Walter Klimecki

IL-13 is a central mediator of allergic inflammation. The single nucleotide polymorphism IL13-1112C>T (rs1800925) is associated with allergic phenotypes in ethnically distinct populations, but the underlying mechanism(s) remain unknown. Using in vivo, in vitro, and in silico analysis, we show that the IL13-1112T allele enhanced IL13 promoter activity in primary human and murine CD4(+) Th2 lymphocytes. Increased expression of IL13-1112T in Th2 cells was associated with the creation of a Yin-Yang 1 binding site that overlapped a STAT motif involved in negative regulation of IL13 expression and attenuated STAT6-mediated transcriptional repression. Because IL-13 secretion was increased in IL13-1112TT homozygotes, we propose that increased expression of IL13-1112T in vivo may underlie its association with susceptibility to allergic inflammation. Interestingly, IL13-1112T had opposite transcriptional effects in nonpolarized CD4(+) T cells, paralleled by distinct patterns of DNA-protein interactions at the IL13 promoter. Our findings suggest the nuclear milieu dictates the functional outcome of genetic variation.

BEAS-2B, an immortalized, human lung epithelial cell line, has been used to model pulmonary epithelial function for over 30 years. The BEAS-2B phenotype can be modulated by culture conditions that include the presence or absence of fetal bovine serum (FBS). The popularity of BEAS-2B as a model of arsenic toxicology, and the common use of BEAS-2B cultured both with and without FBS, led us to investigate the impact of FBS on BEAS-2B in the context of arsenic toxicology. Comparison of genome-wide gene expression in BEAS-2B cultured with or without FBS revealed altered expression in several biological pathways, including those related to carcinogenesis and energy metabolism. Real-time measurements of oxygen consumption and glycolysis in BEAS-2B demonstrated that FBS culture conditions were associated with a 1.4-fold increase in total glycolytic capacity, a 1.9-fold increase in basal respiration, a 2.0-fold increase in oxygen consumed for ATP production and a 2.8-fold increase in maximal respiration, compared with BEAS-2B cultured without FBS. Comparisons of the transcriptome changes in BEAS-2B resulting from FBS exposure to the transcriptome changes resulting from exposure to 1 μM sodium arsenite revealed that mRNA levels of 43% of the arsenite-modulated genes were also modulated by FBS. Cytotoxicity studies revealed that BEAS-2B cells exposed to 5% FBS for 8 weeks were almost 5 times more sensitive to arsenite cytotoxicity than non-FBS-exposed BEAS-2B cells. Phenotype changes induced in BEAS-2B by FBS suggest that culture conditions should be carefully considered when using BEAS-2B as an experimental model of arsenic toxicity.

PMID: 16918516;Abstract:

Background: Caspase recruitment domain protein (CARD) 4 has been recently identified as an intracellular pattern recognition receptor that interacts with muropeptides found in common Gram-negative bacteria. We therefore aimed to explore whether the previously observed inverse association between exposure to microbial products and asthma and allergies in childhood is modified by genetic variation in CARD4. Methods: We genotyped 668 children [mean age 9.3 (SD 1.5) years] enrolled in the cross-sectional ALEX study for seven haplotype tagging single nucleotide polymorphisms in CARD4. We studied the association of asthma, hay fever and allergen-specific serum immunoglobulin E with exposure to a farming environment and with levels of endotoxin and muramic acid measured in house dust samples. We tested whether these associations differed between the genotypes of the polymorphisms under study. Results: A strong protective effect of a farming environment on allergies was only found in children homozygous for the T allele in CARD4/-21596, but not in children carrying the minor allele (C). Among the former, farmers' children had a significantly lower frequency of sensitization against pollen (5.8%), hay fever (1.7%) and atopic asthma symptoms (1.7%) compared with children not living on a farm (19.4%, 13.0% and 7.6%, P 0.01, 0.01 and 0.05, respectively). Conversely, no significant difference in prevalence of these phenotypes by farming status was found among children with a C allele in CARD4/-21596 (14.3%, 7.1% and 8.0%vs 16.5%, 9.0% and 5.7%, respectively). Conclusion: Polymorphisms in CARD4 significantly modify the protective effect of exposure to a farming environment. © 2006 The Authors.

PMID: 15096567;PMCID: PMC1867471;Abstract:

Infection with mucosotropic human papillomavirus (HPV) is the necessary cause of cervical intraepithelial neoplasia. Several epidemiological studies suggest that HPV viral load can be a risk factor of cervical dysplasia. The purpose of the present study was to evaluate a methodology to determine HPV viral load of eight oncogenic HPV types (16, 18, 31, 39, 45, 51, 52, and 58). The quantitation assay is based on a high-throughput real-time PCR. The E6-E7 region of HPV types 16, 18, 45, and 51 were used to amplify specific DNA sequences and cloned into a plasmid vector. The constructs for HPV types 16, 18, 45, and 51, and the whole genome for HPV types 31, 39, 52, and 58 were quantitated using a limiting dilution analysis and used to create standard curves. Type-specific HPV clones were used to determine specificity, linearity, and intra- and inter-assay variation. The sensitivity of our assay covered a dynamic range of up to seven orders of magnitude with a coefficient of intra-assay variation less than 6% and the inter-assay variation less than 20%. No cross-reactivity was observed on any of the type-specific standard curves when phylogenetically close HPV types were used as templates. Our real-time PCR methodologies are highly reproducible, sensitive, and specific over a sevenfold magnitude dynamic range. Copyright © American Society for Investigative Pathology and the Association for Molecular Pathology.