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The purpose of this study was to utilize secondary analysis to identify the impact of challenging life events on the inner strength of women in El Salvador. The women interviewed lived in a rural area repatriated following the civil war that ended in 1992. The original ethnographic study examined health beliefs and practices of these Salvadoran women. The data were collected through face-to face interviews over a period of one month. The experience of inner strength was evident in the original data, leading to the examination by a secondary analysis. Findings from the secondary analysis revealed the relationship between the women's descriptions of their lifeways and the six attributes of inner strength as described by Dingley, Roux, and Bush (2000). Findings provided further clarification and meaning to the concept of inner strength in women. Implications for practice resulting from this analysis facilitate understanding of behaviors affecting health and illness and provide a rich and holistic understanding of the culture in Salvadoran women. [ABSTRACT FROM AUTHOR]

Appaloosa horses are predisposed to equine recurrent uveitis (ERU), an immune-mediated disease characterized by recurring inflammation of the uveal tract in the eye, which is the leading cause of blindness in horses. Nine genetic markers from the ECA1 region responsible for the spotted coat color of Appaloosa horses, and 13 microsatellites spanning the equine major histocompatibility complex (ELA) on ECA20, were evaluated for association with ERU in a group of 53 Appaloosa ERU cases and 43 healthy Appaloosa controls. Three markers were significantly associated (corrected P-value <0.05): a SNP within intron 11 of the TRPM1 gene on ECA1, an ELA class I microsatellite located near the boundary of the ELA class III and class II regions and an ELA class II microsatellite located in intron 1 of the DRA gene. Association between these three genetic markers and the ERU phenotype was confirmed in a second population of 24 insidious ERU Appaloosa cases and 16 Appaloosa controls. The relative odds of being an ERU case for each allele of these three markers were estimated by fitting a logistic mixed model with each of the associated markers independently and with all three markers simultaneously. The risk model using these markers classified ~80% of ERU cases and 75% of controls in the second population as moderate or high risk, and low risk respectively. Future studies to refine the associations at ECA1 and ELA loci and identify functional variants could uncover alleles conferring susceptibility to ERU in Appaloosa horses., (© 2014 Stichting International Foundation for Animal Genetics.)

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 β-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., (© 2013 The Authors. Veterinary Dermatology © 2013 ESVD and ACVD.)

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&#95;T and SNP43&#95;G were fixed in Pietrain and Sinclair pig breeds. SNP64&#95;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., (Copyright © Taylor & Francis Group, LLC)

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.

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., (Copyright © 2010 Elsevier B.V. All rights reserved.)

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.

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.

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.

Effective management of patients after the implantation of mechanical cardiac valves includes correct recognition of each valve and its related complications. Herein, we present the case of a patient who had undergone implantation of a floating-disc Beall-Surgitool mitral valve in 1976 and developed multiple valve-related complications. Over 30 years and in multiple medical centers, the device was mistakenly assumed to be a "ball" valve. The correct identification of the prosthesis led to the recognition of valvular failure, and the patient underwent its replacement with an On-X bileaflet carbon valve. Pathologic and microscopic examination of the explanted Beall valve showed massive pannus formation that extended over the sewing cuff on the atrial and ventricular side, preventing complete disc closure; disrupted fabric coating of the sewing ring, with exposure of the underlying metal; and a marked inflammatory reaction. We report one of the longest intervals on record between the implantation and replacement of a Beall-Surgitool valve.

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.

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.

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.

Recently, we established and phenotypically characterized an immortalized porcine olfactory bulb neuroblast cell line, OBGF400 (1). To facilitate the future application of these cells in studies of neurological dysfunctions and neuronal pathogen interactions, a comprehensive knowledge of their genomic variability and overall gene expression capacity was pursued. Accordingly, the OBGF400 cells were subjected to karyotyping and more extensive transcriptome analyses. Cytogenetic characterization of these cells revealed a genetic mosaicism of neuronal hyperdiploidy. A direct comparison of the OBGF400 cell transcriptome pattern, generated by utilizing the Affymetrix GeneChip(R) Porcine Genome Array, to that of a non-neural, porcine epithelial cell line facilitated the identification of 831 probe sets preferentially hybridized by the neuroblast transcripts. Subsequent functional annotation of these OBGF400 RNAs using the Database for Annotation, Visualization and Integrated Discovery 2008 enabled their allocation to the corresponding gene ontology biological process term, thereby assisting the recognition of key elements involved in the regulation of neuronal signal transduction and neurogenesis.

