Association of twelve candidate gene polymorphisms and response to challenge with Salmonella enteritidis in poultry

Breeding for disease resistance to Salmonella enteritidis (SE) could be an effective approach to control Salmonella in poultry. The candidate gene approach is a useful method to investigate genes that are involved in genetic resistance. In this study, 12 candidate genes that are involved in the pathogenesis of Salmonella infection were investigated using five different genetic groups of meat-type chicken. The genes were natural resistance associated macrophage protein 1 (SLC11A1, previously known as NRAMP1), inhibitor of apoptosis protein 1 (IAP1), prosaposin (PSAP), Caspase-1 (CASP1), inducible nitric oxide production (iNOS), interferon-gamma (IFNG), interleukin-2 (IL2), immunoglobulin light chain (IGL), ZOV3, and transforming growth factors B2, B3 and B4 (TGFB2, B3 and B4). In total, 117 birds of all groups were challenged with SE at the age of 3 weeks. In all birds at 7-day post-infection SE load in caecum content, spleen and liver were quantified. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assays were used to genotype all animals for each gene. Overall we found the most significant associations with caecum content, nine of 12 genes showed a significant association (SLC11A1, IAP1, PSAP, CASP1, iNOS, IL2, IGL, TGFB2 and TGFB4). For liver, five genes (SLC11A1, CASP1, IL2, IGL, and TGFB4) and for spleen, only one gene (TGFB3) showed a significant association with SE load. By showing associations of 12 PCR-RFLP assays with SE load after a pathogen challenge, this study confirmed the polygenic nature of disease resistance to SE.     

Interleukin 10 regulates inflammatory cytokine synthesis to protect against lipopolysaccharide-induced abortion and fetal growth restriction in mice

Interleukin 10 (IL10) is a potent immune-regulating cytokine and inhibitor of inflammatory cytokine synthesis. To evaluate the anti-inflammatory role of IL10 in pregnancy, the response of genetically IL10-deficient mice to low-dose lipopolysaccharide (LPS)-induced abortion was examined. When IL10-null mutant C57Bl/6 (Il10(-/-)) and control (Il10(+/+)) mice were administered low-dose LPS on Day 9.5 of gestation, IL10 deficiency predisposed to fetal loss accompanied by growth restriction in remaining viable fetuses, with an approximately 10-fold reduction in the threshold dose for 100% abortion. After LPS administration, inflammatory cytokines tumor necrosis factor-alpha (TNFA) and IL6 were markedly increased in serum, uterine, and conceptus tissues in Il10(-/-) mice compared with Il10(+/+) mice, with elevated local synthesis of Tnfa and Il6 mRNAs in the gestational tissues. IL1A and IL12p40 were similarly elevated in serum and gestational tissues, whereas interferon gamma (IFNG) and soluble TNFRII content were unchanged in the absence of IL10. Recombinant IL10 rescued the increased susceptibility to LPS-induced fetal loss in Il10(-/-) mice but did not improve outcomes in Il10(+/+) mice. IL10 genotype also influenced the responsiveness of mice to a TNFA antagonist, etanercept. Fetal loss in Il10(-/-) mice was partly alleviated by moderate or high doses of etanercept, whereas Il10(+/+) mice were refractory to high-dose etanercept, consistent with attenuation by IL10 status of TNFA bioavailability after etanercept treatment. These data show that IL10 modulates resistance to inflammatory stimuli by downregulating expression of proinflammatory cytokines TNFA, IL6, IL1A, and IL12, acting to protect against inflammation-induced pathology in the implantation site.     

Maturation of dendritic cell precursors into IL12-producing DCs by J-LEAPS immunogens

LEAPS (ligand epitope antigen presentation system) vaccines consist of a peptide containing a major histocompatibility antigen binding peptide conjugated to an immune cell binding ligand (ICBL) such as the ‘J’ peptide from beta-2-microglobulin. Treatment of monocytes, monocytes plus GMCSF, or monocytes plus GMCSF and IL4 with JgD (containing a peptide from gD of herpes simplex virus type 1) or JH (with a peptide from HIV p17 gag protein) was sufficient to promote their maturation into Interleukin 12 producing dendritic cells. JgD-dendritic cells supported allotypic activation of T cells to produce Th1-related cytokines.     

