High-fat meal increases peripheral blood mononuclear cell pro-inflammatory cytokine expression in African-American women.

African-American (AA) women have elevated predominance of inflammatory diseases concurrent with local inflammation resulting in compromised metabolic function. The purpose of the study was 2-fold: 1) to examine the gene and protein expression of pro- and anti-inflammatory cytokine secretion by peripheral blood mononuclear cells (PBMC) obtained from AA and Caucasian-American (CA) women in response to an acute high-fat meal; and 2) to explore the influence of race (AA vs. CA) on PBMC reactivity. Ten AA and 11 CA women consumed a high-fat meal with baseline and 4 h postprandial venous blood draws. PBMCs were incubated for 3 h then messenger RNA expression and supernatant protein concentration was used to examine inflammatory profiles. All women had a postprandial increase in interleukin (IL)-8 gene expression, IL-8 protein concentration, and tumor necrosis factor alpha (TNF-α) protein concentration (P < 0.05). AA women had a postprandial increase in IL-6, IL-8, and TNF-α protein concentration (P < 0.05). AA women had higher postprandial IL-1ß protein concentration and IL-8 gene expression compared with CA women (P < 0.05). Our data uncovers the specific impact of race and time on pro-inflammatory PBMC (IL-1ß, IL-6, IL-8, and TNF-α) expression profiles in response to an acute high-fat meal challenge. Novelty: African Americans have higher predominance of inflammatory disease. We explored the potential race impact on peripheral blood mononuclear cell reactivity in response to a meal. A pro-inflammatory response to an acute high-fat meal with race impact was observed possibly contributing to health disparities impacting African-American women.

Assessment of Interleukin-8 in Bronchial Asthma in Iraq.

It has been approved that neutrophils are responsible for many inflammatory lung diseases, such as acute respiratory distress syndrome, chronic obstructive pulmonary disease, and asthma. It is well documented that the CXC chemokine interleukin-8 (IL-8) plays a key role as a potent neutrophil recruiting and activating factor. Asthma is one of the most common major non-contagious diseases and has a substantial impact on the patient's quality of life. The current evidence suggests that asthma is a complex multifactorial disorder, and its etiology is increasingly attributed to interactions between genetic susceptibility, host factors, and environmental exposures. IL-8 plays an important role in respiratory diseases and is a known regulator of pulmonary inflammation and immunity, induced phagocytosis, and promoted angiogenesis. This study aimed to investigate the IL-8 gene expression in blood samples of bronchial asthma patients. Therefore, the blood samples were taken from two groups of participants, including the group of patients with asthma (n=100) in the age range of20-61years and the group of healthy individuals (n=50).The obtained results indicated that the expression of IL-8 mRNA in the group of asthma patients was three times higher than that in the group of healthy individuals. Therefore, it is suggested that the antagonism of IL-8 could be a potent therapeutic strategy in the treatment of asthma.

Development of fibrin hydrogel-based in vitro bioassay system for assessment of skin permeability to and pro-inflammatory activity mediated by zinc ion released from nanoparticles.

Nanoparticles (NPs) are promising products in industry and medicine due to their unique physicochemical properties. In particular, zinc oxide (ZnO) NPs are extensively incorporated into sunscreens to protect the skin from exposure to ultraviolet radiation. However, there are several health concerns about skin penetration and the resultant toxicity. As methodologies for evaluating NP toxicity are under development, it is difficult to fully assess the toxicity of ZnO NPs toward humans. In this study, we developed a platform to simultaneously detect skin permeability to and pro-inflammatory activity mediated by zinc ion released from NPs. First, we generated a stable reporter cell line expressing green fluorescent protein (GFP) under the control of interleukin-8 (IL-8) promoter activity. The expression levels of GFP induced by zinc reflected the endogenous IL-8 expression levels and the pro-inflammatory responses. Next, we found that fibrin hydrogel can reproduce permeability to zinc ion of a human skin equivalent model and is therefore a promising material to assess skin permeability to zinc ion. Then, we constructed a fibrin hydrogel-based in vitro bioassay system for the simultaneous detection of skin permeability to and pro-inflammatory activity mediated by zinc ion released from NPs by using a stable reporter cell line and a fibrin hydrogel layer. This bioassay system is a promising in vitro permeation test due to its technical simplicity and good predictability. Overall, we believe that our bioassay system can be widely used in the cosmetics and pharmaceutical industries.

