The need for central and peripheral tolerance in the B cell repertoire

The immune system normally avoids producing antibodies that react with autologous ("self") antigens by censoring self-reactive T and B cells. Unlike the T cell repertoire, antibody diversity is generated within the B cell repertoire in two phases; the first occurs by gene rearrangement in primary lymphoid organs, and the second phase involves antigen-driven hypermutation in peripheral lymphoid organs. The possibility that distinct cellular mechanisms may impose self tolerance at these two different phases of B cell diversification may explain recent findings in transgenic mouse models, in which self-reactive B cells appear to be silenced both by functional inactivation and by physical elimination.     

Direct polyclonal activation of human B lymphocytes by the acquired immune deficiency syndrome virus

When B lymphocytes from normal human peripheral blood were incubated for 1 hour with the retrovirus that causes the acquired immune deficiency syndrome (AIDS), the B cells showed marked proliferation and differentiation. Proliferative responses to the virus peaked on day 4 and appeared to be independent of accessory cells. This finding was repeated with three separate viral isolates, one of which was from a patient from Zaire. The magnitude of the observed responses was comparable to that seen with standard polyclonal B-cell activators. This phenomenon may be at least partially responsible for the polyclonal B-cell activation seen in patients with AIDS.     

A primitive T cell-independent mechanism of intestinal mucosal IgA responses to commensal bacteria

The immunoglobulin A (IgA) is produced to defend mucosal surfaces from environmental organisms, but host defenses against the very heavy load of intestinal commensal microorganisms are poorly understood. The IgA against intestinal commensal bacterial antigens was analyzed; it was not simply "natural antibody" but was specifically induced and responded to antigenic changes within an established gut flora. In contrast to IgA responses against exotoxins, a significant proportion of this specific anti-commensal IgA induction was through a pathway that was independent of T cell help and of follicular lymphoid tissue organization, which may reflect an evolutionarily primitive form of specific immune defense.     

鸵鸟皮肤抗菌肽对雏鸡免疫器官指数及T淋巴细胞数量的影响初探

为了观察非洲鸵鸟皮肤抗菌肽对雏鸡免疫器官指数及T淋巴细胞数量的影响,进而研究其对雏鸡免疫系统的调节能力.选取50只1日龄健康雏鸡,随机分为两组:试验组(T)从1日龄开始每天在饮水中加入浓度为1μg/ml的鸵鸟皮肤抗菌肽提取物,对照组(C)饮水中不添加抗菌肽提取物,分别于1、4、7、10、17日龄时随机抽取5只雏鸡称重后处死,采取免疫器官称重并运用ANAE染色方法测定免疫器官T淋巴细胞数量.结果发现抗菌肽能够提高10-17日龄雏鸡体重,提高4～7日龄雏鸡的免疫器官指数及7日龄免疫器官的T淋巴细胞数量(P<0.05).结果表明饮水中添加非洲鸵鸟皮肤抗菌肽可以促进雏鸡免疫器官发育,增强机体的细胞免疫.     

Human monoclonals from antigen-specific selection of B lymphocytes and transformation by EBV

Hybridoma technology has made it possible to prepare monoclonal antibodies with the use of murine lymphocytes. Attempts to extend this technology to the human level, however, have met with difficulties. A method has been developed for making human monoclonal antibodies of predetermined specificity. Biotinylated antigens (human thyroglobulin or tetanus toxoid) were incubated with human B lymphocytes from peripheral blood. The lymphocytes to which the antigens bound were selected by fluorescence-activated cell sorting. Positively selected (high fluorescence) and negatively selected (low fluorescence) cells were then transformed with Epstein-Barr virus (EBV) and grown in microculture wells. All wells from the positively selected fraction produced antigen-specific antibody (95 to 1800 nanogram-equivalents per milliliter), whereas fewer than 6% of the wells from negatively selected fraction made any detectable antibody (less than 10 nanogram-equivalents per milliliter). When the positively selected EBV-transformed cells were cultured in limiting dilution, clones were obtained that made antigen-specific monoclonal antibodies. By this method, monoclonal antibodies to both foreign antigens and autoantigens can be prepared from the normal human B-cell repertoire.     