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.

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.

Interleukin (IL)-4 has been shown to induce protection in porcine vascular endothelial cells (ECs) from killing by human complement. This protection is dependent on the PI3K/Akt signaling pathway. In this study, we investigated mechanisms downstream of Akt and found that activation of the lipid biosynthesis pathway is required for protection from complement in ECs treated with IL-4. Cells incubated with IL-4 for 48 hours contained increased fatty acids and phospholipids but cholesterol was not increased when compared with medium-treated controls. The transcription factor SREBP-1, which regulates fatty acid synthesis, was found to be activated in extracts of ECs incubated with IL-4 for 6 hours. Finally, induction of protection from complement killing with IL-4 was fully prevented by the presence of the SREBP inhibitor 25-OH cholesterol. This study showed that IL-4 induces lipid biosynthesis in porcine ECs through activation of SREBP-1 and that the activation of this pathway is critical for IL-4 to induce protection of porcine ECs from killing by human complement. Further study of these mechanisms may provide new strategies for the prevention of complement-mediated vascular injury as it occurs in xenograft rejection.

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.

To determine the chromosomal locations for genes expressed in porcine Peyer's patches, polymerase chain reaction-based mapping of expressed sequence tags (ESTs) isolated from a porcine Peyer's patch-specific cDNA library was performed across a 6500-rad swine radiation hybrid panel. A total of 116 ESTs were mapped with LOD scores >6.0, and another 11 ESTs had LOD scores between 5.0 and 6.0. Of these 127 ESTs, 63% matched known genes (

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.

Introduction: Giant aneurysms that develop in native coronary arteries or saphenous vein grafts are morphologically defined as abnormally expanded outpouching vascular structures >4 cm in diameter. The location, morphology, and content of giant aneurysms account for adverse cardiovascular effects., Methods: Two cases of giant aneurysms were studied comprehensively by noninvasive and invasive cardiac methods and subsequent histopathology. The first patient had a giant aneurysm that developed over a course of several years in a saphenous vein graft whereas the second patient had a giant aneurysm occurring within a native coronary artery. Accompanying clinical and angiographic findings are described., Results: Atherosclerosis and thrombosis were among the prominent histopathological findings., Conclusions: Atherosclerosis and associated thrombosis within giant aneurysms result in obstruction of flow, distal embolization, and development of acute coronary syndromes including recurrent ischemic chest pain, unstable angina, and acute myocardial infarction. The options for clinical management of giant coronary or vein graft aneurysms include surgical excision, percutaneous coil occlusion and stent deployment, or medical approach.

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.

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.

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.

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.

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.

Objective: To investigate the health practices and lifeways of rural villagers in a remote area of El Salvador who had been displaced by the recent civil war. The purpose of the study was to explore their view on health and experiences of loss during the war., Design: Ethnography (Spradley, 1980, 1999; Agar, 1996)., Method: The participants included any resident of three rural Salvadoran villages who were 18 years of age and over. Participants included nine families, with a total of twelve participants. Data collection included participant observation, audiotaped interviews, demographic information, and field notes. One of the Spanish-speaking key informants acted as the interpreter. The content of all data was analyzed for recurrent themes., Findings: All nine families were displaced to refugee camps in Honduras during the civil war. Two cultural themes that emerged from the data were: 1) War: "We lost everything; we had to leave running," and 2) Health: "It's in God's hands.", Conclusions: It is a challenge to encourage culture-specific care that acknowledges Salvadoran herbal remedies, strength of spirit, and a belief that a Supreme Being controls their lives. The health practices of the participants were shaped by their experiences of suffering from loss of family members during the war, displacement from their homes, and lack of potable water and environmental sanitation., Implications: To make a positive impact and effect change on health services in these rural areas, efforts should be directed toward democratic and community-based social and economic development within the context of the cultural system. Recent earthquakes (2001) have intensified the need for improvement in environmental factors including potable water.