Clostridium perfringens beta2 toxin induced in vitro oxidative damage and its toxic assessment in porcine small intestinal epithelial cell lines.

Clostridium perfringens beta2 (CPB2), a key virulence factor, is produced by C. perfringens type C that is the main pathogenic microorganism causing diarrhea in piglets. However, little is known concerning the toxic damage effect of CPB2 on intestinal cells of piglets. In present study, CPB2 toxin obtained by genetic recombination technology was evaluated for its cytotoxicity property using the intestinal porcine epithelial (IPEC-J2) cells, which aims to attempt to understand and explain its mechanism of action in porcine small intestinal epithelial cells. IPEC-J2 cells were treated with different concentrations of CPB2 toxin (5, 10, 20, 30, 40, and 50 µg/mL), and MTT assay results showed that the cell viability of CPB2-treated IPEC-J2 cells decreased in a dose-dependent manner. Lactate dehydrogenase (LDH) assay results revealed that CPB2 significantly increased the LDH release, relative to the control. The expression of tumor necrosis factor α (TNF-α) and interleukin 8 (IL-8) gradually increased, while the expression of interleukin 10 (IL-10) gradually decreased in IPEC-J2 cells with increasing concentration of CPB2 (10-30 µg/mL), as analyzed by quantitative real-time PCR (RT-qPCR). Also, CPB2 increased the content of intracellular reactive oxygen species (ROS) and decreased mitochondrial membrane potential (ΔΨm) of IPEC-J2 cells. Western blot and immunofluorescence results demonstrate that CPB2 decreased the expression of zonula occludens (ZO-1), claudin12 (CLDN12) and occludin (OCLN) in IPEC-J2 cells. In addition, CPB2 increased Bax expression, and inhibited Bcl-2 and Bcl-xL expression, as measured by Western blot. Considering all of these findings, it was concluded that CPB2 toxin shows significant cytotoxicity, cell growth inhibition and increase in cell permeability in IPEC-J2 cells in a concentration-dependent manner, thus leading to abnormal cell apoptosis and functions in porcine small intestinal epithelial cells.

The impact of organic vs. inorganic selenium on dairy goat productivity and expression of selected genes in milk somatic cells.

The aim of this study was to determine the effect of diet supplemented with selenized yeast (Se-yeast) on milk yield and milk composition of goats and expression of casein and mammary-gland-immune system genes in milk somatic cells (MSC). Twenty-four dairy goats in their second to fourth lactations were divided into control and experimental groups, balanced according to lactation number and breed (Polish White or Fawn Improved). Morning milk and blood samples were collected four times during lactation (on the 21st, 70th, 120th, 180th day after kidding). The control and experimental groups were fed diets with 0.7 mg inorganic Se/goat/day (sodium selenite) or 0.6 mg organic Se/goat/day (selenized yeast), respectively. Milk, fat and protein yields during lactation as well as average somatic cell count, fat, protein and lactose contents in milk were evaluated. Microelements in milk and blood serum and biochemical parameters in blood serum were determined at the beginning and the end of the experiment. The expression levels of the genes encoding αS1-casein (CSN1S1), αS2-casein (CSN1S2), κ-casein (CSN3), interleukin 8 (IL-8), serum amyloid A3 (SAA3), interleukin 1ß (IL-1ß), bactenecin 7.5 (BAC7.5), bactenecin 5 (BAC5), ß2-defensin (GBD2), hepcidin (HAMP), chemokine 4 (CCL4), tumour necrosis factor α (TNFα), toll-like receptor 2 (TLR2), cathelicidin-7 (MAP34) and cathelicidin-6 (MAP28) were determined in MSC. Milk, fat, and protein yields were higher and somatic cell count (SCC expressed as natural logarithm) was lower in the milk of goats fed organic Se. The Se concentration in milk was twice as high in the organic vs. inorganic treatment groups at the end of the experiment, while there were no differences in studied biochemical parameters between groups. The transcript levels of CSN1S2 and BAC7.5 were higher and IL-8 was lower in MSC of Se-yeast treated groups. Such results may indicate better health status of mammary glands of goats treated with organic Se as well as positive impact of selenized yeast on the goat's milk composition. Differences in the IL-1ß and IL-8 transcript levels were also noted between the stages of lactation, with the highest expression at the peak of lactation (day 70), highlighting the metabolic burden at this time. We concluded that the Se-yeast supplementation improved the productivity and health status of goats and could have significant economic impact on farmer's income.