B细胞活化因子及调节性T细胞在免疫性血小板减少症发病中的作用

目的 了解B细胞活化因子(BAFF)及调节性T(Treg)细胞在免疫性血小板减少症(ITP)发病中的作用.方法 采集34例ITP及28例健康对照者外周血,用流式细胞术检测外周血Treg细胞数,ELISA检测血浆BAFF水平.结果 ITP患者外周血Treg细胞/CD4+细胞比值低于对照者[(1.55±1.13)％ vs (2.73±0.83)％,P＜0.01],而血浆BAFF水平高于对照者[(1 426.14±1 280.67) μg/L vs (644.29±307.97) μg/L,P＜0.01].结论 BAFF与Treg细胞可能存在相互作用,从而参与ITP发病.     

金针菇菇脚对肉鸡T、B淋巴细胞免疫功能的影响

为探索不同含量金针菇菇脚(Flammulina velutipes stembase,FVS)添加到肉鸡饲粮中对肉鸡生产性能以及T、B淋巴细胞免疫功能的影响,采用1日龄健康AA肉鸡270只,随机分为5组,每组3个重复,每个重复18只。试验分为:对照组(基础日粮);抗生素组(基础日粮+0.05%黄霉素);低剂量组(基础日粮+2%FVS);中剂量组(基础日粮+4%FVS);高剂量组(基础日粮+6%FVS)。试验为期42d,检测指标为:生产性能,免疫器官指数,外周血T、B淋巴细胞增殖率(MTT法测定),血清细胞因子IL-2、IL-4、IFN-γ含量测定(ELISA法测定),血清免疫球蛋白IG-A、IG-G、IG-M效价测定(ELISA法测定)。结果显示:各剂量FVS均能提高肉鸡饲喂前期平均日增重,平均日采食量,降低料肉比,提高肉鸡成活率;2%FVS组21日龄胸腺指数及42日龄法氏囊指数显著高于对照组(P0.05),4%FVS组胸腺指数显著高于对照组(P0.05),6%FVS组在42日龄胸腺指数及脾脏指数均显著高于对照组(P0.05);在第22~28、29~35、36~42日龄,各剂量FVS组T、B细胞增殖率显著高于对照组(P0.05);在第22~28、36~42日龄,各剂量FVS均可显著提高细胞因子IL-2的含量(P0.05);各剂量FVS能显著提高22~28、36~42日龄肉鸡血清免疫球蛋白IG-A效价(P0.05)以及7~14、22~28日龄IG-G效价(P0.05)。表明日粮中添加FVS可提高肉鸡生产性能、免疫器官指数,增强肉鸡的T、B淋巴细胞免疫功能。     

Mitosis: induction by cultures of human peripheral lymphocytes

Ribosomal RNA extracted from peripheral lymphocytes, which had been recently stimulated by specific antigens to which the donor was sensitized, is capable of promoting transformation and mitosis when added to cultures of autologous unstimulated lymphocytes.     

Dendritic cell apoptosis in the maintenance of immune tolerance

Apoptosis in the immune system is critical for maintaining self-tolerance and preventing autoimmunity. Nevertheless, inhibiting apoptosis in lymphocytes is not alone sufficient to break self-tolerance, suggesting the involvement of other cell types. We investigated whether apoptosis in dendritic cells (DCs) helps regulate self-tolerance by generating transgenic mice expressing the baculoviral caspase inhibitor, p35, in DCs (DC-p35). DC-p35 mice displayed defective DC apoptosis, resulting in their accumulation and, in turn, chronic lymphocyte activation and systemic autoimmune manifestations. The observation that a defect in DC apoptosis can independently lead to autoimmunity is consistent with a central role for these cells in maintaining immune self-tolerance.     

正常猪外周血淋巴细胞cDNA文库构建及EST初步分析

【目的】构建猪淋巴细胞基因文库,初步绘制正常猪外周血淋巴细胞的基因表达谱,为进一步筛选免疫相关基因提供平台。【方法】以mRNA为模板,经反转录酶催化,在体外反转录成cDNA,与质粒载体连接后转化大肠杆菌,进一步扩增后,获得猪外周血淋巴细胞全部mRNA信息的cDNA克隆集合,并进行序列分析和注释。【结果】构建了正常猪外周血淋巴细胞cDNA文库,获得库容量为1．2×10^6PFU／mL、重组率为93．3％、85％插入片段处于750～2000bp的高质量文库;随机测定1100条ESTs,经拼接和聚类,获得152条高表达的基因重叠群（Contigs）和619条低表达基因——单拷贝的EST（Singletons）;经序列比对分析,发现23．3％ESTs为与任何物种都没有匹配的新基因,75．9％ESTs为与猪没有匹配的新基因。用GO数据库对获得的EST进行基因功能分类和KEGG路径图分析,发现丝裂原活化蛋白激酶途径（Mitogenn—activated protein kinase,MAPK）、钙信号通路、胰岛素信号通路、脂肪细胞因子信号通路、Toll—like受体信号通路、B细胞受体信号通路和T细胞受体信号通路的基因均有较高表达。【结论】大部分的猪外周血淋巴细胞基因尚未被分离和鉴定出来,但这些基因mRNA转录和表达丰度均较高,且在猪外周血淋巴细胞中起着非常重要的作用。     

