Genomic Approaches to Study Innate Immune Response to Salmonella Enteritidis Infection in Chickens

Salmonella enterica serovar Enteritidis (SE) is one of the most common food-borne pathogens that cause human salmonellosis. Contamination of consumed poultry products continues to be a global threat to public health. Genetic resistance using genomic approach provides a promising solution to controlling SE infection in poultry. The mechanism of SE resistance in chickens remains elusive. Three different approaches, microarray techology, gene silencing, and computational gene analysis, have been utilized to study SE-induced transcriptional changes of host immune response in the chicken. A whole genome chicken 44K microarray was used to analyze the transcriptome of heterophils from SE-resistant (line A) and SE-susceptible chickens (line B) with/without in vitro SE stimulation. Many differentially expressed immune-related genes were found in the SE-infected to non-infected comparison, where more immune-related genes were down-regulated in line B than line A. These results suggested a similar Toll-like receptor (TLR) regulatory network might exist in heterophils of both lines, and provided strong candidates for further investigating SE resistance and susceptibility in chickens. In the gene silencing study, small interfering RNAs (siRNA) were used to specifically inhibit the expression of NFkB1 in the chicken HD11 macrophage cell line with SE challenge. Genes related to the NF-kB signaling pathway were selected to examine the effect of NFkB1 inhibition on TLR pathway. With 36% inhibition of NFkB1 expression, the results showed an increased expression of TLR4 and interleukin (IL)-6 following SE challenge and suggested a likely inhibitory regulation of NFkB1 on TLR signal pathway. Finally, two novel chicken C-type lectin-like receptors were identified and annotated to chicken CD69 and CD94/NKG2-like with multiple evidences generated by computational (in-silico) sequence analysis. Both genes located in a region on chicken chromosome 1 that is syntenic to mammalian Nature Killer Receptor Complex (NKC) region, which may have existed before the divergence between mammals and aves. While siRNA lays the foundation of using loss-of-function approach on testifying gene-gene interactions, in-silico analysis aids in gathering information of unknown genes of great interest. Both approaches provide great potential to use for down-stream analysis following microarray study

Blood lipid profiles and peripheral blood mononuclear cell cholesterol metabolism gene expression in patients with and without methotrexate treatment

<p>Abstract</p> <p>Background</p> <p>Methotrexate (MTX) is the most commonly prescribed disease-modifying antirheumatic drug (DMARD) in rheumatoid arthritis. ATP-binding cassette transporter-A1 (ABCA1) and 27-Hydroxylase (HY27) are known antiatherogenic proteins that promote cellular cholesterol efflux. In THP-1 macrophages, MTX can promote the reversal of cholesterol transport, limit foam cell formation and also reverse COX-2 inhibitor-mediated downregulation of ABCA1. Despite its antiatherogenic potential <it>in vitro</it>, the impact of clinical use of low-dose MTX on cholesterol metabolism in humans is unknown. Objective of the study was to examine whether clinical MTX use is associated with altered blood lipids and/or <it>ABCA1/HY27 </it>expressions.</p> <p>Methods</p> <p>In all, 100 rheumatoid arthritis subjects were recruited from a medical center in central Taiwan. Plasma lipid profiles and peripheral blood mononuclear cell <it>HY27 </it>and <it>ABCA1 </it>expressions were compared between subjects taking MTX (MTX+) and other disease-modifying antirheumatic drugs (DMARDs) (MTX-). Dietary intake was assessed by a registered dietician.</p> <p>Results</p> <p>Though no difference observed in the blood lipids between MTX+ and MTX- subjects, the expressions of <it>ABCA1 </it>and <it>HY27 </it>were significantly elevated in MTX+ subjects (n = 67) compared to MTX- subjects (n = 32, p < 0.05). ABCA expression correlated with MTX doses (r = 0.205, p = 0.042), and MTX+ subjects are more likely to have increased <it>HY27 </it>compared to MTX- subjects (OR = 2.5, p = 0.038). Prevalence of dyslipidemia and overweight, and dietary fat/cholesterol intake were lower than that of the age-matched population. Although no differences were observed in the blood lipids, the potential impacts of MTX on cholesterol metabolism should not be overlooked and the atheroprotective effects from MTX induced <it>HY27 </it>and <it>ABCA1 </it>expressions may still be present in those persons with pre-existing dyslipidemia.</p> <p>Conclusions</p> <p>We demonstrated novel findings on the increased gene expressions of atheroprotective protein <it>HY27 </it>and <it>ABCA1 </it>in human peripheral blood mononuclear cells (PBMCs) with clinical use of low-dose MTX. Whether MTX induced <it>HY27 </it>and <it>ABCA1 </it>expressions can protect against cardiovascular disease in patients with chronic inflammation through the facilitation of cholesterol export remains to be established. Further studies on the impacts of low-dose MTX on hypercholesterolemic patients are underway.</p