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.

In an effort to identify genes that have a major effect on macrophage function during viral infection, we employed differential display reverse transcription (DDRT)-PCR to capture expressed sequence tags (ESTs) of swine alveolar macrophages infected by the porcine reproductive and respiratory syndrome virus (PRRSV). Sequence analyses showed that approximately 60% of these ESTs had significant similarity (> or =93%) to known pig ESTs or genes or matched sequences from other species with homology > or =80%. To determine chromosomal localization, PCR-based mapping was performed across either swine somatic cell hybrid or radiation hybrid panels. A total of 48 porcine viral response ESTs were mapped via the swine somatic cell panel or the INRA-Minnesota porcine Radiation Hybrid (IMpRH) panel (LOD > 6.0). Northern blot analyses confirmed PRRSV-induced altered transcript expression for several ESTs, including a 2'-5' oligoadenylate synthetase and a putative dual-specificity phosphatase. These virus-response ESTs represent good candidate genes for understanding PRRSV pathogenesis and for dissecting host genes which may have major effect on disease resistance.

The role of TNFalpha in regulating apoptotic signaling was investigated during subacute, low-dose (5.0 mg/kg) dimethylnitrosamine (DMN)-induced hepatotoxicity. In TNFalpha receptor (TNFR) intact (wild-type, WT) mice following 4 and 7 DMN exposures, hepatic transcripts for TNFalpha and TNFR-1 were elevated as compared to vehicle controls. DMN hepatotoxicity in WT and TNFR-1/TNFR-2 double knockout (DKO) mice were then compared over a 7-d exposure period. Liver RNA was isolated to measure hepatic expression of TNFalpha/Fas-related genes and the Bcl-2 family of genes that impact apoptosis. Hepatic mRNA levels for Fas, the apoptosis-promoting gene Bax, and the anti-apoptotic gene, Bcl-X(L), were up regulated following 4 and 7 DMN exposures in both WT and TNFR DKO mice as compared to vehicle controls. Notably, hepatic transcript levels for Bax were higher in TNFR DKO mice treated with DMN compared to identically treated WT mice. However, we detected approximately equal DMN-induced apoptotic degradation of liver DNA following 1, 4, and 7 exposures in WT and TNFR DKO mice. Taken together, these data show DMN-induced hepatic TNFalpha expression and suggest that TNFR-1 signaling may be up regulated following 4 and 7 daily DMN exposures. However, TNFalpha is not required for apoptotic signaling at the mRNA transcript level within the liver and instead may actually decrease Bax production.

The impact of porcine reproductive and respiratory syndrome virus (PRRSV) infection on porcine alveolar macrophages (Mo) was examined by differential display reverse transcription PCR (DDRT-PCR). A PRRSV-induced expressed gene tag (EST) was used to isolate and identify a single cDNA clone from a library prepared from porcine peripheral blood. Rapid amplification of cDNA ends (RACE) was employed to clone a 1.5 kb fragment at the 5' end of the mRNA. DNA sequencing identified an open reading frame (ORF) of 2820 bp. Deduced amino acid sequence revealed the eight conserved domains characteristic of the DEAD/H box protein superfamily. The putative porcine RNA helicase induced by virus (RHIV -1) showed 84% amino acid similarity to human retinoic acid-induced gene (RIG-I). Porcine RHIV -1 transcripts were ubiquitously expressed in various pig tissues, while in PRRSV-infected pigs, higher expression was observed in several tissues persistent for PRRSV. These data indicate the association of PRRSV genome replication with enhaced host cell RNA helicase gene expression. Finally, the RHIV -1 gene was localized on porcine chromosome 10q13 between markers SSC25A02 and SWR334 via somatic cell panel and radiation hybrid (RH) mapping strategies., (Copyright 2000 Academic Press.)

