Altered expression of antimicrobial peptide genes in the skin of dogs with atopic dermatitis and other inflammatory skin conditions.

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.

The Cryptosporidium parvum transcriptome during in vitro development.

Cryptosporidiosis is caused by an obligate intracellular parasite that has eluded global transcriptional or proteomic analysis of the intracellular developmental stages. The transcript abundance for 3,302 genes (87%) of the Cryptosporidium parvum protein coding genome was elucidated over a 72 hr infection within HCT8 cells using Real Time-PCR. The parasite had detectable transcription of all genes in vitro within at least one time point tested, and adjacent genes were not co-regulated. Five genes were not detected within the first 24 hr of infection, one containing two AP2 domains. The fewest genes detected were at 2 hr post infection, while 30% (985) of the genes have their highest expression at 48 and/or 72 hr. Nine expression clusters were formed over the entire 72 hr time course and indicate patterns of transcriptional increases at each of the 7 time points collected except 36 hr, including genes paralleling parasite 18S rRNA transcript levels. Clustering within only the first 24 hr of infection indicates spikes in expression at each of the 4 time points, a group paralleling 18S rRNA transcript levels, and a cluster with peaks at both 6 and 24 hr. All genes were classified into 18 functional categories, which were unequally distributed across clusters. Expression of metabolic, ribosomal and proteasome proteins did not parallel 18S rRNA levels indicating distinct biochemical profiles during developmental stage progression. Proteins involved in translation are over-represented at 6 hr, while structural proteins are over-represented at 12 hr. Standardization methods identified 107 genes with <80% at a single of its total expression at a single time point over 72 hr. This comprehensive transcriptome of the intracellular stages of C. parvum provides insight for understanding its complex development following parasitization of intestinal epithelial cells.

Association of the porcine transforming growth factor beta type I receptor (TGFBR1) gene with growth and carcass traits.

BACKGROUND: Growth and carcass traits are of great economic importance in livestock production. A large number of quantitative trait loci (QTL) have been identified for growth and carcass traits on porcine chromosome one (SSC1). A key positional candidate for this chromosomal region is TGFBR1 (transforming growth factor beta type I receptor). This gene plays a key role in inherited disorders at cardiovascular, craniofacial, neurocognitive, and skeletal development in mammals. RESULTS: In this study, 27 polymorphic SNPs in the porcine TGFBR1 gene were identified on the University of Illinois Yorkshire × Meishan resource population. Three SNPs (SNP3, SNP43, SNP64) representing major polymorphic patterns of the 27 SNPs in F1 and F0 individuals of the Illinois population were selected for analyses of QTL association and genetic diversity. An association analysis for growth and carcass traits was completed using these three representative SNPs in the Illinois population with 298 F2 individuals and a large commercial population of 1008 animals. The results indicate that the TGFBR1 gene polymorphism (SNP64) is significantly associated (p < 0.05) with growth rates including average daily gains between birth and 56 kg (p = 0.049), between 5.5 and 56 kg (p = 0.024), between 35 and 56 kg (p = 0.021). Significant associations (p < 0.05) were also identified between TGFBR1 gene polymorphisms (SNP3/SNP43) and carcass traits including loin-eye-area (p = 0.022) in the Illinois population, and back-fat thickness (p = 0.0009), lean percentage (p = 0.0023) and muscle color (p = 0.021) in the commercial population. These three SNPs were also used to genotype a diverse panel of 130 animals representing 11 pig breeds. Alleles SNP3_T and SNP43_G were fixed in Pietrain and Sinclair pig breeds. SNP64_G allele was uniquely identified in Chinese Meishan pigs. Strong evidence of association (p < 0.01) between both SNP3 and SNP64 alleles and reproductive traits including gestation length and number of corpora lutea were also observed in the Illinois population. CONCLUSION: This study gives the first evidence of association between the porcine TGFBR1 gene and traits of economic importance and provides support for using TGFBR1 markers for pig breeding and selection programs. The genetic diversities in different pig breeds would be helpful to understand the genetic background and migration of the porcine TGFBR1 gene.

Proteolytic products of the porcine reproductive and respiratory syndrome virus nsp2 replicase protein.

