Early time course of change in angiogenic proteins in human skeletal muscle and vascular cells with endurance training.

Angiogenic, mitochondrial, and related transcriptional proteins were assessed in human skeletal muscle and isolated vascular cells during the early phase of endurance training. Thigh muscle biopsies were obtained in healthy young subjects, after one acute bout (n = 9) and after 3, 5, 7, and 14 days (n = 9) of cycle ergometer training. Whole muscle homogenates were analyzed for angiogenic, mitochondrial, and regulatory mRNA and protein levels. Angiogenic proteins were determined in muscle-derived endothelial cells and pericytes sorted by fluorescence-activated cell sorting. Acute exercise induced an increase in whole muscle mRNA of peroxisome proliferator-activated receptor gamma coactivator 1α (4.5-fold; P = .002) and vascular endothelial growth factor (VEGF) (2.4-fold; P = .001) at 2 hours post. After 14 days of training, there was an increase in CD31 protein (63%; P = .010) in whole muscle indicating capillary growth. There was also an increase in muscle VEGF receptor 2 (VEGFR2) (1.5-fold; P = .013), in OXPHOS proteins (complex I, II, IV, V; 1.4- to 1.9-fold; P < .05) after 14 days of training and an increase in estrogen-related receptorα protein (1.5-fold; P = .039) at 14 days compared to 5 days of training. Both endothelial cells and pericytes expressed VEGF and other angiogenic factors at the protein level but with a distinctively lower expression of VEGFR2 and thrombospondin-1 (TSP-1) in pericytes. The findings illustrate that initiation of capillary and mitochondrial adaptations occurs within 14 days of training and suggest that sustained changes in angiogenic proteins including VEGF and TSP-1 are moderate in whole muscle and vascular cells.

Rapid whole blood assay using flow cytometry for measuring phagocytic activity of chicken leukocytes.

Phagocytic activity of leukocytes in whole blood was assessed as a potential immune competence trait in chickens. A flow cytometry based whole blood phagocytosis (WBP) assay was set up and evaluated using blood from chickens homozygous for four different MHC haplotypes, B12, B15, B19 and B21. Fluorescent latex beads and two serotypes of fluorescently labelled heat-killed bacteria (Salmonella Infantis and Salmonella. Typhimurium) were evaluated as phagocytic targets. In addition, the opsonophagocytic potential (OPp) of individual sera from the birds was included in a phagocytosis assay using the HD11 chicken macrophage cell line. Results showed that both serotypes of bacteria but not the latex beads were effectively phagocytosed by leukocytes in the whole blood cultures. Differences were observed in the phagocytic capacity of monocytes and thrombocyte/lymphocytes, respectively between the different MHC lines. No significant differences on the OPp of serum was identified between MHC lines. In addition, for both phagocytic activity of leukocytes and OPp of serum large variations between individuals were observed within MHC haplotypes. No significant relationships were observed between the phagocytic activity of leukocytes and serum OPp or Salmonella-specific IgY levels. In conclusion, our results suggest that the WBP assay, using a no-lyse no-wash single staining method, is a rapid and convenient method to assess phagocytic functions of different leukocyte populations.

CD107a as a marker of activation in chicken cytotoxic T cells.

The study aimed to evaluate cell surface mobilisation of CD107a as a general activation marker on chicken cytotoxic T cells (CTL). Experiments comprised establishment of an in vitro model for activation-induced CD107a mobilisation and design of a marker panel for the detection of CD107a mobilisation on chicken CTL isolated from different tissues. Moreover, CD107a mobilisation was analysed on CTL isolated from airways of infectious bronchitis virus (IBV)-infected birds direct ex vivo and upon in vitro stimulation. Results showed that phorbol 12-myristate 13-acetate (PMA) in combination with ionomycin was a consistent inducer of CD107a cell surface mobilisation on chicken CTL in a 4h cell culture model. In chickens experimentally infected with IBV, higher frequencies of CTL isolated from respiratory tissues were positive for CD107a on the cell surface compared to those from uninfected control chickens indicating in vivo activation. Moreover, upon in vitro PMA+ ionomycin stimulation, higher proportions of CTL isolated from the airways of IBV-infected chickens showed CD107a mobilisation compared to those from uninfected control chickens. Monitoring of CD107a cell surface mobilisation may thus be a useful tool for studies of chicken CTL cytolytic potential both in vivo and in vitro.

