Lymphocyte egress from thymus and peripheral lymphoid organs is dependent on S1P receptor 1

Adaptive immunity depends on T-cell exit from the thymus and T and B cells travelling between secondary lymphoid organs to survey for antigens. After activation in lymphoid organs, T cells must again return to circulation to reach sites of infection; however, the mechanisms regulating lymphoid organ exit are unknown. An immunosuppressant drug, FTY720, inhibits lymphocyte emigration from lymphoid organs, and phosphorylated FTY720 binds and activates four of the five known sphingosine-1-phosphate (S1P) receptors. However, the role of S1P receptors in normal immune cell trafficking is unclear. Here we show that in mice whose haematopoietic cells lack a single S1P receptor (S1P1; also known as Edg1) there are no T cells in the periphery because mature T cells are unable to exit the thymus. Although B cells are present in peripheral lymphoid organs, they are severely deficient in blood and lymph. Adoptive cell transfer experiments establish an intrinsic requirement for S1P1 in T and B cells for lymphoid organ egress. Furthermore, S1P1-dependent chemotactic responsiveness is strongly upregulated in T-cell development before exit from the thymus, whereas S1P1 is downregulated during peripheral lymphocyte activation, and this is associated with retention in lymphoid organs. We find that FTY720 treatment downregulates S1P1, creating a temporary pharmacological S1P1-null state in lymphocytes, providing an explanation for the mechanism of FTY720-induced lymphocyte sequestration. These findings establish that S1P1 is essential for lymphocyte recirculation and that it regulates egress from both thymus and peripheral lymphoid organs.     

PD-1 expression on HIV-specific T cells is associated with T-cell exhaustion and disease progression

Functional impairment of T cells is characteristic of many chronic mouse and human viral infections. The inhibitory receptor programmed death 1 (PD-1; also known as PDCD1), a negative regulator of activated T cells, is markedly upregulated on the surface of exhausted virus-specific CD8 T cells in mice. Blockade of this pathway using antibodies against the PD ligand 1 (PD-L1, also known as CD274) restores CD8 T-cell function and reduces viral load. To investigate the role of PD-1 in a chronic human viral infection, we examined PD-1 expression on human immunodeficiency virus (HIV)-specific CD8 T cells in 71 clade-C-infected people who were naive to anti-HIV treatments, using ten major histocompatibility complex (MHC) class I tetramers specific for frequently targeted epitopes. Here we report that PD-1 is significantly upregulated on these cells, and expression correlates with impaired HIV-specific CD8 T-cell function as well as predictors of disease progression: positively with plasma viral load and inversely with CD4 T-cell count. PD-1 expression on CD4 T cells likewise showed a positive correlation with viral load and an inverse correlation with CD4 T-cell count, and blockade of the pathway augmented HIV-specific CD4 and CD8 T-cell function. These data indicate that the immunoregulatory PD-1/PD-L1 pathway is operative during a persistent viral infection in humans, and define a reversible defect in HIV-specific T-cell function. Moreover, this pathway of reversible T-cell impairment provides a potential target for enhancing the function of exhausted T cells in chronic HIV infection.     

病毒性心肌疾病的基因时空表达特征

目的 研究病毒性心肌炎到扩张型心肌病全病程基因时空表达谱动力学特征.方法 用含有8 000个基因克隆的基因芯片检测CVB3反复感染Balb/c小鼠后第0,7,21天及第3,6,9个月心肌组织基因表达,并用SOM对差异性表达的基因进行聚类分析.结果（1）第10,14,15,19,20类基因在第7天时表达上调,第21,22类基因在第21天表达上调,而第23,24类基因在第7、21天表达均上调;（2）第6,7,11,12类基因在第7天时表达下调,第4,5类基因在第21天表达下调,而第2,3,8类基因则在第7,21天表达均下调;（3）第1,16类基因在第7天时表达下调,而第21天后则表现为上调;（4）有些基因如转移生长因子β结合蛋白、β-2微球蛋白,CD 53抗原却表现为持续性表达上调,而α-1微球蛋,ninjurin 1,舒血管素27,cytokine inducible SH2-containing protein 2则出现持续性表达下调.结论 建立了小鼠CVB病毒性心肌炎及扩张型心肌病全病程基因表达谱动力学模式,为进一步了解其分子发病机制提供全新的视野.     