Tumor necrosis factor receptor knockout (TNFR KO) mice were used to examine the role of tumor necrosis factor-alpha (TNFalpha) signaling during acute hepatotoxicant exposure. Mice were exposed intraperitoneally (ip) to either vehicle, phosphate-buffered saline (PBS), or dimethylnitrosamine (DMN, 100 mg/kg) for 24 h. Histological evaluation showed that DMN-treated TNFR-2 KO mice had increased liver damage compared to wild type (WT), TNFR-1 KO, or TNFR double KO (DKO) mice. Also, 3 of 8 TNFR-2 KO mice died following DMN treatment, suggesting that hepatic TNFR-2 signaling produces protective responses that counteract TNFR-1-mediated damage. DMN-induced cellular infiltration was absent in TNFR-1-deficient mice, indicating that infiltrating cells do not exacerbate acute hepatotoxic events. In separate experiments, mice were exposed ip to either DMN (5.0 or 100 mg/kg), carbon tetrachloride (CCl4, 0.3 or 1.0 ml/kg), or corresponding PBS/corn oil controls for 6 or 24 h to compare the hepatic mRNA expression of cytokine- and apoptotic-associated genes. Following 24 h of DMN (100 mg/kg) or 6-24 h of CCl4 treatment, hepatic transcripts for TNFalpha, interferon (IFN)-gamma, IL (interleukin)-1RI, and transforming growth factor (TGF)-betaRII were induced. Hepatotoxicant-treated WT and TNFR DKO mice induced liver transcripts for the pro- and anti-apoptotic genes, Bax and Bcl-X(L), respectively, indicating TNF-independent gene activation. The anti-apoptotic gene, Bfl-1, was highly expressed in CCl4-treated, TNFR-positive strains, but minimally expressed in TNFR DKO mice, suggesting that hepatic Bfl-1 is TNF-regulated. Taken together, these data show that acute hepatotoxicant exposure is followed by upregulation of liver cytokine, cytokine receptor, and apoptotic transcripts, and that TNFalpha regulates various aspects of liver inflammation and injury in a TNFR-specific fashion.

The detailed mechanism(s) by which porcine reproductive and respiratory syndrome virus (PRRSV) impairs alveolar Mo homeostasis and function remains to be elucidated. We used differential display reverse-transcription PCR (DDRT-PCR) to identify molecular genetic changes within PRRSV-infected Mo over a 24 h post infection period. From over 4000 DDRT-PCR amplicons examined, 19 porcine-derived DDRT-PCR products induced by PRRSV were identified and cloned. Northern blot analysis confirmed that four gene transcripts were induced during PRRSV infection. PRRSV attachment and penetration alone did not induce these gene transcripts. DNA sequence revealed that one PRRSV-induced expressed sequence tag (EST) encoded porcine Mx1, while the remaining 3 clones represented novel ESTs. A full-length cDNA clone for EST G3V16 was obtained from a porcine blood cDNA library. Sequence data suggests that it encodes an ubiquitin-specific protease (UBP) that regulates protein trafficking and degradation. In pigs infected in vivo, upregulated transcript levels were observed for Mx1 and Ubp in lung and tonsils, and for Mx1 in tracheobronchial lymph node (TBLN). These tissues correspond to sites for PRRSV persistence, suggesting that the Mx1 and Ubp genes may play important roles in clinical disease during PRRSV infection., (Copyright 1999 Academic Press.)

