Murine I-A beta chain polymorphism: nucleotide sequences of three allelic I-A beta genes

The polymorphism of immune response genes plays a critical role in determining the immune capabilities of a particular individual. The molecular nature of this polymorphism was studied by examining the structure of the coding portions of three alleles of the I-A beta chain gene, an immune response gene whose protein product constitutes a subunit of the I-A molecule. Comparison of the I-A beta chains encoded by these alleles revealed an amino acid sequence divergence of 5 to 8 percent. The differences were found to be a series of short alterations clustered in the amino terminal half of the polypeptide.     

Role of Na+/H+ exchange by interferon-gamma in enhanced expression of JE and I-A beta genes

The rapid transductional sequences initiated by interferon-gamma (IFN-gamma) on binding to its receptor regulate functional and genomic responses in many cells but are not well defined. Induction of macrophage activation is an example of such functional and genomic changes in response to IFN-gamma. Addition of IFN-gamma to murine macrophages, at activating concentrations, produced rapid (within 60 seconds) alkalinization of the cytosol and a concomitant, rapid influx of 22Na+. Amiloride inhibited the ion fluxes and the accumulation of specific messenger RNA for two genes induced by IFN-gamma (the early gene JE and the beta chain of the class II major histocompatibility complex gene I-A). The data indicate that IFN-gamma initiates rapid exchange of Na+ and H+ by means of the Na+/H+ antiporter and that these amiloride-sensitive ion fluxes are important to some of the genomic effects of IFN-gamma.     

T-cell recognition of Ia molecules selectively altered by a single amino acid substitution

T lymphocytes recognize foreign antigen together with allele-specific determinants on membrane-bound class I and class II (Ia) gene products of the major histocompatibility complex. To identify amino acids of class II molecules critical to this recognition process, the genes encoding the beta chains of the I-Ak molecule were cloned from a wild-type B-cell hybridoma and from an immunoselected variant subline showing distinct serological and T-cell stimulatory properties. Nucleotide sequencing and DNA-mediated gene transfer established that a single base transition (G----A) encoding a change from glutamic acid to lysine at position 67 in the I-Ak beta molecule accounted for all the observed phenotypic changes of the variant cells. These results confirm the importance of residues 62 to 78 in the amino terminal domain of I-A beta for class II-restricted T-cell recognition of antigen and demonstrate the ability of a single substitution in this region to alter this recognition event.     

Structural heterogeneity and functional domains of murine immunoglobulin G Fc receptors

Binding of antibodies to effector cells by way of receptors to their constant regions (Fc receptors) is central to the pathway that leads to clearance of antigens by the immune system. The structure and function of this important class of receptors on immune cells is addressed through the molecular characterization of Fc receptors (FcR) specific for the murine immunoglobulin G isotype. Structural diversity is encoded by two genes that by alternative splicing result in expression of molecules with highly conserved extracellular domains and different transmembrane and intracytoplasmic domains. The proteins encoded by these genes are members of the immunoglobulin supergene family, most homologous to the major histocompatibility complex molecule E beta. Functional reconstitution of ligand binding by transfection of individual FcR genes demonstrates that the requirements for ligand binding are encoded in a single gene. These studies demonstrate the molecular basis for the functional heterogeneity of FcR's, accounting for the possible transduction of different signals in response to a single ligand.     

Depletion of CD4+ T cells in major histocompatibility complex class II-deficient mice

The maturation of T cells in the thymus is dependent on the expression of major histocompatibility complex (MHC) molecules. By disruption of the MHC class II Ab beta gene in embryonic stem cells, mice were generated that lack cell surface expression of class II molecules. These MHC class II-deficient mice were depleted of mature CD4+ T cells and were deficient in cell-mediated immune responses. These results provide genetic evidence that class II molecules are required for the maturation and function of mature CD4+ T cells.     

The NOD mouse: recessive diabetogenic gene in the major histocompatibility complex

Examination of the histocompatibility region of the nonobese diabetic (NOD) mouse with antibodies against class II glycoproteins (products of immune response genes of the major histocompatibility complex I-A and I-E), hybrid T-cell clones, and mixed-lymphocyte cultures and analysis of restriction fragment length polymorphisms indicate that the NOD mouse has a unique class II major histocompatibility complex with no expression of surface I-E, no messenger RNA for I-E alpha, and an I-A not recognized by any monoclonal antibodies or hybrid T-cell clones studied. In crosses of NOD mice with control C3H mice, the development of diabetes was dependent on homozygosity for the NOD mouse's unique major histocompatibility region.     