Restoring function in exhausted CD8 T cells during chronic viral infection

Functional impairment of antigen-specific T cells is a defining characteristic of many chronic infections, but the underlying mechanisms of T-cell dysfunction are not well understood. To address this question, we analysed genes expressed in functionally impaired virus-specific CD8 T cells present in mice chronically infected with lymphocytic choriomeningitis virus (LCMV), and compared these with the gene profile of functional memory CD8 T cells. Here we report that PD-1 (programmed death 1; also known as Pdcd1) was selectively upregulated by the exhausted T cells, and that in vivo administration of antibodies that blocked the interaction of this inhibitory receptor with its ligand, PD-L1 (also known as B7-H1), enhanced T-cell responses. Notably, we found that even in persistently infected mice that were lacking CD4 T-cell help, blockade of the PD-1/PD-L1 inhibitory pathway had a beneficial effect on the 'helpless' CD8 T cells, restoring their ability to undergo proliferation, secrete cytokines, kill infected cells and decrease viral load. Blockade of the CTLA-4 (cytotoxic T-lymphocyte-associated protein 4) inhibitory pathway had no effect on either T-cell function or viral control. These studies identify a specific mechanism of T-cell exhaustion and define a potentially effective immunological strategy for the treatment of chronic viral infections.     

Second messenger role for Mg2+ revealed by human T-cell immunodeficiency

The magnesium ion, Mg(2+), is essential for all life as a cofactor for ATP, polyphosphates such as DNA and RNA, and metabolic enzymes, but whether it plays a part in intracellular signalling (as Ca(2+) does) is unknown. Here we identify mutations in the magnesium transporter gene, MAGT1, in a novel X-linked human immunodeficiency characterized by CD4 lymphopenia, severe chronic viral infections, and defective T-lymphocyte activation. We demonstrate that a rapid transient Mg(2+) influx is induced by antigen receptor stimulation in normal T cells and by growth factor stimulation in non-lymphoid cells. MAGT1 deficiency abrogates the Mg(2+) influx, leading to impaired responses to antigen receptor engagement, including defective activation of phospholipase Cγ1 and a markedly impaired Ca(2+) influx in T cells but not B cells. These observations reveal a role for Mg(2+) as an intracellular second messenger coupling cell-surface receptor activation to intracellular effectors and identify MAGT1 as a possible target for novel therapeutics.     

Viral infection switches non-plasmacytoid dendritic cells into high interferon producers

Type I interferons (IFN-I) are important cytokines linking innate and adaptive immunity. Plasmacytoid dendritic cells make high levels of IFN-I in response to viral infection and are thought to be the major source of the cytokines in vivo. Here, we show that conventional non-plasmacytoid dendritic cells taken from mice infected with a dendritic-cell-tropic strain of lymphocytic choriomeningitis virus make similarly high levels of IFN-I on subsequent culture. Similarly, non-plasmacytoid dendritic cells secrete high levels of IFN-I in response to double-stranded RNA (dsRNA), a major viral signature, when the latter is introduced into the cytoplasm to mimic direct viral infection. This response is partially dependent on the cytosolic dsRNA-binding enzyme protein kinase R and does not require signalling through toll-like receptor (TLR) 3, a surface receptor for dsRNA. Furthermore, we show that sequestration of dsRNA by viral NS1 (refs 6, 7) explains the inability of conventional dendritic cells to produce IFN-I on infection with influenza. Our results suggest that multiple dendritic cell types, not just plasmacytoid cells, can act as specialized interferon-producing cells in certain viral infections, and reveal the existence of a TLR-independent pathway for dendritic cell activation that can be the target of viral interference.     