Gene Expression Profiling of the Local Cecal Response of Genetic Chicken Lines That Differ in Their Susceptibility to Campylobacter jejuni Colonization

Campylobacter jejuni (C. jejuni) is one of the most common causes of human bacterial enteritis worldwide primarily due to contaminated poultry products. Previously, we found a significant difference in C. jejuni colonization in the ceca between two genetically distinct broiler lines (Line A (resistant) has less colony than line B (susceptible) on day 7 post inoculation). We hypothesize that different mechanisms between these two genetic lines may affect their ability to resist C. jejuni colonization in chickens. The molecular mechanisms of the local host response to C. jejuni colonization in chickens have not been well understood. In the present study, to profile the cecal gene expression in the response to C. jejuni colonization and to compare differences between two lines at the molecular level, RNA of ceca from two genetic lines of chickens (A and B) were applied to a chicken whole genome microarray for a pair-comparison between inoculated (I) and non-inoculated (N) chickens within each line and between lines. Our results demonstrated that metabolism process and insulin receptor signaling pathways are key contributors to the different response to C. jejuni colonization between lines A and B. With C. jejuni inoculation, lymphocyte activation and lymphoid organ development functions are important for line A host defenses, while cell differentiation, communication and signaling pathways are important for line B. Interestingly, circadian rhythm appears play a critical role in host response of the more resistant A line to C. jejuni colonization. A dramatic differential host response was observed between these two lines of chickens. The more susceptible line B chickens responded to C. jejuni inoculation with a dramatic up-regulation in lipid, glucose, and amino acid metabolism, which is undoubtedly for use in the response to the colonization with little or no change in immune host defenses. However, in more resistant line A birds the host defense responses were characterized by an up-regulation lymphocyte activation, probably by regulatory T cells and an increased expression of the NLR recognition receptor NALP1. To our knowledge, this is the first time each of these responses has been observed in the avian response to an intestinal bacterial pathogen

Boson Peak in Deeply Cooled Confined Water: A Possible Way to Explore the Existence of the Liquid-to-Liquid Transition in Water

The boson peak in deeply cooled water confined in nanopores is studied with inelastic neutron scattering. We show that in the (P, T) plane, the locus of the emergence of the boson peak is nearly parallel to the Widom line below ∼1600  bar. Above 1600 bar, the situation is different and from this difference the end pressure of the Widom line is estimated. The frequency and width of the boson peak correlate with the density of water, which suggests a method to distinguish the hypothetical “low-density liquid” and “high-density liquid” phases in deeply cooled water.United States. Dept. of Energy (Grant DE-FG02-90ER45429

Gene expression profiling in chicken heterophils with Salmonella enteritidis stimulation using a chicken 44 K Agilent microarray

BACKGROUND: Salmonella enterica serovar Enteritidis (SE) is one of the most common food-borne pathogens that cause human salmonellosis and usually results from the consumption of contaminated poultry products. The mechanism of SE resistance in chickens remains largely unknown. Previously, heterophils isolated from broilers with different genetic backgrounds (SE-resistant [line A] and -susceptible [line B]) have been shown to be important in defending against SE infections. To dissect the interplay between heterophils and SE infection, we utilized large-scale gene expression profiling. RESULTS: The results showed more differentially expressed genes were found between different lines than between infection (SE-treated) and non-infection (control) samples within line. However, the numbers of expressed immune-related genes between these two comparisons were dramatically different. More genes related to immune function were down-regulated in line B than line A. The analysis of the immune-related genes indicated that SE infection induced a stronger, up-regulated gene expression of line heterophils A than line B, and these genes include several components in the Toll-like receptor (TLR) signaling pathway, and genes involved in T-helper cell activation. CONCLUSION: We found: (1) A divergent expression pattern of immune-related genes between lines of different genetic backgrounds. The higher expression of immune-related genes might be more beneficial to enhance host immunity in the resistant line; (2) a similar TLR regulatory network might exist in both lines, where a possible MyD88-independent pathway may participate in the regulation of host innate immunity; (3) the genes exclusively differentially expressed in line A or line B with SE infection provided strong candidates for further investigating SE resistance and susceptibility. These findings have laid the foundation for future studies of TLR pathway regulation and cellular modulation of SE infection in chickens

