鸡B-F2基因真核表达系统的建立与鉴定

鸡B-F2基因编码的主要组织相容性复合体(MHC)I类分子α链在递呈内源性抗原中起重要作用。本研究应用RT-PCR方法从鸡外周血单核细胞中扩增了B-F2基因片段,其大小为1 068 bp。进一步将其定向插入真核表达载体,构建重组质粒pEGFP-N1-B-F2。经脂质体法转染COS7,所表达的α链定位于在细胞的浆膜,同时转染P815细胞,经G418的筛选(0.6 mg/mL)得到稳定表达细胞株。该细胞株经培养10代次后,仍在RT-PCR中能获得相应DNA片段。上述结果表明,B-F2基因在动物真核表达系统得到表达,建立的P815(B-F2)细胞株为进一步研究MHC I类分子α链在免疫应答中作用奠定了基础。     

Expression kinetics of chicken β2-microglobulin and Class I MHC in vitro and in vivo during Marek’s disease viral infections

Marek’s disease virus (MDV) is a highly cell-associated herpesvirus that causes a disease in chickens characterized by tumor formation and immunosuppression. The changes of major histocompatibility complex (MHC) expression in different MDV-infected cells are not completely understood. In this study, we investigated the expression of the Class I MHC and β2-microglobulin (β2m) genes in response to MDV infection at different time points by real-time PCR. In both in vitro and in vivo, the expression levels of Class I MHC and β2m genes were upregulated during early MDV infections in comparison to control cells; We also found that the expression of Class I MHC gene was downregulated in BudR (5-bromo-2′-deoxyuridine)-treated MSB1 cells at 48 h and MDV-infected chicken embryo fibroblast cells (CEF) at 120 and 168 h post infection (hpi); Furthermore, compared to control groups, Class I MHC and β2m expression levels were downregulated in peripheral blood lymphocytes (PBLC) from MDV-infected chickens at 14 and 28 days post infection (dpi); Interestingly, both Class I MHC and β2m gene expression levels increased again in PBLC from MDV RB1B-infected chickens at 35 dpi, in which MDV was in the latent or transformed infection stages. In addition, Class I MHC expression was clearly decreased in MDV-infected CEF at 120 hpi although β2m expression was significantly increased. These changes in Class I MHC and β2m gene expression might provide more insights into host-virus interaction.     

Comprehensive network map of transcriptional activation of chicken type I IFNs and IFN-stimulated genes

Chicken type I interferons (type I IFNs) are key antiviral players of the chicken immune system and mediate the first line of defense against viral pathogens infecting the avian species. Recognition of viral pathogens by specific pattern recognition receptors (PRRs) induce chicken type I IFNs expression followed by their subsequent interaction to IFN receptors and induction of a variety of IFN stimulated antiviral proteins. These antiviral effectors establish the antiviral state in neighboring cells and thus protect the host from infection. Three subtypes of chicken type I IFNs; chIFN-α, chIFN-β, and a recently discovered chIFN-κ have been identified and characterized in chicken. Chicken type I IFNs are activated by various host cell pathways and constitute a major antiviral innate defense in chicken. This review will help to understand the chicken type 1 IFNs, host cellular pathways that are involved in activation of chicken type I IFNs and IFN stimulated antiviral effectors along with the gaps in knowledge which will be important for future investigation. These findings will help us to comprehend the role of chicken type I IFNs and to develop different strategies for controlling viral infection in poultry.     

Functional expression of a bovine major histocompatibility complex class I gene in transgenic mice

Major histocompatibility complex (MHC) class I restricted cellular immune responses play an important role in immunity to intracellular pathogens. By binding antigenic peptides and presenting them to T cells, class I molecules impose significant selection on the targets of immune responses. Candidate vaccine antigens for cellular immune responses should therefore be analysed in the context of MHC class I antigen presentation. Transgenic mice expressing human MHC (HLA) genes provide a useful model for the identification of potential cytotoxic T lymphocyte (CTL) antigens. To facilitate the analysis of candidate CTL vaccines in cattle, we have produced transgenic mice expressing a common bovine MHC (BoLA) class I allele.The functional BoLA-A11 gene, carried on a 7 kb genomic DNA fragment, was used to make transgenic mice by pronuclear microinjection. Three transgenic mouse lines carrying the BoLA-A11 gene were established. Expression of the BoLA-A11 gene was found in RNA and the A11 product could be detected on the surface of spleen and blood cells. Functional analysis of the A11 transgene product, and its ability to act as an antigen presenting molecules in the mouse host will be discussed.     