A genetically humanized mouse model for hepatitis C virus infection

Hepatitis C virus (HCV) remains a major medical problem. Antiviral treatment is only partially effective and a vaccine does not exist. Development of more effective therapies has been hampered by the lack of a suitable small animal model. Although xenotransplantation of immunodeficient mice with human hepatocytes has shown promise, these models are subject to important challenges. Building on the previous observation that CD81 and occludin comprise the minimal human factors required to render mouse cells permissive to HCV entry in vitro, we attempted murine humanization via a genetic approach. Here we show that expression of two human genes is sufficient to allow HCV infection of fully immunocompetent inbred mice. We establish a precedent for applying mouse genetics to dissect viral entry and validate the role of scavenger receptor type B class I for HCV uptake. We demonstrate that HCV can be blocked by passive immunization, as well as showing that a recombinant vaccinia virus vector induces humoral immunity and confers partial protection against heterologous challenge. This system recapitulates a portion of the HCV life cycle in an immunocompetent rodent for the first time, opening opportunities for studying viral pathogenesis and immunity and comprising an effective platform for testing HCV entry inhibitors in vivo.     

Visualizing the generation of memory CD4 T cells in the whole body

It is thought that immunity depends on naive CD4 T cells that proliferate in response to microbial antigens, differentiate into memory cells that produce anti-microbial lymphokines, and migrate to sites of infection. Here we use immunohistology to enumerate individual naive CD4 T cells, specific for a model antigen, in the whole bodies of adult mice. The cells resided exclusively in secondary lymphoid tissues, such as the spleen and lymph nodes, in mice that were not exposed to antigen. After injection of antigen alone into the blood, the T cells proliferated, migrated to the lungs, liver, gut and salivary glands, and then disappeared from these organs. If antigen was injected with the microbial product lipopolysaccharide, proliferation and migration were enhanced, and two populations of memory cells survived for months: one in the lymph nodes that produced the growth factor interleukin-2, and a larger one in the non-lymphoid tissues that produced the anti-microbial lymphokine interferon-gamma. These results show that antigen recognition in the context of infection generates memory cells that are specialized to proliferate in the secondary lymphoid tissues or to fight infection at the site of microbial entry.     

Susceptibility of beta 2-microglobulin-deficient mice to Trypanosoma cruzi infection.

The beta 2-microglobulin (beta 2m) protein associates with the products of the class I major histocompatibility (MHC) loci; this combination functions in the thymic development of and antigen presentation to CD8+ T cells. Mice in which the beta 2m gene has been disrupted by homologous recombination fail to express class I MHC gene products, and therefore lack CD8+ T cells and measurable cytotoxic T-cell responses. However, beta 2m- mice appear to have normal development of both CD4+ alpha/beta T-cell receptor (TCR+) and gamma/delta TCR+ T cells and are not overtly more susceptible than beta 2m+ mice to potential environmental agents of infection or to experimental viral infection. Here we show that beta 2m- mice suffer high parasitaemias and early death when infected with the obligate cytoplasmic protozoan parasite Trypanosoma cruzi. Despite this increased susceptibility, the beta 2m- mice are more responsive than their beta 2m+ littermates in terms of lymphokine production, making higher levels of both interleukin-2 and interferon-gamma in response to mitogen stimulation. In addition, the beta 2m- mice show essentially no inflammatory response in parasite-infected tissues. These results confirm previous experiments on mice depleted of CD8+ cells using antibody treatment in demonstrating the importance of CD8+ T cells in immune protection in T. cruzi infection. They also implicate CD8+ T cells and/or class I MHC molecules in regulation of lymphokine production and recruitment of inflammatory cells.     

