Effects of parturition and dexamethasone on DNA methylation patterns of IFN-γ and IL-4 promoters in CD4+ T-lymphocytes of Holstein dairy cows

This study investigated epigenetic mechanisms by which DNA methylation affects the function of bovine adaptive immune system cells, particularly during the peripartum period, when shifts in type 1 and type 2 immune response (IR) biases are thought to occur. Stimulation of CD4+ T-lymphocytes isolated from 5 Holstein dairy cows before and after parturition with concanavalin A (ConA) and stimulation of CD4+ T-lymphocytes isolated from 3 Holstein dairy cows in mid-lactation with ConA alone or ConA plus dexamethasone (Dex) had significant effects on production of the cytokines interferon gamma (IFN-γ, type 1) and interleukin 4 (IL-4, type 2) that were consistent with DNA methylation profiles of the IFN-γ gene promoter region but not consistent for the IL-4 promoter region. ConA stimulation increased the production of both cytokines before and after parturition. It decreased DNA methylation in the IFN-γ promoter region but increased for IL-4 promoter region. Parturition was associated with an increase in IFN-γ production in ConA-stimulated cells that approached significance. Overall, DNA methylation in both promoter regions increased between the prepartum and postpartum periods, although this did not correlate with secreted cytokine concentrations. Dexamethasone treated cells acted in a manner consistent with the glucocorticoid’s immunosuppressive activity, which mimicked the change at the IFN-γ promoter region observed during parturition. These results support pregnancy as type 2 IR biased, with increases of IFN-γ occurring after parturition and an increase in IL-4 production before calving. It is likely that these changes may be epigenetically controlled.     

Cloning and sequence analysis of IL-2, IL-4 and IFN-γ from Indian Dromedary camels (Camelus dromedarius)

The cDNAs of three cytokines, viz., IL-2, IL-4 and IFN-γ from Dromedary camels were amplified by PCR using Bactrian camel sequences and subsequently cloned for sequence analysis. Relationship based on amino acid sequences revealed that Dromedary camel IL-2 shared 99.5% and 99.3% identity at the nucleotide and amino acid levels with Bactrian camel IL-2. In the case of IL-4, the identity of Dromedary camel was 99.7% and 99.2% at the nucleotide and amino acid levels, respectively with that of Bactrian camel. The Dromedary camel IFN-γ shared 100% identity both at nucleotide and amino acid levels with Bactrian camel IFN-γ. Phylogenetic analysis based on amino acid sequences indicated the close relationship in these cytokine genes between the Dromedary camel and other camelids.     

Construction of DNA vaccines encoding Eimeria acervulina cSZ-2 with chicken IL-2 and IFN-γ and their efficacy against poultry coccidiosis

The study describes vaccination experiments with highly immunogenic sporozoite E. acervulina cSZ-2 co-administered with chicken IL-2 (chIL-2) and interferon-γ (chIFN-γ) to determine their efficacies against homologue challenge. The entire coding sequence of cSZ2, chIL-2 and chIFN-γ were cloned into eukaryotic expression vector pVAX1, constructing DNA vaccines pVAX1-cSZ2, pVAX1-chIL-2, pVAX1-chIFN-γ, pVAX1-cSZ2-chIL-2 and pVAX1-cSZ2-chIFN-γ. The expression of target genes in vivo was detected by RT-PCR and Western blot. Chicken experiments were carried out by vaccinating chickens two times at dose rate of 100 μg intramuscularly. At 28 days of age, all chickens were inoculated orally with 1 × 10⁵ sporulated oocysts of E. acervulina except the unchallenged control group. Seven days after challenge, all chickens were weighted and slaughtered for duodenum collection. The results indicated that these DNA vaccines were successfully constructed and the antigen genes could be expressed effectively in vivo. The findings also demonstrated best synergistic effect of IL-2 with this protein which suggested that co-administration of cytokines with this antigen was a powerful method to enhance immunity by alleviating intestinal lesions, body weight loss and oocyst count imparting partial protection against homologous challenge.     

Upregulation of TNF-α Production by IFN-γ and LPS in Cultured Canine Keratinocytes: Application to Monosaccharides Effects

Activated keratinocytes play a key role in the cutaneous immune system by their interactions with other cell types through the production of cytokines with both autocrine and paracrine activity. But there is little knowledge about epidermal cytokines in the dog. In this study, cultured canine keratinocytes were stimulated by human recombinant interferon γ (IFN-γ) and lipopolysaccharide (LPS) and cell supernatants were tested for tumour necrosis factor α (TNF-α) concentration using a cell viability assay on a murine cell line. We show that IFN-γ in combination with LPS significantly increases TNF-α secretion by canine keratinocytes. The best stimulations were obtained using confluent cultures and the association of IFN-γ (400 ng/ml) and LPS (40 μg/ml). The experimental protocol we describe represents a new method for studying keratinocyte activation and its modulation in the dog. We provide an example of application of our method: the study of the effects of different monosaccharides on canine keratinocyte activation.     