Cytotoxic T lymphocyte responses to Marek's disease herpesvirus-encoded glycoproteins

Cell-mediated immune responses are important for protective immunity to Marek’s disease (MD), especially because MD herpesvirus (MDV) infection is strictly cell-associated in chickens with the exception of the feather follicle epithelium. A system previously developed using reticuloendotheliosis (REV)-transformed cell lines stably expressing individual MDV genes allows the determination of relevant MDV proteins for the induction of cytotoxic T lymphocyte (CTL) responses. To examine the importance of glycoproteins for the induction of CTL, the MDV genes coding for glycoproteins (g) C, D, E, H, I, K, L, and M were stably transfected into the REV-transformed chicken cell lines RECC-CU205 (major histocompatibility complex (MHC): B²¹B²¹) and RECC-CU91 (MHC: B¹⁹B¹⁹). All transfected cell lines were lysed by REV-sensitized, syngeneic splenocytes obtained from MD-resistant N2a (MHC: B²¹B²¹) and MD-susceptible P2a (MHC: B¹⁹B¹⁹) chickens, indicating that the expression of individual MDV glycoproteins did not interfere with antigen processing pathways. Only cell lines expressing gI were recognized by CTL from both N2a and P2a MDV-infected chickens. Cell lines expressing glycoproteins gC and gK, and to a lesser extent, gH, gL, and gM were lysed by syngeneic MDV-sensitized splenocytes from N2a birds but not P2a birds. In contrast, gE was recognized by MDV-sensitized effector cells from the P2a line and not the N2a line. Glycoprotein D was not recognized by either line, with the exception of one marginally significant P2a assay. These results indicate that late viral glycoproteins are relevant for the induction of cell-mediated immunity during MDV infection.     

High interferon type I responses in the lung, plasma and spleen during highly pathogenic H5N1 infection of chicken

ABSTRACT: This study shows that high pathogenic H5N1 influenza virus infection of chicken induced high levels of bioactive interferon type I in the lung (4.3 × 105 U/mg tissue), plasma (1.1 × 105 U/mL), and spleen (9.1 × 105 U/mg tissue). In contrast, a low pathogenic attenuated H5N1 vaccine strain only induced approximately 24 times less IFN in the lung, 441 times less in the spleen and 649 less in the plasma. This was in the same range as a reassortant carrying the HA from the vaccine strain and the remaining genes from the high pathogenic virus. On the other hand, a reassortant virus with the HA from the high pathogenic H5N1 with the remaining genes from the vaccine strain had intermediate levels of IFN. The level of interferon responses related to the viral load, and those in the spleen and blood to the spread of virus to lymphoid tissue, as well as disease severity. In vitro, the viruses did not induce interferon in chicken embryonic fibroblasts, but high levels in splenocytes, with not clear relationship to pathogenicity and virulence. This, and the responses also with inactivated viruses imply the presence of plasmacytoid dendritic cell-like leukocytes within the chicken immune system, possibly responsible for the high interferon responses during H5N1 infection. Our data also indicate that the viral load as well as the cleavability of the HA enabling systemic spread of the virus are two major factors controlling systemic IFN responses in chicken.     

In vivo administration of ligands for chicken toll-like receptors 4 and 21 induces the expression of immune system genes in the spleen

Toll-like receptors (TLRs) are a group of conserved proteins that play an important role in pathogen recognition in addition to the initiation and regulation of innate and adaptive immune responses. To date, several TLRs have been identified in chickens, each recognizing different ligands. TLR stimulation in chickens has been shown to play a role in host-responses to pathogens. However, the mechanisms through which TLRs modulate the chicken immune system have not been well examined. The present study was conducted to characterize the kinetics of responses to TLR4 and TLR21 stimulation in chickens following intramuscular injections of their corresponding ligands, lipopolysaccharide (LPS) and CpG oligodeoxynucleotides (ODNs), respectively. To this end, relative expression of cytokine genes in the spleen was determined at 2, 6, 12 and 24 h after injection of TLR ligands. The results indicated that LPS strongly induced the up-regulation of some immune system genes early on in the response to treatment, including interferon (IFN)-γ, interleukin (IL)-10, and IL-1β. Furthermore, treatment with CpG ODN promoted the up-regulation of major histocompatibility complex (MHC)-II, IFN-γ and IL-10. The response to CpG ODN appeared to be somewhat delayed compared to the response to LPS. Moreover, we found a significant increase in IFN-α gene expression in response to LPS but not CpG ODNs. Future studies may be aimed to further characterize the molecular mechanisms of TLR activation in chickens or to exploit TLR agonists as vaccine adjuvants.     