Lung Defense through IL-8 Carries a Cost of Chronic Lung Remodeling and Impaired Function.

IL-8-dependent inflammation is a hallmark of host lung innate immunity to bacterial pathogens, yet in many human lung diseases, including chronic obstructive pulmonary disease, bronchiectasis, and pulmonary fibrosis, there are progressive, irreversible, pathological changes associated with elevated levels of IL-8 in the lung. To better understand the duality of IL-8-dependent host immunity to bacterial infection and lung pathology, we expressed human IL-8 transgenically in murine bronchial epithelium, and investigated the impact of overexpression on lung bacterial clearance, host immunity, and lung pathology and function. Persistent IL-8 expression in bronchial epithelium resulted in neutrophilia, neutrophil maturation and activation, and chemotaxis. There was enhanced protection against challenge with Pseudomonas aeruginosa, and significant changes in baseline expression of innate and adaptive immunity transcripts for Ccl5, Tlr6, IL-2, and Tlr1. There was increased expression of Tbet and Foxp3 in response to the Pseudomonas antigen OprF, indicating a regulatory T-cell phenotype. However, this enhanced bacterial immunity came at a high price of progressive lung remodeling, with increased inflammation, mucus hypersecretion, and fibrosis. There was increased expression of Ccl3 and reduced expression of Claudin 18 and F11r, with damage to epithelial organization leading to leaky tight junctions, all of which resulted in impaired lung function with reduced compliance, increased resistance, and bronchial hyperreactivity as measured by whole-body plethysmography. These results show that IL-8 overexpression in the bronchial epithelium benefits lung immunity to bacterial infection, but specifically drives lung damage through persistent inflammation, lung remodeling, and damaged tight junctions, leading to impaired lung function.

Antigenic and functional profiles of a Lawsonia intracellularis protein that shows a flagellin-like trait and its immuno-stimulatory assessment.

The obligate intracellular Lawsonia intracellularis (LI), the etiological agent of proliferative enteropathy (PE), is an economically important disease in the swine industry. Due to extreme difficulty of in vitro culture of the pathogen, molecular characterization of protein components of LI that are targets of the immune system, is difficult; thus, the scientific evidence to drive the development of preventive measures is lacking. In this work, we investigated the antigenic and functional characteristics of a putative flagellar-associated protein, LI0570, using in silico computational approaches for epitope prediction and an in vitro protein-based molecular assay. The amino acid sequence of LI0570 exhibited similarities to flagellar-associated proteins in four different bacterial strains. The presence of B cell linear confirmative epitopes of the protein predicted by a bioinformatics tool was validated by western blot analysis using anti-LI mouse hyperimmune serum, which implied that LI0570 induced production of antigen-specific antibodies in vivo. Further, TLR5-stimulating activity and IL-8 cytokine expression produced via downstream signaling were observed in HEK-Blue™-hTLR5 cells stimulated with LI0570. This result indicates that the LI0570 protein can trigger an innate immune response followed by a T-cell-related adaptive immune response in an infected host. Collectively, the data presented here support that the LI0570 protein which shows the antigenic potential could be a useful component of a recombinant vaccine against PE, providing progress toward an effective prevention strategy.

In vitro assessment of silver nanoparticles immunotoxicity.

