RESUMO
BACKGROUND: Reports indicate that human and canine patients with atopic dermatitis (AD) have reduced production of several skin antimicrobial peptides, but more recent data have called those results into question. HYPOTHESIS/OBJECTIVES: To compare the mRNA expression of seven antimicrobial peptide genes in lesional and adjacent nonlesional skin biopsy specimens from dogs with AD with those from normal dogs and from dogs experiencing other inflammatory skin conditions. ANIMALS: Normal dogs and patients with AD or other inflammatory skin conditions were enrolled with owner permission and approval of the Institutional Animal Care and Use Committee. METHODS: Transcripts were measured by quantitative RT-PCR using a standard curve assessment. RESULTS: Normal transcript levels for all seven antimicrobial peptides varied depending on the body site assessed. Transcripts for secretory leukocyte proteinase inhibitor (SLPI) and skin-derived antileucoproteinase (SKALP; also known as elafin) were typically ~10-fold greater in number than transcripts for the canine ß-defensins (CBD)-1, -102, -103, -122 and -124. Transcripts for SKALP, SLPI, CBD-1, CBD-103 and CBD-122 were lower in both lesional and adjacent nonlesional skin from dogs with AD in comparison to normal skin. Transcripts were reduced to a similar extent versus normal dogs in skin of dogs with inflammatory skin conditions from both lesional and nonlesional biopsies, except for CBD-122, which was reduced only in lesional skin. Compared with normal dog skin, transcripts for CBD-102 and CBD-124 were unaffected in dogs with AD. CONCLUSIONS AND CLINICAL IMPORTANCE: Both SKALP and SLPI may be important contributors to skin innate immunity, but their decreased expression in AD patients does not account for increased skin infections compared with other skin conditions.
Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Dermatite Atópica/veterinária , Doenças do Cão/metabolismo , Regulação da Expressão Gênica/fisiologia , Inflamação/veterinária , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Dermatite Atópica/metabolismo , Doenças do Cão/genética , Cães , Feminino , Inflamação/metabolismo , Masculino , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterináriaRESUMO
The major histocompatibility complex (MHC) is a gene dense region with profound effects on the disease phenotype. In many species, characterizations of MHC polymorphisms have focused on identifying allelic haplotypes of the highly polymorphic class I and class II loci through direct immunological approaches such as monoclonal antibodies specific for the major antigens or indirectly through DNA sequence-based approaches. Invariably, these studies fail to assess the broader range of variation at the other loci within the MHC. This study examines variation in the turkey MHC by resequencing 15 interspersed amplicons ( approximately 14 kb) spaced across the MHC-B locus in a representative sampling of 52 commercial birds. Over 200 single nucleotide polymorphisms (SNPs) were identified with high levels of polymorphism (1 SNP/70 bp) and heterozygosity (average minor allele frequency of 0.15). SNP genotypes were used to identify the major haplotypes segregating in the commercial lines. Sequencing of the peptide binding region (PBR, exon 2) of the class IIB loci of select individuals identified 10 PBR alleles/isotypes among the major MHC haplotypes. Examination of pedigreed families provides direct evidence of gene conversion and recombination within the B locus. Results of this study demonstrate the MHC diversity available in commercial flocks and provide genomic resources for studying the effect of this diversity (alleles and/or haplotypes) on disease susceptibility and resistance.
Assuntos
Conversão Gênica/genética , Haplótipos/genética , Complexo Principal de Histocompatibilidade/genética , Polimorfismo de Nucleotídeo Único/genética , Perus/genética , Sequência de Aminoácidos , Animais , Dados de Sequência Molecular , Filogenia , Homologia de Sequência de AminoácidosRESUMO
The cutaneous barrier contains small, cationic antimicrobial peptides that participate in the innate immunity against a wide variety of pathogens. Despite their immune importance, knowledge of canine defensins and their expression is limited primarily to testicular tissue and their relation to coat colour. Studies have shown that the absence of these antimicrobial peptides contribute to increased secondary infections in humans. The goals of this study were to identify defensin and protease inhibitor peptide genes by performing a computer-based iterative screen of the canine genome and to determine whether antimicrobial peptides are expressed in normal canine skin. Reverse transcription-polymerase chain reaction (RT-PCR) was used to test for the expression of several antimicrobial peptides in the skin of five normal dogs. RNA from testis was used for comparison. The iterative screen identified 65 putative antimicrobial peptide genes on nine chromosomes, the majority clustered on chromosomes 16 and 24. Amplification of normal canine skin cDNAs demonstrated expression of antimicrobial peptide genes in five different body sites. These findings will provide a tool for future studies examining the association between antimicrobial gene expression and cutaneous immunity in dogs.
Assuntos
Defensinas/metabolismo , Regulação da Expressão Gênica/fisiologia , Pele/metabolismo , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar/genética , Defensinas/genética , Cães , Perfilação da Expressão GênicaRESUMO
The enteric immune system of swine protects against infectious and noninfectious environmental insults and discriminates ingested nutrients, food, and commensal microflora from pathogenic agents. The molecular and cellular elements of the immune system have been selected over evolutionary time in response to the specific environment of pigs. Thus, models of immune function based on mouse and human need to be applied cautiously in the pig. To better understand how the mucosal immune system of the small intestine accomplishes the conflicting functions of food tolerance and immunity to enteric infection, we used a genomic approach to profile gene expression in the Peyer's patch. More than 40% of mRNA enriched by differential subtraction for Peyer's patch-specific expressed sequences represented genes of unknown function or had no match in GenBank. Microarray analysis and radiation hybrid mapping validated their porcine origin and provided additional insights into putative functions. The abundance of expressed genes of unknown function indicates that a substantial fraction of the immunological and physiological processes of the Peyer's patch remains to be discovered. It further suggests that swine have evolved specialized biochemical and immunological processes in the small intestine. Further elucidation of these processes are expected to provide novel insights into swine enteric mucosal immune function.