Immune gene expression in the spleen of chickens experimentally infected with Ascaridia galli.

Ascaridia galli is a gastrointestinal nematode infecting chickens. Chickens kept in alternative rearing systems or at free-range experience increased risk for infection with resulting high prevalences. A. galli infection causes reduced weight gain, decreased egg production and in severe cases increased mortality. More importantly, the parasitised chickens are more susceptible to secondary infections and their ability to develop vaccine-induced protective immunity against other diseases may be compromised. Detailed information about the immune response to the natural infection may be exploited to enable future vaccine development. In the present study, expression of immune genes in the chicken spleen during an experimental infection with A. galli was investigated using the Fluidigm(®) BioMark™ microfluidic qPCR platform which combines automatic high-throughput with attractive low sample and reagent consumption. Spleenic transcription of immunological genes was compared between infected chickens and non-infected controls at week 2, 6, and 9 p.i. corresponding to different stages of parasite development/maturation. At week 2 p.i. increased expression of IL-13 was observed in infected chickens. Increased expression of MBL, CRP, IFN-α, IL-1ß, IL-8, IL-12ß and IL-18 followed at week 6 p.i. and at both week 6 and 9 p.i. expression of DEFß1 was highly increased in infected chickens. In summary, apart from also earlier reported increased expression of the Th2 signature cytokine IL-13 we observed only few differentially expressed genes at week 2 p.i. which corresponds to the larvae histotrophic phase. In contrast, we observed increased expression of pro-inflammatory cytokines and acute phase proteins in infected chickens, by week 6 p.i. where the larvae re-enter the intestinal lumen. Increased expression of DEFß1 was observed in infected chickens at week 6 p.i. but also at week 9 p.i. which corresponds to a matured stage where adult worms are present in the intestinal lumen.

Chicken mannose-binding lectin function in relation to antibacterial activity towards Salmonella enterica.

Mannose-binding lectin (MBL) is a C-type serum lectin of importance in innate immunity. Low serum concentrations of MBL have been associated with greater susceptibility to infections. In this study, binding of purified chicken MBL (cMBL) to Salmonella enterica subsp. enterica (S. enterica) serotypes B, C1 and D was investigated by flow cytometry, and Staphylococcus aureus (S. aureus) was used for comparison. For S. enterica the C1 serotypes were the only group to exhibit binding to cMBL. Furthermore, functional studies of the role of cMBL in phagocytosis and complement activation were performed. Spiking with cMBL had a dose-dependent effect on the HD11 phagocytic activity of S. enterica subsp. enterica serovar Montevideo, and a more pronounced effect in a carbohydrate competitive assay. This cMBL dose dependency of opsonophagocytic activity by HD11 cells was not observed for S. aureus. No difference in complement-dependent bactericidal activity in serum with high or low cMBL concentrations was found for S. Montevideo. On the other hand, serum with high concentrations of cMBL exhibited a greater bactericidal activity to S. aureus than serum with low concentrations of cMBL. The results presented here emphasise that chicken cMBL exhibits functional similarities with its mammalian counterparts, i.e. playing a role in opsonophagocytosis and complement activation.

The consequence of low mannose-binding lectin plasma concentration in relation to susceptibility to Salmonella Infantis in chickens.

Mannose-binding lectin (MBL) is a key protein in innate immunity. MBL binds to carbohydrates on the surface of pathogens, where it initiates complement activation via the lectin-dependent pathway or facilitates opsonophagocytosis. In vitro studies have shown that human MBL is able to bind to Salmonella, but knowledge in relation to chicken MBL and Salmonella is lacking. In order to study this relation day-old chickens from two selected lines L10H and L10L, differing in MBL serum concentration, were either orally infected with S. Infantis (S.123443) or kept as non-infected controls. The differences between healthy L10H and L10L chicken sublines were more profound than differences caused by the S. Infantis infection. The average daily body weight was higher for L10H than for L10L, regardless of infection, indicating beneficial effects of MBL selection on growth. Salmonella was detected in cloacal swabs and the number of Salmonella positive chickens during the experiment was significantly higher in L10L than L10H, indicating that MBL may affect the magnitude of Salmonella colonisation in day-old chickens. MBL expression was determined in ceca tissue by real-time RT-PCR. L10H chickens showed a significantly higher relative expression than L10L at days 1 and 41 pi, regardless of infection. Finally, flow cytometric analysis of whole blood from infected chickens showed that L10H had a significantly higher count of all assessed leucocyte subsets on day 5 pi, and also a higher count of monocytes on day 12 pi than L10L. No difference was observed between infected and non-infected L10L chicken.