A Fusion Cytokine Coupling GMCSF to IL9 Induces Heterologous Receptor Clustering and STAT1 Hyperactivation through JAK2 Promiscuity

Cytokine receptors are randomly distributed on the cell surface membrane and are activated upon binding of their extracellular ligands to mediate downstream cellular activities. We hypothesized that pharmaceutical clustering of ligand-bound, activated receptors may lead to heretofore unrealized gain-of-function with therapeutically desirable properties. We here describe an engineered bifunctional cytokine borne of the fusion of Granulocyte Macrophage Colony Stimulating Factor (GMCSF) and Interleukin-9 (IL9) (hereafter GIFT9 fusokine) and demonstrate that it chaperones co-clustering of the functionally unrelated GMCSF receptor (GMCSFR) and IL9 receptor (IL9R) on cell surface of target cells. We demonstrate that GIFT9 treatment of MC/9 cells leads to transhyperphosphorylation of IL9R-associated STAT1 by GMCSFR-associated JAK2. We also show that IL9R-associated JAK1 and JAK3 augment phosphorylation of GMCSFR-linked STAT5. The functional relevance of these synergistic JAK/STAT transphosphorylation events translates to an increased mitogenic response by GMCSFR/IL9R-expressing primary marrow mast cells. The notion of inducing heterologous receptor clustering by engineered fusokines such as GIFT9 opens the door to a novel type of biopharmaceutical platform where designer fusokines modulate cell physiology through clustering of targeted receptor complexes.     

Association of cytokine gene polymorphisms with malignant melanoma in Caucasian population

It has been hypothesized that polymorphisms expected to result in functional changes in cytokine genes may influence susceptibility to cancer, including malignant melanoma (MM). Here, we have screened 24 potentially functional polymorphisms in five cytokine genes by PCR-SBT and PCR-SSP methods in 122 MM cell lines derived from Caucasian patients. The polymorphic positions studied were: TNFA −1031, −863, −857, −851, −574, −376, −308, −238, +488; TGFB1 −988, −800, −509, +869, +915, +652, +673, +713, +788; IL10 −1082, −819, −592; IL6 −174; IFNG −333, +874. The frequencies of cytokine genotypes of melanoma tumours were compared with those published for healthy Caucasians. It was found that TNFA −238 GA, TGFB1 −509 CT, −800 GG, IFNG +874 AT, IL6 −174 GG, IL10 −1082 GA genotypes were significantly decreased, while TNFA −238 AA, −857 CC, TGFB1 −509 TT, IFNG +874 AA, IL6 −174 CC, IL10 −1082 AA, −819 TT, −592 AA genotypes were significantly increased, in MM. This suggests that genotypes provisionally associated with low expression of pro-inflammatory and immunomodulatory TNF-α, IFN-γ and IL-6 and anti-inflammatory IL-10 and TGF-β1 could be involved in the mechanisms of cancer progression and escape from immunoserveilance.     

Effect of Co-transfection of Anti-myostatin shRNA Constructs in Caprine Fetal Fibroblast Cells

Knockdown of myostatin gene (MSTN), transforming growth factor-β superfamily, and a negative regulator of the skeletal muscle growth, by RNA interference (RNAi), has been reported to increase muscle mass in mammals. The current study was aimed to cotransfect two anti-MSTN short hairpin RNA (shRNA) constructs in caprine fetal fibroblast cells for transient silencing of MSTN gene. In the present investigation, approximately 89% MSTN silencing was achieved in transiently transfected caprine fetal fibroblast cells by cotransfection of two best out of four anti-MSTN shRNA constructs. Simultaneously, we also monitored the induction of IFN responsive genes (IFN), pro-apoptotic gene (caspase3) and anti-apoptotic gene (MCL-1) due to cotransfection of different anti-MSTN shRNA constructs. We observed induction of 0.66-19.12, 1.04-4.14, 0.50-3.43, and 0.42-1.98 for folds IFN-β, OAS1, caspase3, and MCL-1 genes, respectively (p < 0.05). This RNAi based cotransfection method could provide an alternative strategy of gene knockout and develop stable caprine fetal fibroblast cells. Furthermore, these stable cells can be used as a cell donor for the development of transgenic cloned embryos by somatic cell nuclear transfer (SCNT) technique.     