Differential display reverse transcription-polymerase chain reaction (DDRT-PCR) was used to catalogue altered hepatic transcript expression during dimethylnitrosamine (DMN) exposure in vivo. Mice were administered DMN (1.5 or 5 mg/kg) or vehicle (phosphate-buffered saline) i.p. once daily for up to 7 days, and livers were collected 6 h post-injection. Total RNA was reverse transcribed and cDNA subsets were selectively amplified by PCR. DDRT-PCR products were fractionated on denaturing polyacrylamide gels, and differentially expressed bands were excised, reamplified, and subsequently cloned into a plasmid vector. This study identified 23 cDNAs that were induced and 25 cDNAs that were suppressed during DMN exposure. Altered expression during DMN exposure for cDNA clones was confirmed by Northern blotting, RNase protection, or in situ hybridization analyses. DNA sequence information indicated that four cDNAs suppressed during DMN exposure encode cytochrome P450 isoenzyme-cholesterol 7 alpha-hydroxylase (CYP7), a monokine, a myeloid cell differentiation protein, and mouse major urinary protein (MUP). We further observed a DMN-induced increase in transcripts for complement factor 3 (C3) and serum amyloid A (SAA). In contrast, the remaining differentially expressed transcripts detected by DDRT-PCR during DMN exposure demonstrated no similarity to sequences present in Genbank, suggesting that they may encode previously unreported gene products. In situ hybridization showed MUP transcripts to be expressed by hepatic centrilobular areas that undergo necrosis during subchronic DMN exposure. Thus, the utilization of DDRT-PCR has identified several differentially expressed hepatic mRNAs associated with various doses and stages of DMN exposure.

We assessed the kinetics of inducible nitric oxide synthase (iNOS) mRNA expression and production of nitric oxide (NO) in bovine alveolar macrophages (AMs) stimulated with purified lipopolysaccharide (LPS) from Pasteurella haemolytica strain 12296. The effect of LPS on iNOS gene expression was dose-dependent and was expressed maximally at 24 h after stimulation with 10 micrograms/ml of LPS. Production of NO measured as secreted nitrite in supernatants took place in a time and dose-dependent manner with peak production at 24 h after LPS stimulation. Recombinant bovine gamma interferon (rb gamma IFN) augmented the LPS-induced iNOS gene expression and production of NO. The ability of LPS to induce iNOS gene expression and NO production either alone or in combination with rb gamma IFN was significantly abrogated by polymyxin B. In addition, the iNOS inhibitor NG-monomethyl-Larginine (L-NMMA) significantly inhibited LPS and rb gamma IFN + LPS induced NO production. Our results also demonstrated that NO produced from an exogenous NO donor sodium nitroprusside (SNP), and NO generated from LPS-stimulated AMs (endogenous) caused cytotoxic injury to bovine pulmonary artery endothelial cells in a dose-dependent manner. The cytotoxic injury caused by NO generated from LPS stimulated AMs was inhibited by polymyxin B or L-NMMA. There was a markedly increased concentration of nitrite in the lung lavage fluids of calves following P. haemolytica infection. These findings support a role for NO in the pathogenesis of lung injury in bovine pneumonic pasteurellosis.

In the present study, we have constructed a subtraction cDNA library to identify novel genes induced by IFN-gamma in GM-CSF-derived bone marrow macrophage (m phi). M theta were treated with 50 U/ml IFN-gamma for 40, 70 and 140 min to induce expression of early genes regulated by IFN-gamma, and the M phi were pooled. Poly(A)+RNA was prepared from both unactivated and IFN-gamma-stimulated m theta, and cDNA libraries were constructed in lambda ZAP. Genes expressed in common by both m theta populations were removed by subtraction using biotin-avidin precipitation of hybrid complexes. Further selection was performed by differential screening using cDNA prepared from mRNA of unactivated m phi as a probe, followed by colony hybridization to remove sister clones. Of 17 clones from which sequence information was obtained, two appeared to be identical with the murine genes, C10 (clone GM2B1) and Mac-2 (clone GM2C4) and an additional two clones had high similarity to human cDNAs encoding proteins of unknown function. cDNAs containing sequences which did not match published sequences were used to probe Northern blots prepared from both unstimulated and IFN-gamma-activated GM-CSF- and CSF-1-derived m phi. Five clones (GM1A2, GM1B4, GM1F2, GM2A12 and GM2B8) showed enhanced transcript levels following IFN-gamma treatment of GM-CSF-derived m phi, but demonstrated high constitutive transcript levels in CSF-l-derived m phi. In addition, C10 transcripts were constitutively expressed by GM-CSF-derived m phi, but not by CSF-1-derived m phi, even after activation by IFN-gamma. These data suggest that much of the functional heterogeneity of GM-CSF- and CSF-1-derived m phi resides in the differential expression of early genes specifically induced by IFN-gamma.