The immunologists' debt to the chicken

The immune system of the chicken is an invaluable model for studying basic immunology and has made seminal contributions to fundamental immunological principles. Graft versus host responses and the key role of lymphocytes in adaptive immunity were first described in work with chicken embryos and chickens. 2. Most notably, the bursa of Fabricius provided the first substantive evidence that there are two major lineages of lymphocytes. Bursa-derived lymphocytes, or B cells, make antibodies while thymus-derived, or T cells, are involved in cell-mediated immune responses. 3. Gene conversion, the mechanism used by the chicken to produce its antibody repertoire, was first described in the chicken and requires the unique environment of the bursa. Subsequently it has been shown that some mammals also use gene conversion. 4. The chicken's Major Histocompatibility Complex (MHC), the first non-mammalian MHC to be sequenced, is minimal, compact and some 20-fold smaller than that of mammals. Uniquely, the chicken MHC is strongly associated with resistance to infectious diseases. 5. The first attenuated vaccine was developed by Louis Pasteur against a chicken pathogen, fowl cholera, and the first vaccine against a natural occurring cancer agent, Marek's disease virus, was developed for the chicken. 6. Vaccination of chick embryos on the 18th d of incubation, another breakthrough using chickens, provides protection early after hatching. In ovo vaccination now is widely practised by the poultry industry. 7. Evidence that widespread and intensive vaccination can lead to increased virulence with some pathogens, such as Marek's disease virus and infectious bursal disease virus, was first described with chicken populations. It warns of the need to develop mo resustainable vaccination strategies in future and provides useful lessons for other species, including in the human population. 8. Recombinant DNA technologies now provide the opportunity for the rational design of new vaccines. Such vaccines could contain the protective immunogenic elements from several pathogens and immunomodulatory molecules to direct and enhance immune responses so providing improved protection. The important thing will be to design vaccines that are sustainable and do not drive pathogens to ever-increasing virulence.     

禽类基因工程重组干扰素研究进展

近年来 ,多种禽类干扰素基因已被克隆和在大肠杆菌中表达。禽类基因工程重组干扰素在抗马立克病毒、劳斯肉瘤病毒、新城疫病毒、传染性法氏囊病病毒、传染性支气管炎病毒和禽流感病毒方面效果显著 ,展现出了广阔的应用前景。文章对禽类基因工程重组干扰素的研究进展作了综述     

In situ production of interferon in tissues of chickens exposed as embryos to turkey herpesvirus and Marek's disease virus

Chicken eggs at embryonation day (ED) 18 or newly hatched chicks were inoculated with turkey herpesvirus (HVT), Marek's disease virus (MDV), or virus-free diluent and, at intervals after inoculation, tissue homogenates of virus-exposed and virus-free chickens or chicken embryos were examined for interferon (IFN) activity. Homogenates of lung, thymus and spleen specimens from chickens given HVT at ED 18 had IFN activity. Activity of IFN in the lungs was studied further. Homogenates of lung specimens from chickens exposed to HVT at hatching also had IFN activity, although the concentration of IFN was lower than that in chickens given HVT at ED 18. The pathogenic isolates of MDV (JM-MDV), but not the attenuated (Md11/75C-MDV) or nonpathogenic (SB1-MDV) isolates, inoculated at ED 18 also induced high lung IFN activity. Exposure to a combination of HVT and SB1-MDV induced IFN activity comparable with that in chickens given HVT alone. The IFN activity in homogenates of lung specimens from virus-exposed chickens was species specific and heat and pH stable, but was destroyed by trypsin treatment. Occasionally, low IFN activity also was detected in homogenates of tissue specimens from virus-free chickens or chicken embryos. This IFN activity could have been produced constitutively or may have been induced by substances (inducers) in the environment.     