Identification of a T helper cell-derived lymphokine that activates resting T lymphocytes

A novel lymphokine with apparent molecular size of 10 to 12 kilodaltons is secreted from helper T cell clones within hours after cross-linking their T cell antigen-MHC (major histocompatibility complex) receptors (T3-Ti). This lymphokine, termed interleukin-4A (IL-4A), stimulates resting lymphocytes by binding to a surface component (or components) of the alternative T11 pathway and subsequently by inducing interleukin-2 (IL-2) receptors. The activation process is neither dependent on antigen specificities of the recruited population or the presence of macrophages. It appears, therefore, that IL-4A is a mediator involved in amplifying the T cell immune response.     

Downregulation of L3T4+ cytotoxic T lymphocytes by interleukin-2

Proliferation of activated cytotoxic T lymphocytes (CTLs) that recognize foreign histocompatibility antigens is induced by interleukin-2, a potent immunoregulatory molecule originally described as T cell growth factor. Interleukin-2 (IL-2) is widely used to isolate and induce clonal expansion of CTLs for functional studies in vitro and in vivo. However, in studies with CTLs specific for class I and class II histocompatibility antigens, IL-2 rapidly downregulated the lytic activity of some class II-specific CTLs in a time- and dose-dependent manner. Lytic activity of L3T4+ CTLs specific for the murine class II antigen I-Ek was repeatedly up- and downregulated in vitro by alternate exposure to specific (alloantigen) and nonspecific (recombinant IL-2) signals, respectively. These results demonstrate that some CTLs modulate their functional property (cytolysis) while undergoing IL-2-driven cell proliferation without loss of antigen specificity or ability to revert to a lytic phenotype.     

Organogenic role of B lymphocytes in mucosal immunity

Follicle-associated epithelium (FAE) in the intestinal Peyer's patches contains M cells that deliver pathogens to organized lymphoid tissue. Development of Peyer's patches, FAE, and M cells was found to be impaired in mice that had no B cells. Transgenic expression of membrane-bound immunoglobulin M restored B cells and FAE development. The lack of M cells abrogated infection with a milk-borne retrovirus. Thus, in addition to secretion of antibodies and presentation of antigens, B cells are important for organogenesis of the mucosal immune barriers.     

Linear differentiation of cytotoxic effectors into memory T lymphocytes

A central question in immunology is the origin of long-lived T cell memory that confers protection against recurrent infection. The differentiation of na?ve T cell receptor transgenic CD8+ cells into effector cytotoxic T lymphocytes (CTLs) and memory CD8+ cells was studied. Memory CD8+ cells that were generated after strong antigenic stimulation were the progeny of cytotoxic effectors and retained antigen-specific cytolytic activity 10 weeks after adoptive transfer to antigen-free recipient mice. Thus, potential vaccines based on CTL memory will require the differentiation of na?ve cells into post-effector memory T cells.     

Mitogen-induced replication of woodchuck hepatitis virus in cultured peripheral blood lymphocytes

Peripheral blood lymphocytes (PBLs) isolated from woodchucks chronically infected with the woodchuck hepatitis virus (WHV) carry low levels of nonreplicating WHV DNA. When PBLs from chronic carrier woodchucks were activated in culture with the generalized mitogen lipopolysaccharide (LPS), WHV DNA replication was initiated in cells obtained from one of three animals examined. Intracellular WHV core particles, containing WHV DNA replication intermediates, RNA/DNA hybrid molecules, and an active endogenous DNA polymerase, appeared 3 days after the start of LPS stimulation. After 5 to 7 days of LPS stimulation, WHV DNA-containing particles, which displayed the properties of intact, mature virions, were released into the culture medium. These studies provide evidence for reactivation of a latent WHV infection of circulating lymphoid cells and indicate that the presence of nonreplicating hepadnaviral DNA in lymphoid cells represents a potentially active infection following cellular activation.     

