Walter Klimecki

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.

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: 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.

PMID: 12493003;Abstract:

Under selective pressure from infectious microorganisms, multicellular organisms have evolved immunological defense mechanisms, broadly categorized as innate or adaptive. Recent insights into the complex mechanisms of human innate immunity suggest that genetic variability in genes encoding its components may play a role in the development of asthma and related diseases. As part of a systematic assessment of genetic variability in innate immunity genes, we have thus far have examined 16 genes by resequencing 93 unrelated subjects from three ethnic samples (European American, African American and Hispanic American) and a sample of European American asthmatics. Approaches to discovering and understanding variation and the subsequent implementation of disease association studies are described and illustrated. Although highly conserved across a wide range of species, the innate immune genes we have sequenced demonstrate substantial interindividual variability predominandy in the form of single nucleotide polymorphisms (SNPs). Genetic variation in these genes may play a role in determining susceptibility to a range of common, chronic human diseases which have an inflammatory component. Differences in population history have produced distinctive patterns of SNP allele frequencies, linkage disequilibrium and haplotypes when ethnic groups are compared. These and other factors must be taken into account in the design and analysis of disease association studies.