Marek's disease virus infection in the brain: virus replication, cellular infiltration, and major histocompatibility complex antigen expression

Marek's disease virus (MDV) infection in the brain was studied chronologically after inoculating 3-week-old chickens of two genetic lines with two strains of serotype I MDV representing two pathotypes (v and vv+). Viral replication in the brain was strongly associated with the development of lesions. Three viral antigens (pp38, gB, and meq) were detected in the brain of infected chickens. Marked differences between v and vv+ pathotypes of MDV were identified for level of virus replication, time course of brain lesions, and expression of major histocompatibility complex (MHC) antigens. Two pathologic phenomena (inflammatory and proliferative) were detected in the brain of chickens inoculated with vv+MDV, but only inflammatory lesions were observed in those inoculated with vMDV. Inflammatory lesions, mainly composed of macrophages, CD4+ T cells, and CD8+ T cells, started at 6-10 days postinoculation (dpi) and were transient. Proliferative lesions, characterized by severe infiltrates of CD4+CD8- T cells (blasts), started at 19-26 dpi and persisted. Expression of MHC antigens in endothelial cells and infiltrating cells within the brain was influenced by MDV infection. Upregulation of MHC class II antigen occurred in all treatment groups, although it was more severe in those inoculated with vv+MDV. MHC class I antigen was downregulated only in those groups inoculated with vv+MDV. These results enhance our understanding of the nature and pattern of MDV infection in the brain and help to explain the neurovirulence associated with highly virulent MDV.     

Effect of tumor infiltrating lymphocytes on the expression of MHC molecules in canine transmissible venereal tumor cells

Canine transmissible venereal tumor (CTVT) can be allo-transplanted across major histocompatibility complex barriers. The expression of MHC molecules is usually low in the progression (P) stage and then greatly increases during tumor regression (R). We investigated the effects of tumor infiltrating lymphocytes (TIL) on the expression of MHC molecules of CTVT cells. Isolated, viable CTVT cells were inoculated at each of 12 sites (1 x 10(8) CTVT cells per site) on the back of six, mixed-breed dogs. Tumor masses were collected every 2-3 weeks and prepared for histopathologic, immunocytochemistry, flow cytometry and immunoblotting studies. The level of MHC expression on tumor cells from different stages of growth was measured. Initially, expression of MHC I and II molecules in P phase CTVT was low. Twelve weeks post-inoculation (PI), expression increased dramatically and it continued to increase during R phase. Tumor growth slowed after 12 weeks PI and tumors entered R phase around 17 weeks PI. We hypothesize that CTVT evades host immunosurveillance and grows progressively for 12 weeks, when it becomes vulnerable and subject to the host's anti-tumor immune responses. We further demonstrated that R phase, but not P phase, TIL were closely associated with the over-expression of MHC I and II molecules by CTVT cells. The number and proportion of TIL were higher in R phase tumors. Supernatants, from R phase co-cultures (CTVT+TIL) and TIL only, promoted MHC I and II expression on P phase CTVT cells. After culturing alone for 1 month, expression of MHC classes I and II molecules in R phase CTVT cells decreased to the level of P phase CTVT cells. However, the above-mentioned supernatants restored their expression of MHC I and II molecules. In contrast, supernatants from P phase TIL or CTVT cells increased expression slightly or had no effect. Therefore, TIL, not CTVT cells, produce the effective substance (s) to promote the expression of MHC molecules by the tumor cells. Heat treated supernatant was unable to promote the expression of MHC I and II molecules by CTVT cells. In conclusion, TIL isolated from R phase CTVT secreted a heat-sensitive, soluble substance(s) that triggered over-expression of MHC I and II after 12 weeks PI. This caused the tumor to enter R phase and helped stop CTVT growth. Our findings will facilitate the understanding and further investigation of the mechanisms that initiate host immune surveillance against tumors.     