The measurement of three cytokine transcripts in naïve and sensitized ovine peripheral blood mononuclear cells following in vitro stimulation with bluetongue virus serotype-23

Bluetongue (BT), a serious disease of sheep and some wild ruminants, is caused by bluetongue virus (BTV), a member of the family, Reoviridae. The current research thrust for controlling BT is on development of efficient vaccines, necessitating clear understanding of ovine immunology. At present, comparative studies on cytokine gene expression profiles of na?ve and BTV-sensitized peripheral blood mononuclear cells (PBMC) in sheep have not been clearly understood. In the present study, PBMC from na?ve and BEI-inactivated-saponin-adjuvanted BTV-1 vaccinated sheep were stimulated in vitro with heterologous BTV-23. At various intervals, RT-qPCR was carried out to estimate cytokine (interferon-gamma, interleukin-12 and interleukin-2) mRNA expressions that are linked to cell-mediated immunity. The results showed that PBMC cytokine profiles were relatively increased both temporally and quantitatively in immunized sheep PBMC compared to na?ve ones, suggesting that BTV-1 vaccination may prime immune system that can cross-react with BTV-23 antigens.     

IFN-τ研究进展

干扰素tau(IFN-τ)是反刍动物特有的一种新型Ⅰ型干扰素,通常被认为是反刍动物的妊娠识别信号,在胚胎与子宫内膜的相互作用中调节一系列相关基因的表达。近年来,随着对IFN-τ研究的深入开展,发现其除了参与调节反刍动物妊娠着床外,在其他物种中还具有抗炎、抗病毒、抗肿瘤、免疫调节等多种生理功能。基于此,文章围绕IFN-τ的主要生理作用展开综述。     

Phagolysosome maturation of macrophages was reduced by PE_PGRS 62 protein expressing in Mycobacterium smegmatis and induced in IFN-γ priming

Mycobacterium bovis parasitizes host macrophages and has developed strategies to survive within macrophages. Research on mycobacteria-specific PE_PGRS genes indicates that they code for cell surface proteins that may influence virulence. To further elucidate the molecular pathogenesis of tuberculosis and host response to M. bovis, we explored the mechanisms by which PE_PGRS62 protein increase persistence of mycobacterium within host macrophages. We found that the M. smegmatis strain expressing M. bovis PE_PGRS 62 protein reduced phagolysosome maturation in human macrophages, and significantly decreased the mRNA expression of IL-1β in a dose- and time-dependent. We identified that IFN-γ priming of macrophages immediately prior to infection with PE_PGRS62 expressing M. smegmantis, enhanced the maturation of phagolysosomes and induced IL-1β production both that the protein and mRNA levels and further activated the NF-κB pathway. Overall, we demonstrated that PE_PGRS62 protein altered the immune environment of the host cells, which suggested that the pathogenic PE_PGRS62 protein altering the immune mechanism might be involved in the pathogenesis of mycobacterial disease and hence influenced host cell responses to M. bovis infection.     

Memory T cell proliferative responses and IFN-γ productivity sustain long-lasting efficacy of a Cap-based PCV2 vaccine upon PCV2 natural infection and associated disease

Porcine circovirus type 2 (PCV2) vaccination represents an important measure to cope with PCV2 infection; however, data regarding the modulation of the immune cell compartment are still limited, especially under field conditions. This study is aimed at investigating the features of the cellular immune response in conventional piglets induced by vaccination using a capsid (Cap) protein-based PCV2 vaccine compared to unvaccinated animals when exposed to PCV2 natural infection. Immune reactivity was evaluated by quantifying peripheral cell subsets involved in the anti-viral response and characterizing the interferon-gamma (IFN-γ) secreting cell (SC) responsiveness both in vivo and upon in vitro whole PCV2 recall. The vaccination triggered an early and intense IFN-γ secreting cell response and induced the activation of peripheral lymphocytes. The early increase of IFN-γ SC frequencies resulted in a remarkable and transient tendency to increased IFN-γ productivity in vaccinated pigs. In vaccinated animals, soon before the onset of infection occurred 15-16 weeks post-vaccination, the recalled PCV2-specific immune response was characterized by moderate PCV2-specific IFN-γ secreting cell frequencies and augmented productivity together with reactive CD4⁺CD8⁺ memory T cells. Conversely, upon infection, unvaccinated animals showed very high frequencies of IFN-γ secreting cells and a tendency to lower productivity, which paralleled with effector CD4^–CD8⁺ cytotoxic cell responsiveness. The study shows that PCV2 vaccination induces a long-lasting immunity sustained by memory T cells and IFN-γ secreting cells that potentially played a role in preventing the onset of infection; the extent and duration of this reactivity can be an important feature for evaluating the protective immunity induced by vaccination.     

