD27⁻ T cells only reside within the CD45RAT ⁻(memory or primed) T cell subset. Cells with this phenotype have characteristics of specialized effector T cells according to their cytokine secretion profiles and the expression of tissue-specific adhesion molecules. This subset was previously found to be increased in certain diseases that are associated with immune activation. Therefore we analyzed CD27 expression of peripheral blood and CSF T cells in MS patients. Within the CD4⁺ T cell subset no differences were seen between MS patients and controls in proportions of CD45RA⁻CD27⁻ cells. However, when the CD3⁺ T cell compartment was analyzed, CD27⁻ cells were also found within the CD45RA⁺ subset. These cells, most likely CD8⁺, are significantly reduced in PBL and CSF of MS patients as compared with OND patients. In MS and OND groups the level of CD27⁻ cells in peripheral blood correlated significantly with that in CSF, indicating a balanced migration of CD27⁻ cells between the two compartments. In OIND patients, however, this equilibrium was lost. The correlation of the level of CD27⁺ cells with the amount of intrathecally produced IgG in MS patients may suggest that CD27⁺ cells are responsible for B cell help in this disease.