Dengue-1 Envelope Protein Domain III along with PELC and CpG Oligodeoxynucleotides Synergistically Enhances Immune Responses

The major weaknesses of subunit vaccines are their low immunogenicity and poor efficacy. Adjuvants can help to overcome some of these inherent defects with subunit vaccines. Here, we evaluated the efficacy of the newly developed water-in-oil-in-water multiphase emulsion system, termed PELC, in potentiating the protective capacity of dengue-1 envelope protein domain III. Unlike aluminum phosphate, dengue-1 envelope protein domain III formulated with PELC plus CpG oligodeoxynucleotides induced neutralizing antibodies against dengue-1 virus and increased the splenocyte secretion of IFN-γ after in vitro re-stimulation. The induced antibodies contained both the IgG1 and IgG2a subclasses. A rapid anamnestic neutralizing antibody response against a live dengue virus challenge was elicited at week 26 after the first immunization. These results demonstrate that PELC plus CpG oligodeoxynucleotides broaden the dengue-1 envelope protein domain III-specific immune responses. PELC plus CpG oligodeoxynucleotides is a promising adjuvant for recombinant protein based vaccination against dengue virus

Decreased Circulating Endothelial Progenitor Cell Levels and Function in Patients with Nonalcoholic Fatty Liver Disease

OBJECTIVES: Nonalcoholic fatty liver disease (NAFLD) is associated with advanced atherosclerosis and a higher risk of cardiovascular disease. Increasing evidence suggests that injured endothelial monolayer is regenerated by circulating bone marrow derived-endothelial progenitor cells (EPCs), and levels of circulating EPCs reflect vascular repair capacity. However, the relation between NAFLD and EPC remains unclear. Here, we tested the hypothesis that patients with nonalcoholic fatty liver disease (NAFLD) might have decreased endothelial progenitor cell (EPC) levels and attenuated EPC function. METHODS AND RESULTS: A total of 312 consecutive patients undergoing elective coronary angiography because of suspected coronary artery disease were screened and received examinations of abdominal ultrasonography between July 2009 and November 2010. Finally, 34 patients with an ultrasonographic diagnosis of NAFLD, and 68 age- and sex-matched controls without NAFLD were enrolled. Flow cytometry with quantification of EPC markers (defined as CD34(+), CD34(+)KDR(+), and CD34(+)KDR(+)CD133(+)) in peripheral blood samples was used to assess circulating EPC numbers. The adhesive function, and migration, and tube formation capacities of EPCs were also determined in NAFLD patients and controls. Patients with NAFLD had a significantly higher incidence of metabolic syndrome, previous myocardial infarction, hyperuricemia, and higher waist circumference, body mass index, fasting glucose and triglyceride levels. In addition, patients with NAFLD had significantly decreased circulating EPC levels (all P<0.05), attenuated EPC functions, and enhanced systemic inflammation compared to controls. Multivariate logistic regression analysis showed that circulating EPC level (CD34(+)KDR(+) [cells/10(5) events]) was an independent reverse predictor of NAFLD (Odds ratio: 0.78; 95% confidence interval: 0.69-0.89, P<0.001). CONCLUSIONS: NAFLD patients have decreased circulating EPC numbers and functions than those without NAFLD, which may be one of the mechanisms to explain atherosclerotic disease progression and enhanced cardiovascular risk in patients with NAFLD

Inhibition of NF-kB 1 (NF-kBp50) by RNA interference in chicken macrophage HD11 cell line challenged with Salmonellaenteritidis

The NF-kB pathway plays an important role in regulating the immunity response in animals. In this study, small interfering RNAs (siRNA) were used to specifically inhibit NF-kB 1 expression and to elucidate the role of NF-kB in the signal transduction pathway of the Salmonella challenge in the chicken HD11 cell line. The cells were transfected with either NF-kB 1 siRNA, glyceraldehyde 3-phosphate dehydrogenase siRNA (positive control) or the negative control siRNA for 24 h, followed by Salmonella enteritidis (SE) challenge or non-challenge for 1 h and 4 h. Eight candidate genes related to the signal pathway of SE challenge were selected to examine the effect of NF-kB 1 inhibition on their expressions by mRNA quantification. The results showed that, with a 36% inhibition of NF-kB 1 expression, gene expression of both Toll-like receptor (TLR) 4 and interleukin (IL)-6 was consistently and significantly increased at both 1 h and 4 h following SE challenge, whereas the gene expression of MyD88 and IL-1β was increased at 1 h and 4 h, respectively. These findings suggest a likely inhibitory regulation by NF-kB 1, and could lay the foundation for studying the gene network of the innate immune response of SE infection in chickens