A randomized phase II trial of induction chemotherapy followed by cisplatin chronotherapy versus constant rate delivery combined with radiotherapy

Chronotherapy is no longer a novel concept in cancer treatment after approximately 20 years of development. Many clinical trials have provided strong supporting evidence that chronomodulated treatment yields better results than a traditional dosage regimen. This study aimed to evaluate the adverse reactions, effect on immune functions, and therapeutic efficacy of chronomodulated infusion versus flat intermittent infusion of cisplatin (DDP) combined with intensity-modulated radiation therapy (IMRT) in patients with locoregionally advanced nasopharyngeal carcinoma (NPC). A total of 148 patients with biopsy-diagnosed untreated stage III-IVb NPC were randomly assigned to undergo two cycles of chronomodulated infusion (study group) or flat intermittent infusion (control group) of DDP (100 mg/m2 on day 1, 21 days/cycle) synchronized with radical radiotherapy. Patients in the study group received chronomodulated infusion, with peak delivery of DDP at 16:00 pm. Patients in the control group received a routine constant rate of infusion. Both groups were treated with the same radiotherapy techniques. Over a median follow-up of 20 months, the study group had better outcomes for adverse effects and immune functions compared with the control group. During the phase of concurrent chemoradiotherapy, the incidence of nausea, vomiting, and oral mucositis in the study and control groups was 66.7% and 79.5% (p < 0.05), 47.9% and 71.2% (p < 0.05), and 73.9% and 87.7% (p < 0.05), respectively. There was no significant difference in 2-year overall survival, progression-free survival, and distant metastasis-free survival between the two groups (p > 0.05). Chronochemotherapy significantly reduced the incidence of adverse reactions and enhanced the tolerance for treatment without affecting survival. It is worth mentioning that reduced destruction of immune function is a novel area of exploration in chronotherapy research.     

Evaluation of Interferon Response Induced by Anti-Myostatin shRNA Constructs in Goat (Capra hircus) Fetal Fibroblasts by Quantitative Real TIME-Polymerase Chain Reaction

RNAi is an evolutionary conserved, highly efficient, and cost effective technique of gene silencing. It holds considerable promise and success has been achieved both in vitro and in vivo experiments. However, it is not devoid of undesirable side effects as dsRNA can trigger the immune response and can also cause non-specific off-target gene silencing. In the present study, silencing of myostatin gene, a negative regulator of myogenesis, was evaluated in caprine fetal fibroblasts using three different shRNA constructs. Out of these three constructs, two constructs sh1 and sh2 showed, 72% and 50% reduction (p < 0.05) of myostatin mRNA, respectively. Efficient suppression (42–86%) of MSTN gene (p < 0.05) was achieved even by reducing the concentration of shRNA constructs. The induction of classical interferon stimulated gene (Oligoandenylate Synthetase-1, OAS-1) was studied to analyze the immune response against shRNAs. Notably, a reduction in the potency of shRNAs to induce interferon response was observed at lower concentration for OAS1 gene. The results obtained in the study would be helpful in the abrogation of the bystander effects of RNAi for long term stable expression of anti-MSTN expression constructs in the muscle.     

Histopathological and cellular studies of a case of cutaneous radiation syndrome after accidental chronic exposure to a cesium source.

This study was designed for the histopathological, cellular and biochemical characterization of a skin lesion removed surgically from a young male several months after accidental exposure to cesium-137, with an emphasis on expression of transforming growth factor beta1 (TGFB1) and tumor necrosis factor alpha (TNFA) and the occurrence of apoptosis. Under a hypertrophic epidermis, a highly inhomogeneous inflammatory dermis was observed, together with fibroblastic proliferation in necrotic areas. Immunostaining revealed overexpression of TGFB1 and TNFA inside the keratinocytes of the hypertrophic epidermis as well as in the cytoplasm of the fibroblasts and connective tissue of the mixed fibrotic and necrotic dermis. Inside this dermis, the TUNEL assay revealed areas containing numerous apoptotic fibroblasts next to areas of normal viable cells. Overexpression of TGFB1 was found in the conditioned medium and cellular fractions of both hypertrophic keratinocytes and fibrotic fibroblasts. This overexpression lasted for at least three passages in tissue culture. The present observations were consistent with the central role of TGFB1 in the determination of chronic radiation-induced damage to the skin and a significant involvement of TNFA. In addition, programmed cell death appeared to take place during the remodeling of the mixed fibrotic and necrotic tissue.     