Tumor necrosis factor-alpha (TNF-alpha) is a powerful macrophage-derived proinflammatory cytokine, via both direct effects on host tissues as well as indirectly through the induction of other proinflammatory mediators, including interleukin- (IL) 1 beta and IL-6. Activation of murine bone marrow-derived macrophages (BMDM phi) with lipopolysaccharide (LPS) causes rapid expression of TNF-alpha, which as an autocrine factor enhances BMDM phi function through IL-1 beta and IL-6 production. In this study, we have examined the specific transcriptional inhibition of BMDM phi TNF-alpha using novel enantiomeric carbocyclic nucleoside analogues. BMDM phi were derived in vitro from murine bone marrow progenitors using colony stimulating factor-1 and treated with combinations of LPS (1-100 nG/ml) and the enantiomeric carbocyclic nucleoside (10-100 microM) analogues MDL 201, 112 (9-[(1S,3R)-cis-cyclopentan-3-ol]adenine); MDL 201,451 (9-[1R,3S)-cis-cyclopentan-3-ol]adenine); MDL 201,449 (9-[(1R,3R)-trans-cyclopentan-3-ol]adenine) and MDL 201,484 (9-[(1S,3S)-trans-cyclopentan-3-ol]adenine). Northern blot analysis showed that MDL 201,449 was the most effective agent in vitro at selectively inhibiting TNF-alpha. MDL 201,449 reduced TNF-alpha mRNA levels by nearly 50% for up to 4 hr after the simultaneous addition of LPS and the synthetic agent. In contrast, mRNA and secreted protein levels for IL-1 beta (measured by the D10.S bioassay) and mRNA for TNF-alpha receptor p60 and TNF-alpha receptor p80 were not significantly affected. Carbocyclic nucleoside analogues were effective when added to BMDM phi up-to 2 hr after LPS treatment and at concentrations as low as 10 microM. Regulation of BMDM phi IL-6 by carbocyclic nucleoside analogues in response to LPS appears to be both concentration and time dependent, because IL-6 mRNA and secreted protein levels were inhibited at only high drug concentrations (100 microM) and effective only at longer exposure times (+4 hr of incubation) to LPS. These data support the concept that M phi-derived proinflammatory cytokine gene expression is differentially, rather than coordinately, regulated by selective signal transduction and/or molecular pathways. Enantiomeric carbocyclic nucleoside analogues that specifically inhibit TNF-alpha may have therapeutic potential in inflammatory diseases, such as systemic inflammatory response syndrome, where TNF-alpha has been shown to have an important role in initiating the early stages of disease.

Molecular genotyping of swine major histocompatibility complex SLA-DQB and SLA-DRB genes using polymerase chain reaction (PCR)-based amplification is described. Locus-specific oligonucleotide primers were designed for the analysis of expressed SLA genes by reverse transcription-polymerase chain reaction (RT-PCR). RT-PCR products were sequenced, and the information gained was used to design primers for PCR genotyping of the exon 2 (beta 1) region from genomic DNA templates. A single segregating amplification product was detected for both DQB and DRB in all animals. PCR products were digested with restriction enzymes. Seven SLA-DQB PCR-restriction fragment length polymorphism (RFLP) pattern types were observed for both HaeIII and RsaI that defined 14 SLA-DQB alleles. A total of seven SLA-DRB PCR-RFLP pattern types were defined using MspI (3 RFLP pattern types) and RsaI (6 RFLP pattern types). In order to demonstrate their universal utility, the primers were tested on genomic DNA samples from 10 different swine breeds. No breed-specific alleles were observed. These results show that locus-specific oligonucleotide primers and RFLP analysis provide a simple and rapid method for genotyping expressed SLA-DQB and SLA-DRB from genomic DNA.

