Walter Klimecki

PMID: 15201134;Abstract:

TOLL-like receptor 10 (TLR10) is the most recently identified human homolog of the Drosophila TOLL protein. In humans, the TOLL-like receptors recognize pathogen-associated molecular patterns (PAMPs) as part of innate immune host defenses. Localized to chromosome 4p14, the specific ligands and functions of TLR10 are currently unknown, although it is expressed in lung and in B-lymphocytes. TLR10 is a potential asthma candidate gene because early life innate immune responses to ubiquitous inhaled allergens and PAMPs may influence asthma susceptibility. Resequencing in 47 subjects revealed a total of 78 single nucleotide polymorphisms (SNPS) (1 SNP per 106 bp) of which only 11 had been previously published. A significant association (p = 0.02) between two SNPs (C.+1031G>A, c.+2322A>G) and physician-diagnosed asthma was observed in a case control study (517 cases, 519 control subjects) of European American subjects nested within the Nurses' Health Study cohort. The association for these same two SNPs (p ≤ 0.015) replicated in an independent family based cohort, where a measure of airway hyperresponsiveness (PC20) was also associated (p = 0.026 for c.+1031G>A). Consistent association in two independent samples and association with an intermediate phenotype provides strong support for TLR10 genetic variation contributing to asthma risk.

PMID: 20654299;Abstract:

An organotypic culture (OTC) of a human keratinocyte cell line (HaCaT) over a human fibroblast-embedded collagen gel was used to model human epidermis in arsenicism, a syndrome that currently lacks valid experimental models. Keratinocytes were exposed acutely or chronically to a mixture of arsenate (0.5 μM), monomethylarsonic acid (MMA; 0.5 μM) and dimethylarsinic acid (DMA; 1.5 μM), or to the individual components of the mixture. OTCs were assayed for microscopic morphology, the proliferating cell marker, Ki-67, labelling and cytokeratin expression. Acute exposures resulted in an epidermal phenotype that accurately modelled early human lesions, including hyperkeratosis, acanthosis and keratin 16 induction. Chronic exposures resulted in a de-differentiated epidermal phenotype with focal nests of keratinocytes growing into the collagen gel. The keratin 8/18 pair was induced by either acute or chronic arsenic exposure, as was the proliferating cell marker, Ki-67. Exposure of keratinocytes to individual arsenic compounds demonstrated that all arsenic mixture-induced changes could be duplicated by exposure to arsenate alone. In contrast, MMA and DMA were inactive. This study establishes OTC as a useful model of arsenicism, and implicates inorganic arsenic as the ultimate carcinogen.

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.

PMID: 12160736;Abstract:

Interleukin-10 (IL-10) is a cytokine that seems to function as a downregulator of the innate (nonadaptive) immune system. Approximately three-quarters of interindividual variability in human IL-10 levels has been attributed to genetic variation, and there is evidence suggesting a potential role for IL-10 in a range of human diseases. To provide a basis for haplotype analysis and future disease association studies, we characterized genetic variation in IL10 by sequencing all exons, and 2.5 kb of the 5′- and the 3′-flanking region in a panel of DNA samples from 24 African Americans, 23 European Americans, and 24 Hispanic Americans. The region sequenced was found to contain 28 single-nucleotide polymorphisms (SNPs), 16 with frequency > 2% and 14 with frequency > 5%. All SNPs with frequency > 5% were present in subjects from all three populations. No SNP caused amino acid changes. Differences in pairwise linkage-disequilibrium (LD) patterns and in SNP and haplotype frequency distributions among the three populations may be of potential importance for disease association studies.

PMID: 16159630;Abstract:

Background: Most complex diseases are the result of interactions between polymorphisms in the genome and environmental exposures. Objective: We sought to investigate the previously reported association between a polymorphism in the promoter region of CD14 (CD14/-260C→T) and serum IgE levels in relation to the environment to which children are exposed. Methods: In 624 children living in 2 rural communities in Europe, we compared total and specific serum IgE levels between the genotypes of CD14/-260 in relation to exposure to animals and in relation to house dust endotoxin. Results: We found that the C allele of CD14/-260 was associated with higher levels of both total and specific serum IgE to aeroallergens in children with regular contact with pets, whereas an association in the opposite direction was found in children with regular contact with stable animals. This modifying effect of animal exposure was not explained by levels of house dust endotoxin. However, in children with high levels of house dust endotoxin, the C allele was associated with less specific IgE, independently from animal exposure. Conclusion: Because CD14 is a pattern recognition receptor for microbial molecules, the results suggest that the type and concentrations of such molecules present in the environment strongly determine the direction of the association between CD14/-260 and serum markers of atopy. © 2005 American Academy of Allergy, Asthma and Immunology.