Effects of dietary 25-hydroxyvitamin D3 supplementation on growth performance,immune function and antioxidative capacity in weaned piglets

The study evaluated the effects of different doses of 25-hydroxyvitamin D₃ (25(OH)D₃) on growth performance, immune function and antioxidative capacity in piglets. In a 21-d trial, 35 weaned pigs were divided into five groups and diets were supplemented with 5.5 (control), 43.0, 80.5, 118.0 and 155.5 μg 25(OH)D₃/kg, respectively. No treatment effects were observed for average daily gain, average daily feed intake and feed to gain ratio. Increasing dietary 25(OH)D₃ levels increased serum 25(OH)D₃ concentrations linearly (p < 0.01), decreased the frequency of CD3+CD4+ and CD3+CD8+ T cells (p < 0.01), and the serum level of complement component 3 (p < 0.05). Supplementation of 80.5 and 118.0 μg 25(OH)D₃/kg enhanced the activity of serum glutathione peroxidase (p < 0.05) and addition of 43.0 μg 25(OH)D₃/kg increased the malondialdehyde concentration (p < 0.05). Overall, feeding high-dose 25(OH)D₃ to weaned pigs partly improved immune functions and the antioxidative capacity.     

The role of cytokines in toxoplasmosis

Infection withToxoplasma gondii is normally asymptomatic, but as a consequence of the AIDS epidemic the incidence of symptomatic disease and especially toxoplasmic encephalitis (TE) has grown in frequency. The high frequency of adverse reactions to conventional therapeutic regimens for toxoplasmosis highlight the need to develop new strategies for the management of this disease. The importance of cytokines in resistance againstT. gondii has been shown primarily in murine models of toxoplasmosis and a number of cytokines (e.g., IFN-, TNF-, IL-2 and IL-12) have been proposed for trials in patients with TE. One mechanism by which these cytokines produce their effects is through stimulation of macrophages and/or NK cells. However, there are problems with immunological intervention in immunocompromised patients with TE since the infection is present primarily in the central nervous system (CNS), an immunoprivileged site, and because certain cytokines may down regulate the immune response. While much valuable information has been obtained from studies conducted in immunocompetent strains of mice their relevance to an immunocompromised host is unknown. The development of genetically altered mice with immune deficiencies offers promising new models that may allow for more rational development of new treatment regimens.Abbreviations AIDS Acquired Immunodeficiency Syndrome - CNS Central Nervous System - GM-CSF Granulo cyte-macrophage colony stimulating factor - HIV Human Immunodeficiency Virus - IL Interleukin - IFN Interferon - LAK Lymphokine Activated Killer - LPS Lipopolysacharide - MIP Macrophage Inflammatory Protein - NK Natural Killer - PCR Polymerase chain reaction - SCID Severe Combined Immunodeficiency - TG Toxoplasmic encephalitis - TGF Transforming Growth Factor - TNF Tumor Necrosis Factor     

Genetic variants in transforming growth factor-β gene (TGFB1) affect susceptibility to schizophrenia

Immense body of evidence indicates that dysfunction of immune system is implicated in the etiology of schizophrenia. The immune theory of schizophrenia is supported by alterations in cytokine profile in the brain and peripheral blood. Given the strong genetic background of schizophrenia, it might be assumed that aberrant production of cytokines might be the consequence of genetic factors. This study aimed at investigating the association between schizophrenia susceptibility and selected functional polymorphisms in genes encoding cytokines including: interleukin-2 (IL2 ?330T>G, rs2069756), interleukin-6 (IL-6 ?174G>C, rs1800795), interferon-γ (IFNG +874T>A, rs2430561) as well as for the first time transforming growth factor-β1 (TGFB1 +869T>C, rs1800470 and +916G>C, rs1800471). We recruited 151 subjects with schizophrenia and 279 controls. There was a significant difference in the genotype distribution and allelic frequency of the TGFB1 +869T>C between patients with schizophrenia and healthy controls (p < 0.05). The risk of schizophrenia was more than two-fold higher in carriers of T allele (CT+TT genotypes) than individuals with CC genotype. Given documented gender differences in incidence of schizophrenia, we conducted separate analyses of male and female participants. We have shown that the association was significant in females, while in males it reached a trend toward statistical significance. To the best of our knowledge, it is the first report showing the association between TGFB1 +869T>C polymorphism and schizophrenia.     