Natural killer cells act as rheostats modulating antiviral T cells

Antiviral T cells are thought to regulate whether hepatitis C virus (HCV) and human immunodeficiency virus (HIV) infections result in viral control, asymptomatic persistence or severe disease, although the reasons for these different outcomes remain unclear. Recent genetic evidence, however, has indicated a correlation between certain natural killer (NK)-cell receptors and progression of both HIV and HCV infection, implying that NK cells have a role in these T-cell-associated diseases. Although direct NK-cell-mediated lysis of virus-infected cells may contribute to antiviral defence during some virus infections--especially murine cytomegalovirus (MCMV) infections in mice and perhaps HIV in humans--NK cells have also been suspected of having immunoregulatory functions. For instance, NK cells may indirectly regulate T-cell responses by lysing MCMV-infected antigen-presenting cells. In contrast to MCMV, lymphocytic choriomeningitis virus (LCMV) infection in mice seems to be resistant to any direct antiviral effects of NK cells. Here we examine the roles of NK cells in regulating T-cell-dependent viral persistence and immunopathology in mice infected with LCMV, an established model for HIV and HCV infections in humans. We describe a three-way interaction, whereby activated NK cells cytolytically eliminate activated CD4 T cells that affect CD8 T-cell function and exhaustion. At high virus doses, NK cells prevented fatal pathology while enabling T-cell exhaustion and viral persistence, but at medium doses NK cells paradoxically facilitated lethal T-cell-mediated pathology. Thus, NK cells can act as rheostats, regulating CD4 T-cell-mediated support for the antiviral CD8 T cells that control viral pathogenesis and persistence.     

Massive infection and loss of memory CD4+ T cells in multiple tissues during acute SIV infection

It has recently been established that both acute human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) infections are accompanied by a dramatic and selective loss of memory CD4+ T cells predominantly from the mucosal surfaces. The mechanism underlying this depletion of memory CD4+ T cells (that is, T-helper cells specific to previously encountered pathogens) has not been defined. Using highly sensitive, quantitative polymerase chain reaction together with precise sorting of different subsets of CD4+ T cells in various tissues, we show that this loss is explained by a massive infection of memory CD4+ T cells by the virus. Specifically, 30-60% of CD4+ memory T cells throughout the body are infected by SIV at the peak of infection, and most of these infected cells disappear within four days. Furthermore, our data demonstrate that the depletion of memory CD4+ T cells occurs to a similar extent in all tissues. As a consequence, over one-half of all memory CD4+ T cells in SIV-infected macaques are destroyed directly by viral infection during the acute phase-an insult that certainly heralds subsequent immunodeficiency. Our findings point to the importance of reducing the cell-associated viral load during acute infection through therapeutic or vaccination strategies.     

鲟源豚鼠气单胞菌分离与强弱毒株病原生物学比较研究

从患慢性出血败血综合症的杂交鲟和达氏鳇病鱼标本中分离到一株优势菌,优势率80%,经回感和重分离确定为致病菌,并经生理生化和16SrDNA检测为豚鼠气单胞菌,命名为ZZF071029（简称ZF1029）,以豚鼠气单胞菌弱毒株ZF1029和强毒株XL2-T为研究对象,进行了菌落形态学、运动性实验、溶血性实验、小鼠致死试验、角膜毒力实验、药物敏感性实验等病原生物学相关实验。实验结果证明,病原菌运动性、溶血性、小鼠致死力、角膜毒力与菌株毒力和致病力正相关,并与所致杂交鲟和达氏鳇出血败血综合症急、慢性程度和致死率一致。     

Mu-opioid receptor desensitization by beta-arrestin-2 determines morphine tolerance but not dependence

Morphine is a powerful pain reliever, but also a potent inducer of tolerance and dependence. The development of opiate tolerance occurs on continued use of the drug such that the amount of drug required to elicit pain relief must be increased to compensate for diminished responsiveness. In many systems, decreased responsiveness to agonists has been correlated with the desensitization of G-protein-coupled receptors. In vitro evidence indicates that this process involves phosphorylation of G-protein-coupled receptors and subsequent binding of regulatory proteins called beta-arrestins. Using a knockout mouse lacking beta-arrestin-2 (beta arr2-/-), we have assessed the contribution of desensitization of the mu-opioid receptor to the development of morphine antinociceptive tolerance and the subsequent onset of physical dependence. Here we show that in mice lacking beta-arrestin-2, desensitization of the mu-opioid receptor does not occur after chronic morphine treatment, and that these animals fail to develop antinociceptive tolerance. However, the deletion of beta-arrestin-2 does not prevent the chronic morphine-induced up-regulation of adenylyl cyclase activity, a cellular marker of dependence, and the mutant mice still become physically dependent on the drug.     