Assuntos
Imunidade nas Mucosas/genética , Mucosa Intestinal/imunologia , Intestino Delgado/imunologia , Sus scrofa/genética , Animais , Mapeamento Cromossômico , Mapeamento de Sequências Contíguas , Etiquetas de Sequências Expressas , Perfilação da Expressão Gênica , Genômica , Análise em Microsséries , Modelos Biológicos , Nódulos Linfáticos Agregados/metabolismo , Mapeamento de Híbridos Radioativos , Sus scrofa/imunologiaRESUMO
Both trimellitic anhydride (TMA), a small molecular weight chemical, and ovalbumin (OVA), a reference protein allergen, cause asthma with eosinophilia. To test the hypothesis that different allergens elicit symptoms of asthma via different effector pathways, gene expression was compared in lungs of Balb/c mice sensitized with either TMA or OVA, followed by intratracheal challenge with TMA conjugated to mouse serum albumin (TMA-MSA) or OVA, respectively. Sensitized animals challenged with mouse serum albumin (MSA) alone were controls. Seventy-two hours after challenge, lung eosinophil peroxidase indicated that both allergens caused the same significant change in eosinophilia. Total RNA was isolated from lung lobes of 6-8 animals in each of four treatment groups and hybridized to Affymetrix U74Av2 GeneChips. False discovery rates (q-values) were calculated from an overall F test to identify candidate genes with differences in expression for the four groups. Using a q-value cutoff of 0.1, 853 probe sets had significantly different expression across the four treatment groups. Of these 853 probe sets, 376 genes had an Experimental/Control ratio of greater than 1.2 or less than 1/1.2 for either OVA- or TMA-treated animals, and 249 of the 376 genes were uniquely up- or down-regulated for OVA or TMA (i.e., differentially expressed with the allergen). qRT-PCR analysis of selected transcripts confirmed the gene expression analysis. Increases in both arginase transcript and enzyme activity were significantly greater in OVA-induced asthma compared to TMA-induced asthma. These data suggest that pathways of arginine metabolism and the importance of nitric oxide may differ in OVA- and TMA-induced asthma.
Assuntos
Alérgenos/farmacologia , Arginase/metabolismo , Asma/enzimologia , Eosinofilia/enzimologia , Ovalbumina/farmacologia , Anidridos Ftálicos/farmacologia , Alérgenos/administração & dosagem , Animais , Arginase/genética , Asma/induzido quimicamente , Asma/imunologia , Modelos Animais de Doenças , Eosinofilia/induzido quimicamente , Eosinofilia/imunologia , Eosinófilos/efeitos dos fármacos , Eosinófilos/enzimologia , Eosinófilos/imunologia , Feminino , Expressão Gênica/efeitos dos fármacos , Intubação Intratraqueal , Camundongos , Camundongos Endogâmicos BALB C , Procedimentos Analíticos em Microchip , Ovalbumina/administração & dosagem , Peroxidase/metabolismo , Anidridos Ftálicos/administração & dosagem , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Maps of the canine genome are now developing rapidly. Most of the markers on the current integrated canine radiation hybrid/genetic linkage/cytogenetic map are highly polymorphic microsatellite (type II) markers that are very useful for mapping disease loci. However, there is still an urgent need for the mapping of gene-based (type I) markers that are required for comparative mapping, as well as identifying candidate genes for disease loci that have been genetically mapped. We constructed an adult brain cDNA library as a resource to increase the number of gene-based markers on the canine genome map. Eighty-one percent of the 2700 sequenced expressed sequence tags (ESTs) represented unique sequences. The canine brain ESTs were compared with sequences in public databases to identify putative canine orthologs of human genes. One hundred nine of the canine ESTs were mapped on the latest canine radiation hybrid (RH) panel to determine the location of the respective canine gene. The addition of these new gene-based markers revealed three conserved segments (CS) between human and canine genomes previously detected by fluorescence in situ hybridization (FISH), but not by RH mapping. In addition, five new CS between dog and human were identified that had not been detected previously by RH mapping or FISH. This work has increased the number of gene-based markers on the canine RH map by approximately 30% and indicates the benefit to be gained by increasing the gene content of the current canine comparative map.
Assuntos
Encéfalo/metabolismo , Etiquetas de Sequências Expressas , Mapeamento de Híbridos Radioativos , Animais , DNA Complementar , Cães , Análise de Sequência de DNARESUMO
Within the naval medical center construct, the disparate disciplines encompassed within ancillary services lend themselves to formal quality analysis and process improvement. This analysis uses the Six Sigma approach. Error rates were investigated and calculated for various processes within ancillary services at Naval Medical Center, San Diego. These were translated into the common metric of defects per million opportunities (DPMO). DPMO rates vary between 21.5 and 420,000. These correspond to Sigma values from 1.7 to approaching 6. Rates vary with biological complexity of the system and the degree of automation available. Some ancillary services translate well into a Six Sigma schema. Systems with high potential patient risk if performed poorly and those amenable to second checking and computer oversight may be candidates for such optimization. This should be undertaken in a local environment conducive to individual error reporting, and in a corporate environment with the will and funding to support the transition.