Immunological basis of differences in disease resistance in the chicken

Genetic resistance to diseases is a multigenic trait governed mainly by the immune system and its interactions with many physiologic and environmental factors. In the adaptive immunity, T cell and B cell responses, the specific recognition of antigens and interactions between antigen presenting cells, T cells and B cells are crucial. It occurs through a network of mediator proteins such as the molecules of the major histocompatibility complex (MHC), T cell receptors, immunoglobulins and secreted proteins such as the cytokines and antibodies. The diversity of these proteins that mainly is due to an intrinsic polymorphism of the genes causes phenotypic variation in disease resistance. The well-known linkage of MHC polymorphism and Marek's disease resistance difference represents a classic model revealing immunological factors in resistance differences and diversity of mediator molecules. The molecular bases in any resistance variation to infectious pathogens are vaguely understood. This paper presents a review of the major immune mediators involved in resistance and susceptibility to infectious diseases and their functional mechanisms in the chicken. The genetic interaction of disease resistance with production traits and the environment is mentioned.     

Detection of protein binding to a glucocorticoid response element-like sequence in a chicken major histocompatibility complex class II promoter

The glucocorticoid response element in gene promoters mediates regulation of gene expression by glucocorticoids. The major histocompatibility (MHC) class II genes, crucial for immunoresponsiveness, are among those modulated by glucocorticoids. A GRE-like sequence has been located in the promoter of a chicken MHC class II promoter. DNase footprinting revealed protein binding by the GRE-like sequence when nuclear extract from chicken T or B cell lines were used. Gel shift assays detected multiple binding activities in the lymphocyte cell lines, but little binding in the macrophage cell line. Relative band intensity differed among the lymphocyte cell lines. By using a mutant GRE oligonucleotide, most of the binding activities were demonstrated to be specific to the GRE. This study suggests a role of the GRE-like sequence in regulating chicken MHC class II genes and provides further evidence for the previously reported influence of glucocorticoids on chicken MHC class II expression which may be the molecular basis of glucocorticoid immunomodulation.     

Effect of Propionibacterium acnes-containing immunostimulant on interferon-gamma (IFNγ) production in the neonatal foal

Production of the Th1 cytokine interferon gamma (IFNγ) is associated with resistance to intracellular pathogens, including Rhodococcus equi. While neonatal foals are initially deficient in IFNγ production, expression of this cytokine increases throughout their first year of life. This is presumably the result of stimulation by environmental antigens including pathogen associated molecular patterns (PAMPS) signaling through toll-like receptors (TLR). This increased expression of IFNγ is likewise associated with an age-related resistance to R. equi infection. While immunostimulants containing PAMPS have been administered to adult horses in an attempt to modify their immune response, the effect of these materials on IFNγ expression in foals is unknown. The main objective of this study was to determine the effect of administering a commercial immunomostimulant EqStim? (Propionibacterium acnes) on IFNγ production measured using intracellular staining (IFNγ) and RT-PCR.     

Immune responses against Marek's disease virus

It is more than a century since Marek's disease (MD) was first reported in chickens and since then there have been concerted efforts to better understand this disease, its causative agent and various approaches for control of this disease. Recently, there have been several outbreaks of the disease in various regions, due to the evolving nature of MD virus (MDV), which necessitates the implementation of improved prophylactic approaches. It is therefore essential to better understand the interactions between chickens and the virus. The chicken immune system is directly involved in controlling the entry and the spread of the virus. It employs two distinct but interrelated mechanisms to tackle viral invasion. Innate defense mechanisms comprise secretion of soluble factors as well as cells such as macrophages and natural killer cells as the first line of defense. These innate responses provide the adaptive arm of the immune system including antibody- and cell-mediated immune responses to be tailored more specifically against MDV. In addition to the immune system, genetic and epigenetic mechanisms contribute to the outcome of MDV infection in chickens. This review discusses our current understanding of immune responses elicited against MDV and genetic factors that contribute to the nature of the response.     

Down-regulation of MHC class II receptors of DH82 cells, following infection with Ehrlichia canis

In order to evaluate whether infection with E. canis alters the expression of major histocompatibility complex (MHC) class I and/or MHC class II receptors, and by doing so alters the immune response to the organism, flow cytometry was performed on DH82 cells infected with Ehrlichia canis (90% infection) and on uninfected DH82 cells of the same passage, using anti-canine MHC class I and II antibodies. MHC class II expression was evident in 47.6 and 46.2% (mean 46.9%) of uninfected DH82 cells using two different anti-MHC class II antibodies, while no MHC class II expression was evident in DH82 cells infected with E. canis. The present results indicate that infection of DH82 macrophages with E. canis down-regulates their MHC class II receptors. These results suggest a possible mechanism by which E. canis evades the immune system.