This study aimed to characterize unwanted immune effects of nanoparticles (NP) using THP-1 cells, human whole blood and enriched peripheral blood monocytes. Commercially available silver NP (AgNP < 100 nm, also confirmed by Single Particle Extinction and Scattering) were used as prototypical NP. Cells were treated with AgNP alone or in combination with classical immune stimuli (i.e. LPS, PHA, PWM) and cytokine assessed; in addition, CD54 and CD86 expression was evaluated in THP-1 cells. AgNP alone induced dose-related IL-8 production in all models, with higher response observed in THP-1 cells, possibly connected to different protein corona formation in bovine versus human serum. AgNP potentiated LPS-induced IL-8 and TNF-α, but not LPS-induced IL-10. AgNP alone induced slight increase in IL-4, and no change in IFN-γ production. While responses to PHA in term of IL-4 and IFN-γ production were not affected, increased PWM-induced IL-4 and IFN-γ production were observed, suggesting potentiation of humoral response. Reduction in PHA-induced IL-10 was observed. Overall, results indicate immunostimulatory effects. THP-1 cells work as well as primary cells, representing a useful and practical alternative, with the awareness that from a physiological point of view the whole blood assay is the one that comes closest to reality.

Assessment of the Role of Metabolic Determinants on the Relationship between Insulin Sensitivity and Secretion.

BACKGROUND: Insulin secretion correlates inversely with insulin sensitivity, which may suggest the existence of a crosstalk between peripheral organs and pancreas. Such interaction might be mediated through glucose oxidation that may drive the release of circulating factors with action on insulin secretion. AIM: To evaluate the association between whole-body carbohydrate oxidation and circulating factors with insulin secretion to consecutive oral glucose loading in non-diabetic individuals. METHODS: Carbohydrate oxidation was measured after an overnight fast and for 6 hours after two 3-h apart 75-g oral glucose tolerance tests (OGTT) in 53 participants (24/29 males/females; 34±9 y; 27±4 kg/m2). Insulin secretion was estimated by deconvolution of serum C-peptide concentration, ß cell function by mathematical modelling and insulin sensitivity from an OGTT. Circulating lactate, free-fatty acids (FFA) and candidate chemokines were assessed before and after OGTT. The effect of recombinant RANTES (regulated on activation, normal T cell expressed and secreted) and IL8 (interleukin 8) on insulin secretion from isolated mice islets was also measured. RESULTS: Carbohydrate oxidation assessed over the 6-h period did not relate with insulin secretion (r = -0.11; p = 0.45) or ß cell function indexes. Circulating lactate and FFA showed no association with 6-h insulin secretion. Circulating chemokines concentration increased upon oral glucose stimulation. Insulin secretion associated with plasma IL6 (r = 0.35; p<0.05), RANTES (r = 0.30; p<0.05) and IL8 (r = 0.41; p<0.05) determined at 60 min OGTT. IL8 was independently associated with in vivo insulin secretion; however, it did not affect in vitro insulin secretion. CONCLUSION: Whole-body carbohydrate oxidation appears to have no influence on insulin secretion or putative circulating mediators. IL8 may be a potential factor influencing insulin secretion.

Assessment of a panel of interleukin-8 reporter lung epithelial cell lines to monitor the pro-inflammatory response following zinc oxide nanoparticle exposure under different cell culture conditions.

BACKGROUND: Stably transfected lung epithelial reporter cell lines pose an advantageous alternative to replace complex experimental techniques to monitor the pro-inflammatory response following nanoparticle (NP) exposure. Previously, reporter cell lines have been used under submerged culture conditions, however, their potential usefulness in combination with air-liquid interface (ALI) exposures is currently unknown. Therefore, the aim of the present study was to compare a panel of interleukin-8 promoter (pIL8)-reporter cell lines (i.e. green or red fluorescent protein (GFP, RFP), and luciferase (Luc)), originating from A549 lung epithelial type II-like cells cells, following NPs exposure under both submerged and ALI conditions. METHODS: All cell lines were exposed to zinc oxide (ZnO) NPs at 0.6 and 6.2 µg/cm(2) for 3 and 16 hours under both submerged and ALI conditions. Following physicochemical characterization, the cytotoxic profile of the ZnO-NPs was determined for each exposure scenario. Expression of IL-8 from all cell types was analyzed at the promoter level and compared to the mRNA (qRT-PCR) and protein level (ELISA). RESULTS: In summary, each reporter cell line detected acute pro-inflammatory effects following ZnO exposure under each condition tested. The pIL8-Luc cell line was the most sensitive in terms of reporter signal strength and onset velocity following TNF-α treatment. Both pIL8-GFP and pIL8-RFP also showed a marked signal induction in response to TNF-α, although only after 16 hrs. In terms of ZnO-NP-induced cytotoxicity pIL8-RFP cells were the most affected, whilst the pIL8-Luc were found the least responsive. CONCLUSIONS: In conclusion, the use of fluorescence-based reporter cell lines can provide a useful tool in screening the pro-inflammatory response following NP exposure in both submerged and ALI cell cultures.