The nsp2 replicase protein of porcine reproductive and respiratory syndrome virus (PRRSV) was recently demonstrated to be processed from its precursor by the PL2 protease at or near the G(1196)|G(1197) dipeptide in transfected CHO cells. Here the proteolytic cleavage of PRRSV nsp2 was further investigated in virally infected MARC-145 cells by using two recombinant PRRSVs expressing epitope-tagged nsp2. The data revealed that PRRSV nsp2 exists as different isoforms, termed nsp2a, nsp2b, nsp2c, nsp2d, nsp2e, and nsp2f, during PRRSV infection. Moreover, on the basis of deletion mutagenesis and antibody probing, these nsp2 species appeared to share the same N terminus but to differ in their C termini. The largest protein, nsp2a, corresponded to the nsp2 product identified in transfected CHO cells. nsp2b and nsp2c were processed within or near the transmembrane (TM) region, presumably at or near the conserved sites G(981)|G(982) and G(828)|G(829)|G(830), respectively. The C termini for nsp2d, -e, and -f were mapped within the nsp2 middle hypervariable region, but no conserved cleavage sites could be definitively predicted. The larger nsp2 species emerged almost simultaneously in the early stage of PRRSV infection. Pulse-chase analysis revealed that all six nsp2 species were relatively stable and had low turnover rates. Deletion mutagenesis revealed that the smaller nsp2 species (e.g., nsp2d, nsp2e, and nsp2f) were not essential for viral replication in cell culture. Lastly, we identified a cellular chaperone, named heat shock 70-kDa protein 5 (HSPA5), that was strongly associated with nsp2, which may have important implications for PRRSV replication. Overall, these findings indicate that PRRSV nsp2 is increasingly emerging as a multifunctional protein and may have a profound impact on PRRSV replication and viral pathogenesis.

Activation of NF-κB pathway and TNF-α are involved in the cytotoxicity of anthrax lethal toxin in bovine BoMac macrophages.

Anthrax lethal toxin (LeTx) is an important virulence factor of Bacillus anthracis and causes illness and lethality for both animals and humans. Because species demonstrate varied sensitivity to anthrax intoxication, we investigated signaling pathways involved in anthrax LeTx cytotoxicity using a bovine macrophage cell line (BoMac). We found that bovine macrophages are sensitive to LeTx as displayed by a concentration-dependent increase in cell death. LeTx induced the degradation of I-κB and increased the nuclear translocation of NF-κB in BoMac cells. Blocking NF-κB activation with either chemical inhibitors or a dominant negative super-repressor I-κBαm eliminated LeTx-induced cell death. LeTx-induced production of TNF-α that contributed dramatically to cellular cytotoxicity. Inhibiting NF-κB activation eliminated TNF-α release and decreased cytotoxicity. The caspase pathway was also important for cytotoxicity as specific inhibitors abrogated LeTx-induced cell death. Taken together, our results show that activation of the NF-κB pathway and TNF-α production contribute to the cytotoxicity of anthrax LeTx in bovine macrophages.

Expression and regulation of IL-22 by bovine peripheral blood gamma/delta T cells.

IL-22 is a novel T and NK cell cytokine that belongs to the IL-10 cytokine family. Here we report the identification of a bovine IL-22 ortholog that is expressed by mitogen activated bovine peripheral blood gamma/delta T cells. The full-length bovine IL-22 cDNA contained a 68 bp 5'-untranslated region (UTR), a 570-bp open reading frame, and a 480-bp 3'-UTR. The deduced pre-IL-22 has 190 amino acid residues containing a secretory signal peptide from amino acids 1-33 and several potential N-glycosylation sites. The mature protein is predicted to be a secreted, alpha-helical molecule. The bovine IL-22 gene is approximately 7.5 kb in length and is comprised of five introns and six exons, and the first exon is non-coding. Computer analysis and gel shift assay showed that the -1132 and -879 region in the 5' upstream gene sequence contained putative transcription factor binding sites for STATx, Sox-5/9, Sp1, Ik-1, and AREB6. Mutagenesis of STATx and Sox5/9 binding sites decreased promoter functionality by approximately 50%, suggesting their importance in transcription regulation of IL-22. Expression of IL-22 transcripts induced by various mitogens indicated existence of two regulatory control pathways in gamma/delta T cells; IL-2 or PMA treatment induced a slow accumulation of IL-22 mRNA without affecting the maximum induction pathway, whereas ConA treatment rapidly induced a limited amount of IL-22 transcripts. Similar maximal levels of IL-22 transcripts could be induced in gamma/delta T cells and alpha/beta T cells. We conclude that bovine gamma/delta T cells are important sources of IL-22 and suggest a role for this cytokine in regulating immune responses at mucosal surfaces, including the gut.

IL-4 induces protection of vascular endothelial cells against killing by complement and melittin through lipid biosynthesis.