正常猪外周血淋巴细胞cDNA文库部分EST序列的电子克隆及验证

【目的】探讨从EST序列电子克隆全长基因序列的有效性,为今后快速克隆和研究免疫相关基因提供新方法。【方法】以正常猪外周血淋巴细胞cDNA文库测序所得的EST序列作为起始材料,应用生物信息库对文库中的部分EST序列进行电子克隆,并用RT-PCR验证其准确性。【结果】电子克隆正常猪外周血淋巴细胞cDNA文库序列,共获得20条片段重叠群（Contigs）,经基因注释,证实其均得到了有效延伸,具有完整开放阅读框（ORF）,其中已知的猪基因序列14条,猪新基因序列6条;从已知猪基因序列和未知猪新基因序列中各抽取1条感兴趣序列（ESTcontig110和ESTcontig133）进行全长cDNA预测,发现电子克隆所得序列ESTcontig110和ESTcontig133的预期扩增片段（ORF）与实际扩增片段（ORF）基本一致,核苷酸相似性在98.0%以上,氨基酸相似性在93.0%以上。而对ESTcontig133的ORF序列（猪的60S核糖体蛋白L18a亚基）进行基本特性及结构预测,发现ORF133蛋白为非分泌型蛋白,由6个折叠和6个螺旋结构组成,含有8个不同的磷酸化位点。【结论】从EST序列电子克隆全长基因是可行的,为今后快速克隆和研究免疫相关基因提供了参考。     

HIV-1 production from infected peripheral blood T cells after HTLV-I induced mitogenic stimulation

The human immunodeficiency virus type 1 (HIV-1) and human T-cell leukemia virus type I (HTLV-I) are two distinct human retroviruses that infect T cells. Recent epidemiologic studies have identified a cohort of individuals that are coinfected with both viruses. It is reported here that human peripheral blood leukocytes infected with HIV-1 in vitro can be induced to produce large quantities of HIV-1 after mitogenic stimulation by noninfectious HTLV-I virions. It is also shown that HTLV-I virions may exert this effect prior to, immediately following, or well after the cells are infected with HIV-1. These results provide further impetus for epidemiologic studies of dually infected individuals to determine whether HTLV-I may act as a cofactor for acquired immunodeficiency syndrome (AIDS).     

Detection of human cytomegalovirus in peripheral blood lymphocytes in a natural infection

In situ hybridization was used to detect human cytomegalovirus (HCMV) in the peripheral blood mononuclear cells of some naturally infected (seropositive) individuals. A subpopulation of cells hybridized specifically to a portion of the HCMV genome that is heavily transcribed during the immediate-early period of infection. The hybridization signal was markedly reduced by base hydrolysis and ribonuclease, and therefore the probe appears to be detecting viral RNA. A fluorescence-activated cell sorter was used to select lymphocytes bearing the OKT4 and OKT8 markers. Hybridization with the HCMV probe revealed a higher proportion of positive cells in the OKT4 than in the OKT8 subset. This observation specifically identifies lymphocytes as a cell population involved in natural HCMV infection and suggests that lymphocytes may be a reservoir for maintaining infection and may also serve as a vehicle for its spread by blood transfusion.     

Altered K+ channel expression in abnormal T lymphocytes from mice with the lpr gene mutation

The observation that voltage-dependent K+ channels are required for activation of human T lymphocytes suggests that pathological conditions involving abnormal mitogen responses might be reflected in ion channel abnormalities. Gigaohm seal techniques were used to study T cells from MRL/MpJ-lpr/lpr mice; these mice develop generalized lymphoproliferation of functionally and phenotypically abnormal T cells and a disease resembling human systemic lupus erythematosus. The number and predominant type of K+ channels in T cells from these mice differ dramatically from those in T cells from control strains and a congenic strain lacking the lpr gene locus. Thus an abnormal pattern of ion channel expression has now been associated with a genetic defect in cells of the immune system.     

T7噬菌体DNA的提取及其反向遗传拯救方法的建立

从T7噬菌体培养液中粗提噬菌体颗粒,经热裂解后用苯酚、氯仿抽提进而获得纯净的T7噬菌体DNA。用PCR、酶切法鉴定T7噬菌体DNA的完整性。通过对不同感受态细菌浓度、T7噬菌体DNA用量、电转化电压条件的优化,建立了T7噬菌体反向遗传拯救方法。结果显示,提取的DNA结构完整,能够被特异性酶切割,多克隆位点序列正确。T7噬菌体的反向遗传拯救方法最优化条件为200 ng T7噬菌体DNA、1 ml 5×109感受态细菌、1.5 kV电转化电压,在此条件下获得的拯救效率为3.5×105 PFU/ng(DNA)。