Immunological Adjuvant Effect of a Water‐Soluble Polysaccharide,CPP, from the Roots of Codonopsis pilosula on the Immune Responses to Ovalbumin in Mice

In this study, one water‐soluble polysaccharide, CPP, was purified from the root of Codonopsis pilosula. The immunomodulatory effect and the adjuvant potential of CPP on the cellular and humoral immune response of ICR mice against ovalbumin (OVA) were investigated. CPP was shown not to be lethal in vivo for mice in doses ranging from 0.5 to 4 mg. ICR Mice were immunized subcutaneously with 0.1 mg of OVA alone or with 0.1 mg of OVA dissolved in saline‐containing aluminum hydroxide gel (Alum) (0.2 mg), QuilA (0.01 and 0.02 mg) or CPP (0.5, 1 or 2 mg) on days 1 and 15. Two weeks later (day 28), concanavalin A (ConA)‐, lipopolysaccharide (LPS)‐, and OVA‐stimulated splenocyte proliferation, and OVA‐specific serum antibodies were measured. CPP significantly enhanced the ConA‐, LPS‐, or OVA‐induced splenocyte proliferation in the OVA‐immunized mice especially at a dose of 1 mg (P<0.05 or P<0.01). The OVA‐specific IgG, IgG1, and IgG2b antibody levels in serum were also significantly enhanced by CPP compared with OVA control group (P<0.05 or P<0.01). The results suggest that CPP could be a safe efficacious adjuvant for use in vaccines against both pathogens and cancer.     

Is peat accumulation in mangrove swamps influenced by the “enzymic latch” mechanism?

Mangrove swamps accumulate a significant amount (45–98 %) of organic carbon in sediments; however, there is a knowledge gap in explaining the mechanism behind this. Through the analysis of substrate samples from a red mangrove (Rhizophora mangle) swamp in southwest Florida, USA, this study investigated whether the “enzymic latch”, which suppresses decomposition in northern peatlands, is prevalent in mangrove soils. Laboratory analyses were performed to investigate the four hypotheses of the “enzymic latch”. Results showed that under aerobic conditions mangroves soil samples have significantly higher phenol oxidase activity (two-fold, p < 0.05), but lower phosphatase activity (?33 %, p < 0.05), β-glucosidase activity (?14 %) and glucosaminidase activity (?11 %) compared to anaerobic conditions. Soil samples supplemented with phenol oxidase showed significantly lower phenolic concentration (?8.3 %, p < 0.01), but increased β-glucosidase (+79 %, p < 0.05), xylosidase (+99 %, p < 0.01) and glucosaminidase (+102 %, p < 0.01) activities. Supplementation of lignin-based solution significantly lowered β-glucosidase (?55 %, p < 0.001), sulphatase (?82 %, p < 0.001) and glucosaminidase (?45 %, p < 0.01) activities. Based on these findings, it is concluded that the “enzymic latch” is highly likely to play a key role in suppressing decomposition rates in red mangrove covered areas of mangrove swamps.     

Increased circulatory levels of lipopolysaccharide (LPS) and zonulin signify novel biomarkers of proinflammation in patients with type 2 diabetes

Emerging data indicate that gut-derived endotoxin (metabolic endotoxemia) may contribute to low-grade systemic inflammation in insulin-resistant states. Specific gut bacteria seem to serve as lipopolysaccharide (LPS) sources and several reports claim a role for increased intestinal permeability in the genesis of metabolic disorders. Therefore, we investigated the serum levels of LPS and zonulin (ZO-1, a marker of gut permeability) along with systemic levels of tumor necrosis factor-α (TNF-α) and Interleukin-6 (IL-6) in patients with type 2 diabetes mellitus (T2DM) compared to control subjects. Study subjects were recruited from the Chennai Urban Rural Epidemiology Study [CURES], Chennai, India. Study group (n = 45 each) comprised of a) subjects with normal glucose tolerance (NGT) and (b) patients with T2DM. LPS, ZO-1, TNF-α, and IL-6 levels were measured by ELISA. Serum levels of LPS [p < 0.05], LPS activity [p < 0.001], ZO-1 [p < 0.001], TNFα [p < 0.001], and IL-6 [p < 0.001] were significantly increased in patients with T2DM compared to control subjects. Pearson correlation analysis revealed that LPS activity was significantly and positively correlated with ZO-1, fasting plasma glucose, 2 h post glucose, HbA1c, serum triglycerides, TNF-α, IL-6, and negatively correlated with HDL cholesterol. Regression analysis showed that increased LPS levels were significantly associated with type 2 diabetes [odds ratio (OR) 13.43, 95 % CI 1.998–18.9; p = 0.003]. In Asian Indians who are considered highly insulin resistant, the circulatory LPS levels, LPS activity, and ZO-1 were significantly increased in patients with type 2 diabetes and showed positive correlation with inflammatory markers and poor glycemic/lipid control.