A BAC clone of MDV strain GX0101 with REV-LTR integration retained its pathogenicity

The complete genome of Marek＇s disease virus （MDV） strain GX0101, which was integrated with the LTR sequences of REV, was cloned in Escherichia coli as a bacterial artificial chromosome （BAC）. BAC vector sequences were introduced into the US2 locus of the MDV genome by homologous recombination. The viral DNA containing the BAC vector was used to transform Escherichia coli strain of DH10B. Then the recombinant virus was successfully rescued by transfection of the recombinant BAC DNA into primary chicken embryo fibroblast （CEF）. This BAC viral clone was named bac-GX0101. When the reconstituted virus was inoculated into 1-day-old birds, visceral tumors could be detected as early as 62 d post infection. There was no difference in growth ability and pathogenicity to birds between the BAC derived virus and its parental virus. The BAC derived virus maintained its oncogenicity and immunosuppressive effects. In conclusion, the complete genome of GX0101 strain was successfully cloned into BAC and the infectious clone was rescued. With the powerful BAC manipulation system, the infectious clone will provide a useful tool for further understanding the functional roles of the inserted REV-LTR sequence in the GX0101 strain of MDV,     

静脉与非静脉吸毒感染成人HIV/AIDS的HAART疗效对比研究

目的:探讨静脉吸毒与非静脉吸毒感染的成人HIV/AIDS患者抗逆转录病毒治疗的疗效及差异.方法:将入选病例按感染途径分为静脉吸毒与非静脉吸毒两组进行临床疗效比较,观察两组患者治疗后0、3、6、12、18、24个月的免疫重建CD4+T淋巴细胞绝对值计数(CD4细胞)变化,观察患者治疗后病毒载量(VL)检测情况,观察治疗前与治疗后患者体力状况的变化,观察治疗后机会性感染的发病情况、死亡率、坚持治疗率.结果:两组治疗前后比较,CD4细胞均明显上升(P<0.01),但两组之间治疗前后对比CD4细胞变化无统计学差异(P>0.05).治疗前后对比两组均发生体力状况改善、机会性感染率下降,两组间无统计学差异.静脉吸毒与非静脉吸毒感染患者坚持治疗率分别为65.12%、81.25%,两组比较有显著性差异(P<0.01).静脉吸毒与非静脉吸毒感染患者的死亡率分别为18.60%、9.38%,两组比较有显著性差异(P<0.01).结论:经过高效抗逆转录病毒疗法(HAART)治疗静脉吸毒与非静脉吸毒感染的成人HIV/AIDS患者可以取得同样的治疗效果,但静脉吸毒感染者的死亡率增高、坚持治疗率低.     

CD4+CD25+ regulatory T cells control Leishmania major persistence and immunity

The long-term persistence of pathogens in a host that is also able to maintain strong resistance to reinfection, referred to as concomitant immunity, is a hallmark of certain infectious diseases, including tuberculosis and leishmaniasis. The ability of pathogens to establish latency in immune individuals often has severe consequences for disease reactivation. Here we show that the persistence of Leishmania major in the skin after healing in resistant C57BL/6 mice is controlled by an endogenous population of CD4+CD25+ regulatory T cells. These cells constitute 5-10% of peripheral CD4+ T cells in naive mice and humans, and suppress several potentially pathogenic responses in vivo, particularly T-cell responses directed against self-antigens. During infection by L. major, CD4+CD25+ T cells accumulate in the dermis, where they suppress-by both interleukin-10-dependent and interleukin-10-independent mechanisms-the ability of CD4+CD25- effector T cells to eliminate the parasite from the site. The sterilizing immunity achieved in mice with impaired IL-10 activity is followed by the loss of immunity to reinfection, indicating that the equilibrium established between effector and regulatory T cells in sites of chronic infection might reflect both parasite and host survival strategies.     