We have shown previously that cytokines IL-4 and IL-13 induce protection in porcine vascular endothelial cells (EC) against killing by the membrane attack complex (MAC) of human complement. This protection is intrinsic, not due to changes in complement regulatory proteins, and requires activation of Akt and sterol receptor element binding protein-1 (SREBP-1), which regulates fatty acid and phospholipid synthesis. Here we report that, compared to EC incubated in medium, IL-4-treated EC had a profound reduction in complement-mediated ATP loss and in killing assessed by vital dye uptake, but only a slight reduction in permeability disruption measured by calcein release. While controls exposed to complement lost mitochondrial membrane potential and subsequently died, protected EC maintained mitochondrial morphology and membrane potential, and remained alive. SREBP-1 and fatty acid synthase activation were required for protection and fatty acid and phospholipid synthesis, including cardiolipin, were increased after IL-4 stimulation, without increase in cholesterol content or cell proliferation. IL-4 also induced protection of EC from killing by the channel forming protein melittin, similar to protection observed for the MAC. We conclude that IL-4 induced activation of Akt/SREBP-1/lipid biosynthesis in EC, resulting in protection against MAC and melittin, in association with mitochondrial protection.

The porcine reproductive and respiratory syndrome virus nsp2 cysteine protease domain possesses both trans- and cis-cleavage activities.

The N terminus of the replicase nonstructural protein 2 (nsp2) of porcine reproductive and respiratory syndrome virus (PRRSV) contains a putative cysteine protease domain (PL2). Previously, we demonstrated that deletion of either the PL2 core domain (amino acids [aa] 47 to 180) or the immediate downstream region (aa 181 to 323) is lethal to the virus. In this study, the PL2 domain was found to encode an active enzyme that mediates efficient processing of nsp2-3 in CHO cells. The PL2 protease possessed both trans- and cis-cleavage activities, which were distinguished by individual point mutations in the protease domain. The minimal size required to maintain these two enzymatic activities included nsp2 aa 47 to 240 (Tyr(47) to Cys(240)) and aa 47 to 323 (Tyr(47) to Leu(323)), respectively. Introduction of targeted amino acid mutations in the protease domain confirmed the importance of the putative Cys(55)- His(124) catalytic motif for nsp2/3 proteolysis in vitro, as were three additional conserved cysteine residues (Cys(111), Cys(142), and Cys(147)). The conserved aspartic acids (e.g., Asp(89)) were essential for the PL2 protease trans-cleavage activity. Reverse genetics revealed that the PL2 trans-cleavage activity played an important role in the PRRSV replication cycle in that mutations that impaired the PL2 protease trans function, but not the cis activity, were detrimental to viral viability. Lastly, the potential nsp2/3 cleavage site was probed. Mutations with the largest impact on in vitro cleavage were at or near the G(1196)|G(1197) dipeptide.

Expression of endogenous antimicrobial peptides in normal canine skin.

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.

Biphasic modulation of apoptotic pathways in Cryptosporidium parvum-infected human intestinal epithelial cells.

The impact of Cryptosporidium parvum infection on host cell gene expression was investigated by microarray analysis with an in vitro model using human ileocecal HCT-8 adenocarcinoma cells. We found changes in 333 (2.6%) transcripts at at least two of the five (6, 12, 24, 48, and 72 h) postinfection time points. Fifty-one of the regulated genes were associated with apoptosis and were grouped into five clusters based on their expression patterns. Early in infection (6 and 12 h), genes with antiapoptotic roles were upregulated and genes with apoptotic roles were downregulated. Later in infection (24, 48, and 72 h), proapoptotic genes were induced and antiapoptotic genes were downregulated, suggesting a biphasic regulation of apoptosis: antiapoptotic state early and moderately proapoptotic state late in infection. This transcriptional profile matched the actual occurrence of apoptosis in the infected cultures. Apoptosis was first detected at 12 h postinfection and increased to a plateau at 24 h, when 20% of infected cells showed nuclear condensation. In contrast, experimental silencing of Bcl-2 induced apoptosis in 50% of infected cells at 12 h postinfection. This resulted in a decrease in the infection rate and a reduction in the accumulation of meront-containing cells. To test the significance of the moderately proapoptotic state late in the infection, we inhibited apoptosis using pancaspase inhibitor Z-VAD-FMK. This treatment also affected the progression of C. parvum infection, as reinfection, normally seen late (24 h to 48 h), did not occur and accumulation of mature meronts was impaired. Control of host apoptosis is complex and crucial to the life of C. parvum. Apoptosis control has at least two components, early inhibition and late moderate promotion. For a successful infection, both aspects appear to be required.