Cytokine gene expression profiles in chicken spleen and intestinal tissues during Ascaridia galli infection.

In the poultry production industry, chickens with access to outdoor areas are exposed to a wide range of parasites e.g. the helminth Ascaridia galli. By real-time quantitative RT-PCR, the relative gene expression of the T helper 1 (Th1) cytokine IFN-γ, the T helper 2 (Th2) cytokine IL-13, the anti-inflammatory cytokines IL-10 and TGF-ß4 and the pro-inflammatory cytokine IL-17F were determined over a period of 3 weeks in A. galli and non-A. galli-infected chickens. A characteristic Th2 response was observed in the jejunum of A. galli-infected chickens with increased expression of IL-13 and decreased expression of IFN-γ from day 14 post infection. At the putative time of larvae invasion into the intestinal mucosa (day 7), an increased expression of IFN-γ, IL-10, and TGF-ß4 was observed in the spleen. At the putative onset of the innate immune response (day 10), a decreased expression of jejunal IFN-γ and IL-13 was observed. Finally, at the expected period of an adaptive immune response (days 14-21) a general decreased expression of IFN-γ and TGF-ß4 in spleen and IFN-γ in jejunum was followed by a decreased expression of IFN-γ and IL-10 at day 21 in caecal tonsils.

Characterization of cellular and humoral immune responses after IBV infection in chicken lines differing in MBL serum concentration.

Chickens from two inbred lines selected for high (L10H) or low (L10L) mannose-binding lectin (MBL) serum concentrations were infected with infectious bronchitis virus (IBV), and innate as well as adaptive immunological parameters were measured throughout the experimental period. Chickens with high MBL serum concentrations were found to have less viral load in the trachea than chickens with low MBL serum concentrations indicating that these chickens were less severely affected by the infection. This study is the first to show that MBL expression is present in the lungs of healthy chickens and that the expression is upregulated at days 3 postinfection (p.i.) in L10H chickens. Furthermore, in the liver of infected chickens, the MBL expression was upregulated at day 7 p.i., despite the fact that the MBL serum concentrations were decreased below baseline at that time point. The number of TCRγδ+CD8α+ cells in the blood of noninfected chickens increased from week 0 to 3 p.i. However, the number of cells was higher in L10H chickens than in L10L chickens throughout the experiment. No increase was observed in the number of TCRγδ+CD8α+ cells in the blood of the infected L10H and L10L chickens. The numbers of B cells at week 3 p.i. were higher for noninfected L10L chickens than for the other chickens. No differences were observed between the infected and noninfected L10H chickens or between the infected L10H and L10L chickens. Furthermore, at week 3 p.i., the number of monocytes was higher in infected and noninfected L10H chickens than in the infected and noninfected L10L chickens. Thus, these results indicate that MBL is produced locally and may be involved in the regulation of the cellular immune response after an IBV infection. However, MBL did not appear to influence the humoral immune response after IBV infection in this study.

Ascaridia galli infection influences the development of both humoral and cell-mediated immunity after Newcastle Disease vaccination in chickens.