Viral persistence in neurons explained by lack of major histocompatibility class I expression

Viruses frequently persist in neurons, suggesting that these cells can evade immune surveillance. In a mouse model, 5 x 10(6) cytotoxic T lymphocytes (CTLs), specific for lymphocytic choriomeningitis virus (LCMV), did not lyse infected neurons or cause immunopathologic injury. In contrast, intracerebral injection of less than 10(3) CTL caused disease and death when viral antigens were expressed on leptomeningeal and choroid plexus cells of the nervous system. The neuronal cell line OBL21 expresses little or no major histocompatibility (MHC) class I surface glycoproteins and when infected with LCMV, resisted lysis by virus-specific CTLs. Expression of MHC heavy chain messenger RNA was limited, but beta 2-microglobulin messenger RNA and protein was made normally. OBL21 cells were made sensitive to CTL lysis by transfection with a fusion gene encoding another MHC class I molecule. Hence, neuronal cells probably evade immune surveillance by failing to express MHC class I molecules.     

A structural framework for deciphering the link between I-Ag7 and autoimmune diabetes

Susceptibility to murine and human insulin-dependent diabetes mellitus correlates strongly with major histocompatibility complex (MHC) class II I-A or HLA-DQ alleles that lack an aspartic acid at position beta57. I-Ag7 lacks this aspartate and is the only class II allele expressed by the nonobese diabetic mouse. The crystal structure of I-Ag7 was determined at 2.6 angstrom resolution as a complex with a high-affinity peptide from the autoantigen glutamic acid decarboxylase (GAD) 65. I-Ag7 has a substantially wider peptide-binding groove around beta57, which accounts for distinct peptide preferences compared with other MHC class II alleles. Loss of Asp(beta57) leads to an oxyanion hole in I-Ag7 that can be filled by peptide carboxyl residues or, perhaps, through interaction with the T cell receptor.     

Molecular analysis of the hotspot of recombination in the murine major histocompatibility complex

Biological and serological assays have been used to define four subregions for the I region of the major histocompatibility complex (MHC) in the order I-A, I-B, I-J, and I-E. The I-J subregion presumably encodes the I-J polypeptide of the elusive T-cell suppressor factors. Restriction enzyme site polymorphisms and DNA sequence analyses of the I region from four recombinant mouse strains were used to localize the putative I-B and I-J subregions to a 1.0-kilobase (kb) region within the E beta gene. Sequencing this region from E beta clones derived from the two mouse strains: B10.A(3R), I-Jb and B10.A(5R), I-Jk initially used to define the I-J subregion revealed that these regions are identical, hence the distinct I-Jb and I-Jk molecules cannot be encoded by this DNA. In addition, the DNA sequence data also refute the earlier mapping of the I-B subregion. Analysis of the DNA sequences of three parental and four I region recombinants reveals that the recombinant events in three of the recombinant strains occurred within a 1-kb region of DNA, supporting the proposition that a hotspot for recombination exists in the I region. The only striking feature of this hotspot is a tetramer repeat (AGGC)n that shows 80 percent homology to the minisatellite sequence which may facilitate recombination in human chromosomes.     

Activation of gamma delta T cells in the primary immune response to Mycobacterium tuberculosis

Although the immunologic role of T cells bearing the conventional alpha beta T cell receptor (TCR) has been well characterized, little is known about the function of the population of T cells bearing the gamma delta TCR. Therefore, the role of gamma delta T cells in the immune response to Mycobacterium tuberculosis (MT) was investigated. The number of TCR gamma delta cells in the draining lymph nodes of mice immunized with MT was greatly increased in comparison with the number of TCR alpha beta cells. Three biochemically distinct gamma delta TCRs were detected. Analyses of cell cycle, of interleukin-2 receptor expression, and of interleukin-2 responsiveness showed that a large proportion of the gamma delta T cells were activated in vivo. TCR gamma delta cells responded to solubilized MT antigens in vitro but, in contrast to MT-specific alpha beta T cells, the response of gamma delta T cells to MT did not require major histocompatability complex class II recognition. These results provide an example of antigen-specific activation of gamma delta T cells in vivo and indicate that gamma delta T cells may have a distinct role in generating a primary immune response to certain microorganisms.     

Requirement for cotolerogenic gene products in the clonal deletion of I-E reactive T cells

T cells that express the T cell receptor V beta 5.2 domain react with the class II major histocompatibility complex (MHC) molecule I-E, and V beta 5.2+ T cells are deleted in mouse strains that express I-E glycoproteins. By examination of genetically defined recombinant inbred (RI) mouse strains, it was found that the deletion was dependent on the expression of I-E and one of a limited number of non-MHC gene products (cotolerogens). The gene encoding one of these cotolerogens maps to chromosome 12 and is linked to the endogenous provirus Mtv-9. These observations suggest that the I-E-mediated and minor lymphocyte-stimulating antigen (Mls)-mediated deletions of alpha beta T cells from the repertoire are similar; both require the expression of a class II MHC glycoprotein and a second non-MHC gene product.     