IFN-τ与妊娠

IFN-τ属于一类功能特化的 IFN,它是反刍动物母体妊娠识别的孕体信号 ,在黄体维持和妊娠建立过程中发挥重要的生物学功能。本文对 IFN- τ的抗溶黄作用及其抗溶黄机制等方面的研究进展进行了系统综述。     

IFN-γ expression and infectivity of Toxoplasma infected tissues are associated with an antibody response against GRA7 in experimentally infected pigs

Toxoplasma gondii, an obligate intracellular parasite, can be transmitted to humans via the consumption of infected meat. However, there are currently no veterinary diagnostic tests available for the screening of animals at slaughter. In the current work, we investigated whether cytokine responses in the blood, and antibody responses against recombinant T. gondii GRA1, GRA7, MIC3 proteins and a chimeric antigen EC2 encoding MIC2-MIC3-SAG1, are associated with the infectivity of porcine tissues after experimental infection with T. gondii. Two weeks after experimental infection of conventional 5-week-old seronegative pigs, an IFN-γ response was detected in the blood, with a kinetic profile that followed the magnitude of the GRA7 antibody response. Antibody responses to GRA1, MIC3 and EC2 were very weak or absent up to 6 weeks post infection. Antibodies against GRA7 occurred in all infected animals and were associated with the presence of the parasite in tissues at euthanasia a few months later, as demonstrated by quantitative real-time PCR and isolation by bio-assay. Remarkably, although brain and heart tissue remained infectious, musculus gastrocnemius and musculus longissimus dorsi were found clear of infectious parasites 6 months after experimental infection. Seropositive response in a GRA7 ELISA indicates a Toxoplasma infection in pigs and is predictive of the presence of infectious cysts in pig heart and brain. This new ELISA is a promising tool to study the prevalence of Toxoplasma infection in pigs. Clearance of the infection in certain pig tissues suggests that the risk assessment of pig meat for human health needs further evaluation.     

Expansion of intracellular IFN-γ positive lymphocytes during Mycoplasma agalactiae infection in sheep

A method to assess the expansion of antigen-specific intracellular IFN-γ positive T cell subsets during the infection will be helpful for a better understanding of mycoplasmal infections physiopathology in the sheep. We analysed the percentage of antigen-specific lymphocytes positive for intracellular IFN-γ during the infection of sheep with Mycoplasma agalactiae by culturing peripheral blood mononuclear cells of infected or uninfected animals with irradiated M. agalactiae. The expansion of antigen-specific IFN-γ positive lymphocytes in infected sheep was initially sustained by CD4⁺ T cells at day 15 after infection, when antigen specific IgG start to be detectable, followed by CD8/IFN-γ double positive cells. γδ T-cells were not expanded at any time point analysed. IFNγ⁺ T cells disappear 60 days after infection, suggesting that antigen specific IFNγ⁺ T cells, mainly detected in the early phase of the disease, could be useful to understand the role of cell-mediated immunity during M. agalactiae infection.     

Expansion of intracellular IFN-γ positive lymphocytes during Mycoplasma agalactiae infection in sheep

A method to assess the expansion of antigen-specific intracellular IFN-γ positive T cell subsets during the infection will be helpful for a better understanding of mycoplasmal infections physiopathology in the sheep. We analysed the percentage of antigen-specific lymphocytes positive for intracellular IFN-γ during the infection of sheep with Mycoplasma agalactiae by culturing peripheral blood mononuclear cells of infected or uninfected animals with irradiated M. agalactiae. The expansion of antigen-specific IFN-γ positive lymphocytes in infected sheep was initially sustained by CD4⁺ T cells at day 15 after infection, when antigen specific IgG start to be detectable, followed by CD8/IFN-γ double positive cells. γδ T-cells were not expanded at any time point analysed. IFNγ⁺ T cells disappear 60 days after infection, suggesting that antigen specific IFNγ⁺ T cells, mainly detected in the early phase of the disease, could be useful to understand the role of cell-mediated immunity during M. agalactiae infection.     