Potent vaccine efficiency is crucial for disease control in both human and livestock vaccination programmes. Free range chickens and chickens with access to outdoor areas have a high risk of infection with parasites including Ascaridia galli, a gastrointestinal nematode with a potential influence on the immunological response to vaccination against other infectious diseases. The purpose of this study was to investigate whether A. galli infection influences vaccine-induced immunity to Newcastle Disease (ND) in chickens from an MHC-characterized inbred line. Chickens were experimentally infected with A. galli at 4 weeks of age or left as non-parasitized controls. At 10 and 13 weeks of age half of the chickens were ND-vaccinated and at 16 weeks of age, all chickens were challenged with a lentogenic strain of Newcastle disease virus (NDV). A. galli infection influenced both humoral and cell-mediated immune responses after ND vaccination. Thus, significantly lower NDV serum titres were found in the A. galli-infected group as compared to the non-parasitized group early after vaccination. In addition, the A. galli-infected chickens showed significantly lower frequencies of NDV-specific T cells in peripheral blood three weeks after the first ND vaccination as compared to non-parasitized chickens. Finally, A. galli significantly increased local mRNA expression of IL-4 and IL-13 and significantly decreased TGF-ß4 expression in the jejunum two weeks after infection with A. galli. At the time of vaccination (six and nine weeks after A. galli infection) the local expression in the jejunum of both IFN-? and IL-10 was significantly decreased in A. galli-infected chickens. Upon challenge with the NDV LaSota strain, viral genomes persisted in the oral cavity for a slightly longer period of time in A. galli-infected vaccinees as compared to non-parasitized vaccinees. However, more work is needed in order to determine if vaccine-induced protective immunity is impaired in A. galli-infected chickens.

Adjuvant effects of mannose-binding lectin ligands on the immune response to infectious bronchitis vaccine in chickens with high or low serum mannose-binding lectin concentrations.

Mannose-binding lectin (MBL) plays a major role in the immune response as a soluble pattern-recognition receptor. MBL deficiency and susceptibility to different types of infections have been subject to extensive studies over the last decades. In humans and chickens, several studies have shown that MBL participates in the protection of hosts against virus infections. Infectious bronchitis (IB) is a highly contagious disease of economic importance in the poultry industry caused by the coronavirus infectious bronchitis virus (IBV). MBL has earlier been described to play a potential role in the pathogenesis of IBV infection and the production of IBV-specific antibodies, which may be exploited in optimising IBV vaccine strategies. The present study shows that MBL has the capability to bind to IBV in vitro. Chickens from two inbred lines (L10H and L10L) selected for high or low MBL serum concentrations, respectively, were vaccinated against IBV with or without the addition of the MBL ligands mannan, chitosan and fructooligosaccharide (FOS). The addition of MBL ligands to the IBV vaccine, especially FOS, enhanced the production of IBV-specific IgG antibody production in L10H chickens, but not L10L chickens after the second vaccination. The addition of FOS to the vaccine also increased the number of circulating CD4+ cells in L10H chickens compared to L10L chickens. The L10H chickens as well as the L10L chickens also showed an increased number of CD4-CD8α-γδ T-cells when an MBL ligand was added to the vaccine, most pronouncedly after the first vaccination. As MBL ligands co-administered with IBV vaccine induced differences between the two chicken lines, these results indirectly suggest that MBL is involved in the immune response to IBV vaccination. Furthermore, the higher antibody response in L10H chickens receiving vaccine and FOS makes FOS a potential adjuvant candidate in an IBV vaccine.

No protection in chickens immunized by the oral or intra-muscular immunization route with Ascaridia galli soluble antigen.

In chickens, the nematode Ascaridia galli is found with prevalences of up to 100% causing economic losses to farmers. No avian nematode vaccines have yet been developed and detailed knowledge about the chicken immune response towards A. galli is therefore of great importance. The objective of this study was to evaluate the induction of protective immune responses to A. galli soluble antigen by different immunization routes. Chickens were immunized with a crude extract of A. galli via an oral or intra-muscular route using cholera toxin B subunit as adjuvant and subsequently challenged with A. galli. Only chickens immunized via the intra-muscular route developed a specific A. galli antibody response. Frequencies of γδ T cells in spleen were higher 7 days after the first immunization in both groups but only significantly so in the intra-muscularly immunized group. In addition, systemic immunization had an effect on both Th1 and Th2 cytokines in caecal tonsils and Meckel's diverticulum. Thus both humoral and cellular immune responses are inducible by soluble A. galli antigen, but in this study no protection against the parasite was achieved.

Chicken mannose-binding lectin (MBL) gene variants with influence on MBL serum concentrations.