Vitamin C Compound Mixtures Prevent Ozone-Induced Oxidative Damage in Human Keratinocytes as Initial Assessment of Pollution Protection.

INTRODUCTION: One of the main functions of cutaneous tissues is to protect our body from the outdoor insults. Ozone (O3) is among the most toxic stressors to which we are continuously exposed and because of its critical location, the skin is one of the most susceptible tissues to the oxidative damaging effect of O3. O3 is not able to penetrate the skin, and although it is not a radical per se, the damage is mainly a consequence of its ability to induce oxidative stress via the formation of lipid peroxidation products. AIM OF STUDY: In this study we investigated the protective effect of defined "antioxidant" mixtures against O3 induced oxidative stress damage in human keratinocytes and understand their underlying mechanism of action. RESULTS: Results showed that the mixtures tested were able to protect human keratinocytes from O3-induced cytotoxicity, inhibition of cellular proliferation, decrease the formation of HNE protein adducts, ROS, and carbonyls levels. Furthermore, we have observed the decreased activation of the redox sensitive transcription factor NF-kB, which is involved in transcribing pro-inflammatory cytokines and therefore constitutes one of the main players associated with O3 induced skin inflammation. Cells exposed to O3 demonstrated a dose dependent increase in p65 subunit nuclear expression as a marker of NF-kB activation, while pre-treatment with the mixtures abolished NF-kB nuclear translocation. In addition, a significant activation of Nrf2 in keratinocytes treated with the mixtures was also observed. CONCLUSION: Overall this study was able to demonstrate a protective effect of the tested compounds versus O3-induced cell damage in human keratinocytes. Pre-treatment with the tested compounds significantly reduced the oxidative damage induced by O3 exposure and this protective effect was correlated to the abolishment of NF-kB nuclear translocation, as well as activation of Nrf2 nuclear translocation activating the downstream defence enzymes involved in cellular detoxification process.

Assessment of CCL2 and CXCL8 chemokines in serum, bronchoalveolar lavage fluid and lung tissue samples from dogs affected with canine idiopathic pulmonary fibrosis.

Canine idiopathic pulmonary fibrosis (CIPF) is a progressive disease of the lung parenchyma that is more prevalent in dogs of the West Highland white terrier (WHWT) breed. Since the chemokines (C-C motif) ligand 2 (CCL2) and (C-X-C motif) ligand 8 (CXCL8) have been implicated in pulmonary fibrosis in humans, the aim of the present study was to investigate whether these same chemokines are involved in the pathogenesis of CIPF. CCL2 and CXCL8 concentrations were measured by ELISA in serum and bronchoalveolar lavage fluid (BALF) from healthy dogs and WHWTs affected with CIPF. Expression of the genes encoding CCL2 and CXCL8 and their respective receptors, namely (C-C motif) receptor 2 (CCR2) and (C-X-C motif) receptor 2 (CXCR2), was compared in unaffected lung tissue and biopsies from dogs affected with CIPF by quantitative PCR and localisation of CCL2 and CXCL8 proteins were determined by immunohistochemistry. Significantly greater CCL2 and CXCL8 concentrations were found in the BALF from WHWTs affected with CIPF, compared with healthy dogs. Significantly greater serum concentrations of CCL2, but not CXCL8, were found in CIPF-affected dogs compared with healthy WHWTs. No differences in relative gene expression for CCL2, CXCL8, CCR2 or CXCR2 were observed when comparing lung biopsies from control dogs and those affected with CIPF. In affected lung tissues, immunolabelling for CCL2 and CXCL8 was observed in bronchial airway epithelial cells in dogs affected with CIPF. The study findings suggest that both CCL2 and CXCL8 are involved in the pathogenesis of CIPF. Further studies are required to determine whether these chemokines might have a clinical use as biomarkers of fibrosis or as targets for therapeutic intervention.

Quantitative assessment of the associations between interleukin-8 polymorphisms and periodontitis susceptibility.