水貂IFN-α、IFN-β及IFN-γmRNA荧光定量RT-PCR检测方法的建立及应用

为采用SYBR GreenⅠ染料建立检测水貂IFN-α、IFN-β和IFN-γmRNA荧光定量RT-PCR检测方法,根据GenBank中登录的MiIFN-α和MiIFN-β、MiIFN-γ和3-磷酸甘油脱氢酶(GAPDH)基因序列,分别设计合成特异性引物,通过RT-PCR扩增MiIFN-α、MiIFN-β、MiIFN-γ和MiGAPDH的基因片段,构建到pMD18-T载体中制备质粒标准品,建立了相应基因mRNA的荧光定量RT-PCR检测方法并建立标准曲线。结果表明,MiGAPDH、MiIFN-α和MiIFN-β和MiIFN-γ基因的Ct值与标准品稀释度在10拷贝质粒/μL~107拷贝质粒/μL内均呈良好的线性关系,相关系数(R2)均为1.00,熔解曲线均呈单一熔解峰,检测下限为10拷贝质粒/μL,重复性试验表明组内、组间变异系数均小于4.5%。临床样品检测结果表明水貂感染肠炎病毒后,MiIFN-α、MiIFN-β和MiIFNγ相对表达水平均在6d达到最高峰值,MiIFN-α相对表达量要比MiIFN-β和MiIFN-γ相对表达量高两个数量级,并且在感染期(4d~6d)MiIFN-α相对表达量明显提高。本研究为水貂IFN mRNA的定量分析提供了有效工具。     

Dose dependent disposition of sulphadimidine and of its N4‐acetyl and hydroxy metabolites in plasma and milk of dairy cows

Summary

The disposition of sulphadimidine (SDM) and of its N₄‐acetyl (N₄‐SDM) and two hydroxyl metabolites, 6‐hydroxymethyl‐ (SCH₂OH) and 5‐hydroxyasulphadimidine (SOH), was studied in plasma and milk of dairy cows following intramuscular or intravenous administration of sulphadimididine‐33.3% at doses of 10, 45, 50, and 100 mg/kg. The main metabolite in plasma as well as in milk was SCH₂OH. The metabolite percentages, the final plasma elimination half‐lives, and the time of peak SDM concentrations in milk are presented for different dosages. The concentrations of SDM and its metabolites in milk ran parallel to those in plasma beyond4 hours p.i. The metabolite concentrations in plasma and milk were lower than those of the parent SDM. Sulphate and glucuronide metabolites could not be detected in milk.

At high doses (45 mg/kg or more) and SDM plasma concentrations exceeding 20 μg/ml, a capacity limited metabolism of SDM to SCH₂OH was noticed, viz, asteady state concentration of SCH₂OH and a biphasic elimination pattern for SDM and SCH,OH in plasma and milk.

The mean ultrafiltrate ratios of the milk to plasma concentrations with respect to SDM, SCH₂OH, SOH, and N₄‐SDM were: 0.69, 0.22, 020, and 0.63, respectively.

The total amount of SDM and its metabolites recovered from the milk after milking twice daily over the whole experimental time was less than 2% of the applied dose.

A bioassay method allowed of detecting qualitatively SDM concentrations exceeding 0.2 μg/ml in plasma or milk. Withholding times for edible tissues and milk are suggested.     

Production of IFN-γ in feline whole blood after incubation with potential T-cell epitopes of the nucleocapsid protein of feline coronavirus

Interferon gamma (IFN-γ) plays an important role in cell mediated responses against mutated feline coronavirus strains (FCoV) involved in the pathogenesis of feline infectious peritonitis (FIP). The aim of this study was to establish a combined in silico and in vitro approach to assess feline leukocyte production of IFN-γ in response to selected peptides of the nucleocapside protein (N) of FCoVs. To this aim, we designed, through a bioinformatic approach, 8 potentially immunogenic peptides from the protein N corresponding to sequences of residues 14, 182, 198 detected only in FCoVs from FIP cats (virulent strains), only in FCoVs from healthy cats (avirulent strains) and both in FIP and in healthy cats (mixed strains). The peptides or a sham solution were incubated with whole blood from 16 cats (7 healthy and 9 with chronic diseases other than FIP) and IFN-γ concentration was measured on plasma using an ELISA system. RT-PCR expression of IFN-γ mRNA was also evaluated after incubation of the peptides or a sham solution with whole blood from 4 clinically healthy cats. The mean plasma concentration of IFN-γ in samples incubated with peptides decreased and the expression of IFN-γmRNA did not change compared with the sham solution, except for some cats with chronic diseases (which probably have a "pre-activated" immune response). These cats responded to "avirulent" or "mixed" peptides by increasing the concentration of IFN-γ and the expression of IFN-γ mRNA. The combined approach employed in this study allowed us to identify potentially immunogenic peptides of FCoV N protein that can modulate the production of IFN-γ especially in cats with a "pre-activated" cell mediated response.     