表达HBHA和hIL-12融合蛋白的重组耻垢分枝杆菌对结核分枝杆菌感染小鼠的免疫治疗作用

评价表达HBHA和hIL12融合蛋白的重组耻垢分枝杆菌用于结核分枝杆菌感染小鼠的免疫治疗效果.结核分枝杆菌H37Rv感染小鼠4周后,用表达HBHA和hIL12融合蛋白的重组耻垢分枝杆菌免疫治疗,检测免疫小鼠肺部荷菌量和组织病理变化.重组耻垢分枝杆菌可有效控制感染小鼠肺部结核分枝杆菌的荷菌量,减轻病理损伤.但在降低肺部荷菌量方面不如化疗药物.表达HBHA和hIL12融合蛋白的重组耻垢分枝杆菌可有效控制结核分枝杆菌在小鼠体内的增殖,可能成为联合化疗药物控制结核病的有效候选疫苗.     

含油污泥微生物堆制处理研究

采用微生物堆制法对延长油田含油污泥进行处理,以除油率为指标,分别研究了不同菌种、接种量、调理剂及堆制时间等因素对处理效果的影响.结果表明:增加堆制时间与接种量,除油率提高.经过35 d堆制处理,YC-1菌与YC-13菌的除油率分别达43.41%和54.02%.YC-1菌采用土作为调理剂时除油率为40.01%,优于采用土+荞麦皮的19.05%,而YC-3菌采用土+荞麦皮作为调理剂时除油率为32.55%,略优于采用土作为调理剂的25.38%.     

Immune control of HIV-1 after early treatment of acute infection

Virus-specific T-helper cells are considered critical for the control of chronic viral infections. Successful treatment of acute HIV-1 infection leads to augmentation of these responses, but whether this enhances immune control has not been determined. We administered one or two supervised treatment interruptions to eight subjects with treated acute infection, with the plan to restart therapy if viral load exceeded 5,000 copies of HIV-1 RNA per millilitre of plasma (the level at which therapy has been typically recommended) for three consecutive weeks, or 50,000 RNA copies per ml at one time. Here we show that, despite rebound in viraemia, all subjects were able to achieve at least a transient steady state off therapy with viral load below 5,000 RNA copies per ml. At present, five out of eight subjects remain off therapy with viral loads of less than 500 RNA copies per ml plasma after a median 6.5 months (range 5-8.7 months). We observed increased virus-specific cytotoxic T lymphocytes and maintained T-helper-cell responses in all. Our data indicate that functional immune responses can be augmented in a chronic viral infection, and provide rationale for immunotherapy in HIV-1 infection.     

Selective expression of an antigen receptor on CD8-bearing T lymphocytes in transgenic mice

The major problem in the study of T-cell development is that of tracking thymocytes of a given specificity. Recent studies have exploited natural correlations between the expression of a particular V beta gene segment and T-cell receptor (TCR) specificity. We and others (refs 5, 6 and M. Davis, personal communication) have taken an alternative approach. We have generated transgenic mice expressing the alpha beta antigen receptor from the cytotoxic T-lymphocyte clone 2C (ref. 7). In transgenic mice of the same haplotype as the 2C clone, the 2C TCR was expressed on 20-95% of peripheral T cells. Very few of these T cells carried the CD4 antigen; the vast majority were CD4-CD8+ and were able to lyse targets with the same specificity as the original 2C clone. These results indicate that the alpha beta heterodimer transfers specificity to recipient cells as expected from earlier studies, and that receptor specificity in T-cell repertoire selection is determined by both alpha beta heterodimer and CD4 or CD8 accessory molecules.