BACKGROUND: This review assesses the associations of interleukin-8 gene (IL-8) -251A/T (rs4073) and -845T/C (rs2227532) polymorphisms with susceptibility to periodontitis. METHODS: Several electronic databases were searched for eligible articles. Twelve studies involving 2,233 cases and 2,655 controls were retrieved and analyzed. Odds ratios (ORs) along with 95% confidence intervals (CIs) were calculated to assess the strength of relationship between the IL-8 polymorphisms and periodontitis risk. RESULTS: No significant association was found for IL-8 -251A/T polymorphism with periodontitis in the overall analysis and stratification by periodontitis type and smoking status. Subgroup analysis by ethnicity revealed that -251A/T T allele and TT genotype were associated with decreased risk of periodontitis in a Brazilian mixed population (T allele versus A allele: OR 0.80, 95% CI 0.68 to 0.94, Pheterogeneity = 0.30; TT versus AA: OR 0.65, 95% CI 0.46 to 0.93, Pheterogeneity = 0.39; TT versus AA/AT: OR 0.58, 95% CI 0.35 to 0.98, Pheterogeneity = 0.01). In addition, -251A/T T allele was associated with increased periodontitis risk in Asians. Pooled estimates showed that the -845T/C polymorphism was associated with periodontitis susceptibility in overall analysis and the chronic periodontitis subgroup. In addition, marginal associations were observed between -845T/C polymorphism and periodontitis in a Brazilian mixed population. Moreover, this association was also confirmed to be significant in Brazilian non-smokers. CONCLUSION: This meta-analysis indicated that both IL-8 -251A/T and -845T/C polymorphisms may be involved in the development of periodontitis in a Brazilian mixed population, whereas the -251A/T allele T appeared to be a risk factor for periodontitis in Asians.

Expression of recombinant human interleukin-8 and its purification using a single buffer system.

Chemokines, a class of small secreted proteins, direct immune cells to their target sites and play an important role in chronic inflammations and allergies. To study their interactions with their cellular receptors or potential inhibitors large quantities of chemokines are required. Here we present a fast and efficient strategy to purify the human chemokine interleukin-8 (IL-8, CXCL8). The chemokine is expressed with a pelB-leader peptide that is cleaved off its N-terminus by an endogenous bacterial peptidase. This yields wild-type 72aa IL-8 with a serine at its N-terminus. IL-8 is recovered in the soluble fraction after lysis while pelB-IL8 fusion protein remains in the pellet. Interleukin-8 is purified via cation exchange chromatography and heparin affinity chromatography using a single inexpensive buffer system. No dialysis or membrane filtration steps are required and the final protein fractions may be used without any desalting steps. The use of 0.5% Triton X-114 in the lysis buffer leads to low endotoxin levels in the resulting protein. The protein can be eluted from the gel filtration column with a variety of buffers and is ready to be used in binding assays and activity assays.

Assessment of bovine mammary chemokine gene expression in response to lipopolysaccharide, lipotechoic acid + peptidoglycan, and CpG oligodeoxynucleotide 2135.

During intramammary infections pathogen associated molecular patterns (PAMPs) induce an inflammatory response, recognized clinically as mastitis. Recognition of PAMPs by mammary cells leads to the production of the pro-inflammatory cytokines, TNF-alpha and IL-1beta. These cytokines augment the secretion of various chemokines that are responsible for directing the host cellular immune response, and consequently the outcome of infection. Previous research has shown that gram-negative and gram-positive bacteria elicit different types of innate immune responses. The purpose of this study, therefore, was to characterize the expression of various chemokine genes in bovine mammary gland explants in response to lipopolysaccharide (LPS), peptidoglycan (PTG) combined with lipotechoic acid (LTA), and CpG oligodeoxynucleotide (CpG-ODN) 2135 representing gram-negative bacteria, gram-positive bacteria, and bacterial DNA, respectively, to determine if these PAMPs induce different chemokine gene expression patterns. Explants from 3 Holstein cows were cultured with 10 microg/mL of LPS, LTA + PTG, or CpG-ODN 2135 for 6 and 24 h. Total RNA was extracted and the expression of CXCL8, MCP-1, MCP-2, MCP-3, MIP1-alpha, and RANTES genes was measured by real-time polymerase chain reaction (RT-PCR). Lipopolysaccharide significantly induced MCP-1, MCP-2, and MCP-3 expression, and slightly increased CXCL8 gene expression. The combined PAMPs, LTA + PTG, on the other hand, significantly induced MCP-1 gene expression, and slightly increased MCP-3 expression. No significant expression differences for any of the chemokine genes were observed in explants stimulated with CpG-ODN 2135. These results demonstrate that PAMPs associated with different mastitis-causing pathogens induce chemokine-specific gene expression patterns that may contribute to different innate immune responses to bacteria.