Inhibition of apoptosis in Cryptosporidium parvum-infected intestinal epithelial cells is dependent on survivin.

Cryptosporidium parvum is an obligate intracellular protozoan capable of causing severe diarrheal disease in a wide variety of mammals, including humans. C. parvum infection has been associated with induction of apoptosis in exposed epithelial cells, and we now demonstrate that apoptosis is restricted to a subset of cells actively infected with C. parvum. Approximately 20% of the infected cells underwent apoptosis within 48 h of infection, suggesting that the majority of the infected cells are rescued from apoptosis. C. parvum infection resulted in low-level activation of multiple members of the caspase family, including caspase-2, -3, -4, -6, -8, and -9. The kinetics of caspase activation correlated with apoptosis over a 48-h time course. Pan caspase inhibitors reduced apoptosis of epithelial cells infected by C. parvum. Furthermore, C. parvum infection inhibited staurosporine-induced apoptosis and caspase-3/7 activation at 24 h and 48 h. Infection with C. parvum led to upregulation of genes encoding inhibitors of apoptosis proteins (IAPs), including c-IAP1, c-IAP2, XIAP, and survivin. Knockdown of survivin gene expression, but not that of c-IAP1, c-IAP2, or XIAP expression, increased caspase-3/7 activity as well as apoptosis of infected cells and decreased C. parvum 18S rRNA levels. These data suggest that the apoptotic response of infected intestinal epithelial cells is actively suppressed by C. parvum via upregulation of survivin, favoring parasite infection.

TNFR1 promoter -329G/T polymorphism results in allele-specific repression of TNFR1 expression.

Tumor necrosis factor (TNF) and the TNF receptor (TNFR) superfamily play very important roles for cell death as well as normal immune regulation. Dysregulation of TNF-TNFR superfamily gene expression will influence many biological processes, and contributes to human diseases, including cancer. We investigated the genetic alterations of the TNF-TNFR superfamily genes in hepatocellular carcinoma (HCC). Several genetic alterations were detected in the 44 TNF-TNFR superfamily genes by sequencing hepatocellular carcinoma DNA samples. In particular, we found that the TNFR1 promoter -329G/T polymorphism was strongly associated with primary HCC (odds ratio [OR]=5.22, p=0.0007). We also observed frequent loss of heterozygosity at the polymorphic TNFR1 -329G/T site in the primary tumor tissues, indicating that the polymorphic TNFR1 -329G/T site is very susceptible to genetic alterations in HCC. Furthermore, in the polymorphic TNFR1 -329G/T site, the T allele resulted in the repression of TNFR1 expression. Therefore, our results suggest that TNFR1 -329G/T polymorphism may play an important role in the development of HCC.

Genomic dissection of mucosal immunobiology in the porcine small intestine.

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.

Arginase activity differs with allergen in the effector phase of ovalbumin- versus trimellitic anhydride-induced asthma.

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.

An interfering RNA protocol for primary porcine alveolar macrophages.

RNA interference (RNAi) is a cellular process of post-transcriptional gene silencing in which a short interfering dsRNA (siRNA, 21-23 nt) targets a homologous mRNA for degradation by ribonuclease. RNAi has been used successfully to inhibit targeted gene expression and viral replication in mammalian cells. In this study we established an RNAi transfection protocol for primary porcine alveolar macrophages and evaluated potential off-target effects of siRNA introduction into these cells. Porcine alveolar macrophages were transfected using a fluorescence-labeled siRNA to compare transfection reagents from different suppliers. Under optimized transfection conditions, up to 95% of macrophages were fluorescent at 12 and 24 h post-transfection using an amine-based transfection reagent. An siRNA targeting GAPDH suppressed macrophage endogenous GAPDH transcript levels as much as 60% through 24h. Further, we did not detect a significant interferon response following siRNA transfection. These data suggest that RNAi will be an efficient and convenient approach for studying loss of gene function in primary porcine alveolar macrophages.

Host intestinal epithelial response to Cryptosporidium parvum.

Cryptosporidium parvum is an obligate intracellular protozoan parasite that is a well-recognized cause of diarrhea in humans and animals throughout the world, and is associated with a substantial degree of morbidity and mortality in patients with the acquired immunodeficiency syndrome (AIDS). C. parvum primarily infects epithelial cells of the gastrointestinal tract, resulting in acute watery diarrhea for which there is no effective therapy. During infection, all parasite development, sexual or asexual, occurs within epithelial cells of the host. This requires a unique and complex association between two distinct eukaryotic organisms. Conversely, due to the intracellular nature of C. parvum, epithelial cells appear to play a key role in activating and communicating with the host immune system. Delineation of the biochemical processes that are regulated within infected epithelial cells is crucial for understanding the pathology of C. parvum infection, the process by which the host clears and ultimately develops resistance to infection, and the development of chemotherapeutic strategies to intercede infections.