Inflammatory responses are accompanied by increased expression of hepatocyte-derived proteins collectively known as acute phase reactants (APR). B6C3F1 female mice were exposed intraperitoneally every 24 hr to either vehicle (PBS) or DMN (5 mg/kg) for up to six exposures. Following a single treatment (acute), liver tissues were collected at 3, 6, 12, and 24 hr post-exposure. The same collection scheme was repeated following the fourth and sixth exposures (subchronic). Total cellular RNA was isolated and Northern blot analyses were performed using 3'-end radiolabeled oligonucleotides specific for serum amyloid A (SAA), serum amyloid P (SAP), and albumin (ALB). SAA transcripts were detected 3 hr after acute DMN exposure, peaked at 6 hr, and rapidly declined to vehicle control levels by 12 hr. No SAA transcripts were observed in vehicle-treated controls. In contrast, SAP transcripts were constitutively expressed in both vehicle and DMN-treated groups throughout the acute exposure period. However, at 3 and 6 hr after DMN exposure, elevated levels of SAP transcripts were observed before returning to control levels at 12 and 24 hr. Expression of albumin transcripts decreased rapidly following acute DMN exposure and remained suppressed throughout the first 24-hr period measured. Serum levels of complement component-3 (C3) increased 2 hr after a single DMN exposure, whereas decreases in serum albumin levels were first observed at 24 hr post-exposure. After four exposures to DMN, SAA transcripts were detected at all time periods measured. Similarly, SAP transcripts in livers of DMN-exposed animals were consistently elevated above vehicle controls. Results after six exposures to DMN were similar, with SAA and SAP transcripts elevated at all time points tested. By comparison, repeated vehicle exposures resulted in a stress-related transient expression of SAA and SAP transcripts. Thus, acute and subchronic DMN exposure resulted in differential APR transcript expression and may serve as useful biomarkers following chemical exposure.

CTP:phosphocholine cytidylyltransferase is the rate-controlling enzyme in phosphatidylcholine biosynthesis and is essential for the survival of eukaryotic cells. The murine cDNA for the cytidylyltransferase was cloned and sequenced. A genomic clone was isolated and the chromosomal location of the Ctpct locus determined by Southern blot hybridization of DNAs from a panel of interspecific backcross progeny derived from matings of [(C57BL/6J x Mus spretus)F1 x C57BL/6J] mice. These data place the Ctpct gene on mouse chromosome 16 between the Smst and Stf-1 genes.

Macrophage populations exhibit a wide range of antigenic and functional phenotypes, including cytokine production, response to immunomodulatory stimuli, and clearance of pathogens. The expanding clinical exploitation of recombinant growth factors and cytokines with the potential to regulate the production and function of peripheral macrophage populations necessitates an increased understanding of the mechanisms by which functionally distinct macrophage populations arise as well as the ramifications of macrophage heterogeneity. The present review summarizes recent data which supports multiple mechanisms by which heterogeneous macrophage populations arise: 1) differential signals experienced within diverse tissue microenvironments; 2) developmentally-staged expression of specific functions; 3) clonal variation of myeloid progenitor cells; and 4) alternate hematopoietic stimulation. These data show that the above processes are not mutually exclusive and that each likely contributes to the observed heterogeneity of peripheral macrophage populations.

Different macrophage preparations were compared for functional capacity in conditions of high prostaglandin E2 (PGE2) or low L-arginine concentrations. Macrophages derived in vitro from bone marrow progenitor cells (bone marrow-derived macrophages, BMDMs) using colony-stimulating factor 1 (CSF-1) as the myelopoietic stimulus displayed a greater sensitivity to PGE2-induced suppression of tumor necrosis factor alpha (TNF-alpha) secretion than did macrophages derived using granulocyte-macrophage colony-stimulating factor (GM-CSF). Neither BMDM population was inhibited by PGE2 for the direct cytolysis of L929 cells (TNF-alpha sensitive), and only GM-CSF-derived macrophages showed decreased killing of TNF-alpha-resistant K562 targets. Exogenous cAMP inhibited TNF-alpha secretion, but not nitrite secretion, by both BMDM populations. GM-CSF-derived macrophages accumulated less cAMP following PGE2 treatment than did CSF-1-derived macrophages. Removing L-arginine from the medium did not inhibit cytotoxicity or PGE2 secretion, but the listeriacidal activity specific to interferon-gamma plus lipopolysaccharide (LPS)-activated GM-CSF-derived macrophages was blocked by removal of L-arginine. Treatment with CSF-1 or GM-CSF alone did not activate the macrophages, but GM-CSF efficiently primed both BMDM populations for augmented TNF-alpha secretion in response to secondary stimulation using LPS. However, GM-CSF augmented the LPS-induced production of nitrite and PGE2 by CSF-1-derived macrophages only. These results demonstrate the potential for differential macrophage function within inflammatory sites based on the hematopoietic stimulus under which the macrophage is derived and the specific conditions present in the lesion.