Quantification of mRNA encoding cytokines and chemokines and assessment of ciliary function in canine tracheal epithelium during infection with canine respiratory coronavirus (CRCoV).

One of the first lines of defence against viral infection is the innate immune response and the induction of antiviral type I interferons (IFNs). However some viruses, including the group 2 coronaviruses, have evolved mechanisms to overcome or circumvent the host antiviral response. Canine respiratory coronavirus (CRCoV) has previously been shown to have a widespread international presence and has been implicated in outbreaks of canine infectious respiratory disease (CIRD). This study aimed to quantify pro-inflammatory cytokine mRNAs following infection of canine air-interface tracheal cultures with CRCoV. Within this system, immunohistochemistry identified ciliated epithelial and goblet cells as positive for CRCoV, identical to naturally infected cases, thus the data obtained would be fully transferable to the situation in vivo. An assay of ciliary function was used to assess potential effects of CRCoV on the mucociliary system. CRCoV was shown to reduce the mRNA levels of the pro-inflammatory cytokines TNF-alpha and IL-6 and the chemokine IL-8 during the 72 h post-inoculation. The mechanism for this is unknown, however the suppression of a key antiviral strategy during a period of physiologic and immunological stress, such as on entry to a kennel, could potentially predispose a dog to further pathogenic challenge and the development of respiratory disease.

A transcriptomic analysis of the stress induced by capture-release health assessment studies in wild dolphins (Tursiops truncatus).

The health of wild bottlenose dolphins (Tursiops truncatus) is typically evaluated by the study of animals that are captured and released back into the wild after examination. The impact of such studies on gene expression in peripheral blood cells was investigated using microarray and quantitative polymerase chain reaction methods. Significantly increased expression was observed in two major classes of genes: (i) energy metabolism, and (ii) responsiveness to stress and trauma, the latter effect suggesting the initiation of an acute-phase response. The value of data obtained in capture/release studies may need to be weighed against the potential physiological impacts of such studies.

Novel perspectives on the pathogenesis of Lonomia obliqua caterpillar envenomation based on assessment of host response by gene expression analysis.

Animal venomous secretions have been explored as source of active substances affecting mammal hemostasis. These active principles impinge on key elements of almost all physiologic pathways and have an enormous potential in the development of new therapeutic drugs. The envenomation caused by the caterpillar Lonomia obliqua (lonomism) is characterized by a hemorrhagic clinical profile. Investigations of caterpillar venom have, in general, involved the isolation and biochemical characterization of active principles related to the pathophysiology of envenomation. In the last few years, these studies focused on the caterpillar's secretions pro-coagulant, fibrin(ogen)olytic, hemolytic, edematogenic and nociceptive activities. Recently, a significant advance was achieved as a result of a transcriptome study, which generated a catalog of putative toxic proteins in the caterpillar venom, giving rise to hypotheses on the molecular basis of pathogenesis which could be experimentally explored. In this investigation, using a microarray methodology, we analyzed the effects of the caterpillar venom on the gene expression profile of cultured human fibroblasts with the aim of gaining insight into genes possibly associated with the clinical manifestations of lonomism. Our hypothesis was that both the direct action L. obliqua venomous proteins on the host as well as an indirect effect caused by alteration in the gene expression pattern in host tissues could function in concert and perhaps synergistically to give rise to the profound symptoms observed during lonomism. Interesting changes in the expression pattern of some genes, such as IL-8, prostaglandin-endoperoxide synthase 2, urokinase-type plasminogen activator receptor and tissue factor, were observed in treated fibroblasts, which could contribute to some of the observed pathological sequela in lonomism. Thus, lonomism appears to be a result of both the previously described direct effects of the venom as well as indirect effects caused by changes in host gene expression profiles. These studies have enhanced our understanding of lonomism and may contribute to insights into more effective treatments.