Mac-1-mediated uptake and killing of Bordetella bronchiseptica by porcine alveolar macrophages.

The role of Mac-1 as a receptor for Bordetella bronchiseptica infection of alveolar macrophages (AMphi) was examined using 6 strains (2 ATCC and 4 pathogenic field isolates) to assess B. bronchiseptica binding, uptake and replication in primary porcine AMphi. All B. bronchiseptica strains were rapidly killed by porcine serum in a dose- and time-dependent manner. However, heat-inactivated porcine serum (HIS) did not demonstrate any bacterial-killing activity, suggesting that complement may have a direct killing activity. All field isolates were more resistant to direct complement-mediated B. bronchiseptica killing. The uptake of B. bronchiseptica into AMphi was inhibited approximately 50% by anti-Mac-1 monoclonal antibodies in the medium. However, B. bronchiseptica phagocytosed in the presence of serum or HIS was not altered by anti-Mac-1 antibodies although more bacteria were internalized by addition of serum or HIS. These data suggest that Mac-1 is a target for direct uptake of B. bronchiseptica via opsonin-independent binding. The phagocytosed B. bronchiseptica, either via direct or serum-mediated binding, were efficiently killed by AMphi within 10 hr postinfection. This demonstrates that Mac-1-mediated B. bronchiseptica uptake is a bacterial killing pathway not leading to productive infections in AMphi.

Characterization and radiation hybrid mapping of expressed sequence tags from the canine brain.

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.

Genome-wide multilocus analysis for immune-mediated complex diseases.

In postgenomic era, searching and identification of disease genes associated with complex diseases are still one of the great challenge for dissecting human complex diseases. To improve the disease gene localization for complex diseases, a group of closely immune-mediated disease loci were overlapped on each chromosome based on previously reported genome-wide scanning data. Interestingly, five overlapping chromosomal regions (1q21, 2q33, 5q31.1-q33.1, 6p21, and 11q13) were identified by co-localizing disease loci for the following diseases: diabetes, asthma, atopic dermatitis, osteoporosis, and inflammatory bowel disease. The development of specific disease was associated with different combinations of disease loci among five overlapped chromosomal regions. Therefore, the analysis of multiple genetic loci should be considered to determine the effects of multiple genes responsible for complex diseases resulting from the influence of multiple genes.

Countervailing influence of tumor necrosis factor-alpha and nitric oxide in endotoxemia.

Tumor necrosis factor-alpha (TNF-alpha), a crucial mediator in sepsis, elicits multiple biologic effects, including intravascular thrombosis and circulatory shock. TNF-alpha exerts its biologic effects through two distinct cell surface receptors, TNF-R1 and TNF-R2. The pathophysiologic interaction between TNF-alpha and nitric oxide (NO) in glomerular thrombosis caused by endotoxemia in rats and wild-type mice (C57BL6) as well as in knockout mice that are deficient in TNF-R1 (R1 -/-), TNF-R2 (R2 -/-), or both receptors (R1R2 -/-) was studied. Administration of lipopolysaccharide (LPS; Escherichia coli endotoxin) resulted in increased NO and TNF-alpha production but failed to induce glomerular thrombosis. Concomitant administration of LPS + NG-nitro-L-arginine methyl ester (L-NAME; an NO synthesis inhibitor) resulted in glomerular thrombosis in rats and in wild-type mice. Intraperitoneal administration of pentoxifylline before LPS inhibited TNF-alpha synthesis and prevented glomerular thrombosis in rats given LPS + L-NAME. In contrast to the results observed in rats and wild-type mice, administration of LPS + L-NAME did not result in glomerular thrombosis in knockout mice with either single or double TNF-alpha receptor deletion. Thus, during endotoxemia, (1) TNF-alpha fosters glomerular thrombosis if there is deficiency of NO synthesis and (2) both TNF-alpha receptors are necessary for TNF-alpha's prothrombogenic action. Clinically, these novel studies suggest that in gram-negative endotoxemia, inhibition of NO synthesis and selective blockade of TNF-alpha receptors may provide unique therapeutic approaches for mitigation of glomerular thrombosis and restitution of vascular tone.