Mannose-binding lectin (MBL) plays a major role in the innate immune defence by activating the lectin complement pathway or by acting as an opsonin. Two forms of MBL have been characterised from several species, but for humans and chickens, only one form of functional MBL has been described. The human MBL2 gene is highly polymorphic, and it causes varying MBL serum levels. Several of the single-nucleotide polymorphisms (SNPs) have been associated with the severity of diseases of bacterial, viral or parasitic origin. Association between various diseases and different MBL serum levels has also been identified in chickens. In this study, two inbred chicken lines (L10L and L10H) which have been selected for low and high MBL levels in serum and four other experimental chicken lines were analysed for polymorphism in the MBL gene. The presence of polymorphisms in the MBL gene was revealed by southern blot analyses, and the differences in the serum concentrations of MBL were found to be of transcriptional origin according to real-time quantitative reverse transcription PCR analysis. Several SNPs were discovered in the promoter and the 5' untranslated region of the chicken MBL gene which resulted in the identification of six different alleles. Mapping of regulatory elements in the promoter region was performed, and SNPs that could affect the MBL serum concentration were identified. One SNP that was found to be located in a TATA box was altered in one of the six alleles only. This allele was associated with low MBL serum concentration.

Crosstalk between innate and adaptive immune responses to infectious bronchitis virus after vaccination and challenge of chickens varying in serum mannose-binding lectin concentrations.

Mannose-binding lectin (MBL), a C-type collectin with structural similarities to C1q, is an innate pattern-recognition molecule that is sequestered to sites of inflammation and infections. MBL selectively binds distinct chemical patterns, including carbohydrates expressed on all kinds of pathogens. The present study shows that serum MBL levels influence the ability of chickens to clear the respiratory tract of virus genomes after an infectious bronchitis virus (IBV) infection. The primary IBV infection induced changes in circulating T-cell populations and in the specific antibody responses. Serum MBL levels also influenced IBV vaccine-induced changes in circulating T-cell populations. Moreover, addition of mannose to an IBV vaccine altered both vaccine-induced changes in circulating T-cell populations and IBV specific vaccine and infection-induced antibody responses in chickens with high serum MBL levels. These data demonstrate that MBL is involved in the regulation of the adaptive immune response to IBV.

MHC expression on spleen lymphocyte subsets in genetically resistant and susceptible chickens infected with Marek's disease virus.

Resistance and susceptibility to Marek's disease (MD) are strongly influenced by the chicken major histocompatibility complex (MHC). In this study, splenic lymphocytes from MD-resistant and MD-susceptible chickens of three MHC genotypes (B21/B21, B19/B21, and B19/B19) were analyzed by flow cytometry for MHC surface expression. In the spleen, constitutive MHC class I surface expression was found to be highest in homozygous B19, lowest in homozygous B21, and intermediate in heterozygous B19/B21 animals. This was observed on CD4(+), CD8(+), and Bu-1(+) splenic lymphocytes. Chickens of all three genotypes were subjected to infection with MD virus (GA strain) and spleen samples from infected as well as MHC-matched negative controls were analyzed at 1, 4, and 8 wk post-infection (p.i.). It was observed that MDV induced an increase in MHC class I expression late in the infection. Thus, MHC class I was increased on the surface of CD4(+) cells from infected chickens of all genotypes at 4 and 8 wk p.i. compared with negative controls. Also, MHC class I expression was increased on CD8(+) cells from infected chickens of all genotypes at 4 and 8 wk p.i., except for the homozygous B19 animals, that showed no increase at 8 wk p.i. MDV-induced differences in MHC class II surface levels were also found. Thus, MHC class II expression was increased on CD4(+) cells from infected B19/21 and B21/B21 chickens at 4 wk p.i., and also at 8 wk p.i. on CD4(+) cells from infected B19/B21 animals. MHC class II expression was increased on CD8(+) cells from infected chickens of all genotypes at 4 wk p.i. These findings suggest that MDV infection in vivo increases the level of MHC surface expression on splenic T cells, indicating a possible role in immunity against MDV.

Mannan-binding lectin (MBL) serum concentration in relation to propagation of infectious bronchitis virus (IBV) in chickens.

Mannan-binding lectin (MBL) is a collectin that mediates activation of the complement system and is of importance for host defenses. In humans low concentrations of MBL in serum have been associated with susceptibility to several viral diseases. To understand the function of MBL in relation to infectious viral diseases two chicken lines were selected for high and low concentrations of MBL in serum for several generations. Offspring from the two sub-lines were subjected to infection with infectious bronchitis virus (IBV) in order to determine their genetic susceptibility to the virus. Results suggested that MBL plays a role in the innate immunity against IBV in the way that it performs an acute phase response, is able to activate complement, and inhibits the propagation of the virus in the trachea.