Macrophages derived in vitro from bone marrow progenitors (bone marrow-derived macrophages, BMDMs) using either macrophage colony-stimulating factor (CSF-1) or granulocyte-macrophage colony-stimulating factor (GM-CSF) as the myelopoietic stimulus display differential functional, morphological, and mRNA phenotypes. The data presented here demonstrate further that CSF-1- and GM-CSF-derived BMDMs differ in immunologic capacity. GM-CSF-derived BMDMs, when compared to CSF-1-derived BMDMs, showed greater cytolytic activity against tumor necrosis factor alpha (TNF-alpha)-resistant, but not TNF-alpha-sensitive, tumor targets. In contrast, CSF-1-derived BMDMs produced nitrite in response to lipopolysaccharide (LPS) alone, whereas GM-CSF-derived BMDMs required interferon gamma plus LPS treatment. The two BMDM populations also showed differential sensitivities to LPS for secretion of TNF-alpha and nitrite, but the maximal inducible amounts of these factors and prostaglandin E2 were similar between the BMDM populations. Lastly, GM-CSF-derived but not CSF-1-derived BMDMs showed an L-arginine-dependent listeriacidal activity. These results show that the functional heterogeneity of CSF-1- and GM-CSF-derived macrophages is limited and appears to result largely from differences in the activational signals required by each BMDM population to elicit a given function.

DMN exposure has been shown to increase macrophage cytotoxic activity against tumor targets both in vitro and in vivo. Since the production and expression of the macrophage-derived cytokine tumor necrosis factor-alpha (TNF-alpha) is associated with such anti-tumor activity, studies were performed to determine whether changes in TNF-alpha gene transcription and biosynthesis resulted following DMN exposure. Thioglycollate-elicited macrophages obtained from DMN-exposed animals displayed enhanced levels of constitutively expressed TNF-alpha transcripts compared to vehicle controls. Northern blot analysis of the time course expression of TNF-alpha following endotoxin (1 microgram/ml) stimulation in vitro showed a significantly greater induction of TNF-alpha transcripts in macrophages from DMN-exposed than control animals, with peak levels detected between 30 and 120 min. Maximum endotoxin-induced TNF-alpha secretion occurred later than the accumulation of the transcripts, with greater secretion observed between 120 and 360 min. In contrast to endotoxin, stimulation with IFN-gamma (100 U/ml) produced no changes in the level of TNF-alpha transcripts. However, stimulation of macrophages with IFN-gamma did greatly enhance the surface expression of membrane-bound TNF-alpha in cells from the DMN-treated animals. Supernatants from media and endotoxin stimulated macrophage were tested for TNF-alpha activity against WEHI-164 cells. In media alone, a five-fold increase in TNF-alpha activity was observed at 6 h in supernatants from macrophage obtained from DMN-exposed animals compared to the vehicle group. Treatment of supernatants with either superoxide dismutase (SOD) or catalase to remove reactive oxygen products did not alter their lytic capacity. However, addition of a neutralizing murine-anti-TNF-alpha antibody reduced the lytic capacity of the supernatants by 90% in both treatment groups. Accumulation of IL-1 beta transcripts gradually increased over the 6 h with a concomitant increase in secreted IL-1 beta that was identical in both DMN and vehicle groups. These results demonstrate that DMN exposure: (1) enhances the expression of TNF-alpha in peripheral macrophages by transcriptional regulatory mechanism(s) and, (2) does not alter the expression or secretion of IL-1 beta.