A live attenuated mutant of Salmonella Montevideo triggers IL-6, IFN-γ and IL-12 cytokines that co-related with humoral and cellular immune responses required for reduction of challenge bacterial load in experimental chickens

A live attenuated Salmonella enterica serovar Montevideo (SM) mutant JOL1599 was constructed by deletion of virulence-associated genes. The protective efficacy and immune response profiles of chickens immunized with JOL1599 were investigated. Immunized chickens demonstrated significant increases in plasma IgG and lymphocyte proliferative responses (P ≤ 0.05). Increased levels of IL-6, INF-γ, and IL-12 were also observed. Immunized birds strongly responded to infection by rapid stimulation of a CD4+ subset of T cells. Organ bacterial recovery assay revealed a significant reduction in the challenge strain among immunized birds. Multiple doses of JOL1599 enhanced the immune responses of the birds as revealed by ascending trends of the immunological profiles. These findings indicate that immunization of chickens with JOL1599 may provide protection against Salmonella Montevideo infection via induction of IL-6, INF-γ, and IL-12 protective cytokines, which in turn triggers conducive humoral and cell-mediated immune responses.     

IFN-γ renders human intestinal epithelial cells responsive to lipopolysaccharide of Vibrio cholerae by down-regulation of DMBT1

Although intestinal epithelial cells (IECs) are continuously exposed to high densities of enteric bacteria, they are not highly responsive to microbe-associated molecular patterns (MAMPs). However, inflammatory cytokines such as interferon-γ (IFN-γ) are potentially capable of priming IECs to enhance responsiveness to MAMPs. In this study, we observed that heat-killed Vibrio cholerae (HKVC) and its lipopolysaccharide (LPS) poorly induced IL-8 production in a human IEC line, HT-29. However, both HKVC and the LPS showed a substantial induction of IL-8 production in IFN-γ-primed HT-29 cells. LPS-induced IL-8 production was proportional to the IFN-γ-priming period and LPS could not induce IL-8 production in the presence of polymyxin B. Moreover, LPS-induced IL-8 production in the IFN-γ-primed HT-29 cells was mediated through signaling pathways requiring p38 kinase and ERK, but not the JNK/SAPK pathway. Since deleted in malignant brain tumor 1 (DMBT1) is known to interact with and antagonize the action of LPS, we hypothesized that IFN-γ enhanced the responsiveness to LPS in HT-29 through down-regulation of DMBT1. We found that IFN-γ indeed attenuated DMBT1 expression at both the mRNA and protein levels in HT-29 cells. Conversely, when the cells were transfected with small interfering RNA to specifically silence DMBT1, IL-8 expression was augmented even in the absence of IFN-γ and the augmentation was further enhanced by treatment with V. cholerae LPS. Since IFN-γ is known to increase IFN-β expression in the IECs, we examined if IFN-β functioned similar to IFN-γ. Although IFN-β alone was able to induce IL-8 expression, it failed to render HT-29 cells responsive to V. cholerae LPS. In conclusion, our study suggests that IFN-γ primes IECs to become responsive to V. cholerae and its LPS by suppressing the expression of DMBT1.     

Effect of β -glucanase and xylanase supplementation of barley- and rye-based diets on caecal microbiota of broiler chickens

1. The aim was to investigate the effect of grain type (barley or rye) and exogenous enzymes (β-glucanase or xylanase) on the composition of chicken caecal microbiota as examined by classical culturing and molecular techniques (fluorescent in-situ hybridisation (FISH) and terminal-restriction fragment-length polymorphism (T-RFLP)). 2. Plate counting revealed higher total numbers of anaerobic bacteria, lactic acid bacteria and yeasts in caecal contents of birds fed with rye-based diets than in birds fed with barley-based diets. 3. As assessed by FISH analysis, the most abundant bacterial groups in the broiler caeca were Clostridium coccoides-Eubacterium rectale followed by Bacteroides sp., Lactobacillus sp./Enterococcus sp., Bifidobacterium sp. and Enterobacteriaceae. For both cereal types, the enzyme supplementation significantly decreased the relative amount of Enterobacteriaceae. 4. The T-RFLP profiles indicated that the caecal microbiota of birds receiving rye-based diets was more diverse than that of birds fed on barley-based diets. 5. Irrespective of the method applied, the results indicate that the cereal type as well as the exogenous enzyme supplementation influence the microbiota in broiler chicken caeca, and may have the effect of reducing potentially pathogenic Enterobacteriaceae populations.