Assessment of the toll-like receptor 4 Asp299Gly, Thr399Ile and interleukin-8 -251 polymorphisms in the risk for the development of distal gastric cancer.

BACKGROUND: The intensity of the inflammation induced by Helicobacter pylori colonization is associated with the development of distal gastric cancer (GC). The host response to H. pylori has been related to genetic polymorphisms that influence both innate and adaptive immune responses.Our aim was to investigate whether the presence of the TLR4 Asp299Gly, TLR4 Thr399Ile and IL-8-251 A/T polymorphisms had any influence in the development of distal GC in a Mexican population. METHODS: We studied 337 patients that were divided in two groups: 78 patients with histologically confirmed distal GC and 259 non-cancer controls. The presence of H. pylori in the control population was defined by positive results of at least two of four diagnostic tests: serology, histology, rapid urease test and culture. Human DNA was purified and genotyped for TLR4 Asp299Gly polymorphism by pyrosequencing, for TLR4 Thr399Ile by PCR-RFLP and for IL8-251 by the amplification refractory mutation system (ARMS)-PCR. RESULTS: The non-cancer control group was found to be in Hardy-Weinberg equilibrium at the polymorphic loci studied (chi-square H-W = 0.58 for IL8-251, 0.42 for TLR4 Asp299Gly and 0.17 for TLR4 Thr399Ile). The frequencies of mutated alleles (homozygous plus heterozygous) were compared between cases and controls. We found no significant difference for TLR4- Asp299Gly [the 7.7% of distal GC patients and 7.7 % non-cancer controls (p = 0.82)] and for TLR4 Thr399Ile [the 1.3% of GC patients and the 5% of the control population (p = 0.2)]. In contrast, for IL-8-251 A/T, 80.77% of the GC patients and 66.4% in the control group age and gender matched had at least one copy of mutated allele (OR = 2.12, 95% CI = 1.1-4.2) (p = 0.023). CONCLUSION: This study showed that the IL8-251*A allele could be related to the development of distal gastric cancer in this Mexican population.

[Assessment of the role of TLR-4 in shear-stress-induced IL-8 gene transcription activation in vascular endothelial cells by gene mutation and gene transfection technology].

This study is to evaluate the role of TLR-4 in lower laminar shear stress-induced interleukin-8 gene transcription activation in vascular endothelial cells by using gene mutation and transfection techniques. RT-PCR, Northern hybridization and immunocytochemical fluorescent staining showed that TLR-4 was expressed in human umbilical vein endothelial cells(HUVEC). When stimulated with 4.2 dyne/cm2 shear stress for 1 hour, an increase of TLR-4 mRNA expression was observed in HUVECs detected by RT-PCR and Northern hybridization. The intracellular domain deletion mutant TLR-4 cDNA (lacking the 155 COOH terminal amino acids of the wild type (TLR-4) and -102 -61 bp DNA sequence in 5'-flanking region of IL-8 gene (IL-8USCS) were isolated from endothelial cells by RT-PCR and PCR. These two DNA fragments were cloned into pcDNA3 and pEGFP1 respectively to construct TLR-4 dominant negative mutant pcDNA3-mTLR4 and IL-8 reporter gene pEGFP1-IL8USCS. ECV304 cells were transfected with the pEGFP1-IL8USCS or co-transfected with pEGFP1-IL8USCS and pcDNA3-mTLR4 by Dosper liposome transfectional reagent and selected by G418, then stimulated with 4.2 dyne/cm2 shear stress for 3 hours. Flow cytometric analysis showed that when exposed to 4.2 dyne/cm2 shear stress for 3 hours, there was a marked increase in the green fluorescent protein expression in pEGFP1-IL8USCS-transfected ECV304 cells. In contrast, there was almost no change in the green fluorescent protein expression in the cells co-transfected with pEGFP1-IL8USCS and pcDNA3-mTLR4 after the stimulation, suggesting the TLR-4 mutant depressed TLR-4 signaling. These experiments suggest that the inflammatory TLR-4/NF-kappa B signaling pathway would probably be involved in flow shear stress-induced IL-8 gene expression in human vascular endothelial cells.