A large deletion within the T-cell receptor beta-chain gene complex in New Zealand white mice

The T-cell receptor beta-chain gene complex contains a duplication of D beta, J beta, and C beta gene segments in mice and man. When DNA from many inbred strains of mice was screened an unusual allele of the beta locus was identified in New Zealand White (NZW) mice. This allele is distinguished by the deletion of an 8.8-kilobase segment of DNA containing C beta 1, D beta 2 and the J beta 2 cluster. Despite the fact that all NZW T-cell receptors must be derived from a single set of beta-chain gene segments, this strain has functional T cells and is phenotypically normal. This deletion of T-cell receptor beta-chain segments occurs in a strain known to contribute to lupus-like autoimmune disease.     

Molecular characterization of the human beta 3-adrenergic receptor

Since the classification of beta-adrenergic receptors (beta-ARs) into beta 1 and beta 2 subtypes, additional beta-ARs have been implicated in the control of various metabolic processes by catecholamines. A human gene has been isolated that encodes a third beta-AR, here referred to as the "beta 3-adrenergic receptor." Exposure of eukaryotic cells transfected with this gene to adrenaline or noradrenaline promotes the accumulation of adenosine 3',5'-monophosphate; only 2 of 11 classical beta-AR blockers efficiently inhibited this effect, whereas two others behaved as beta 3-AR agonists. The potency order of beta-AR agonists for the beta 3-AR correlates with their rank order for stimulating various metabolic processes in tissues where atypical adrenergic sites are thought to exist. In particular, novel beta-AR agonists having high thermogenic, antiobesity, and antidiabetic activities in animal models are among the most potent stimulators of the beta 3-AR.     

h1-calponin基因对五指山小型猪骨髓间充质干细胞体外成骨分化的影响

【目的】通过碱性调宁蛋白（h1-calponin）基因与五指山小型猪骨髓间充质干细胞（bone marrow mesenchymal stem cells,BM MSCs）成骨分化关系的研究,探讨成骨分化机制。【方法】添加成骨分化液对BM MSCs进行成骨分化诱导,采用茜素红染色的方法鉴定细胞成骨分化的程度;通过定量PCR分析h1-calponin基因及成骨相关基因骨桥蛋白（osteopotin,OPN）、骨钙蛋白（osteocalcin,OCN）、核心结合因子α1（core binding factor a1,Cbfα1）及核心结合因子β（core binding factor beta,Cbfβ）表达谱。【结果】①随着诱导时间的延长,茜素红染色矿化结节逐渐增多;②成骨标志基因OPN和OCN表达趋势一致：诱导0至14 d,基因表达量呈增长趋势,诱导14 d表达量最高,而后下降;成骨相关基因Cbfα1和Cbfβ表达量于第7天达到最高峰,之后逐渐下降,但仍高于诱导前;③h1-calponin基因表达量随着诱导的延续逐渐下降;诱导14—21 d时,该基因基本不表达。【结论】h1-calponin基因的表达与五指山小型猪骨髓间充质干细胞体外成骨分化存在着负相关,可能是成骨分化的负调控基因。     

TGF-β/Smad信号通路在Follistatin调节鸭骨骼肌卫星细胞增殖过程中的作用机制

【目的】卵泡抑素（Follistatin）能够调节骨骼肌肥大和脂肪沉积,可促进骨骼肌卫星细胞增殖。拟采用体外重组 Follistatin处理增殖期的鸭骨骼肌卫星细胞,阐明TGF-β/Smad信号通路在Follistatin调节鸭骨骼肌卫星细胞增殖过程中的作用机制。【方法】以孵化14 d的鸭胚为试验材料,采用差速贴壁的方法分离骨骼肌卫星细胞,待细胞长到70%-80%时,将培养基换成含有浓度分别为0、1、10、100 ng·mL^-1的Follistatin培养基,继续培养36 h后,采用CCK-8检测骨骼肌卫星细胞增殖情况;使用抗pax7抗体染色,DAPI染核,鉴定骨骼肌卫星细胞;采用real-time qPCR方法检测Follistatin对骨骼肌卫星细胞增殖过程中的标记基因PCNA、生肌因子基因MyoD和TGF-β信号通路中TGF-β、Smad2和Smad3的表达的影响。【结果】在倒置显微镜下观察,传代培养12 h鸭骨骼肌细胞一部分未贴壁呈圆形,一部分贴壁呈梭形。24 h后细胞全部贴壁,细胞略有变长。2 d后细胞继续增多,且呈长梭形。3 d后细胞数目增加,个别细胞融合。4 d后细胞数目进一步增加,细胞变粗,个别细胞融合。5 d后有少量细胞开始分化,细胞进一步融合。Pax7免疫荧光染色分析显示,95%以上的细胞中Pax7呈阳性表达;CCK-8检测细胞增殖分析表明,不同浓度的Follistatin处理鸭骨骼肌卫星细胞后,各处理组细胞增殖均显著高于对照组(P＜0.01),且10 ng·mL^-1 Follistatin处理鸭骨骼肌卫星细胞增殖效果最明显,为最佳处理浓度;与对照组相比,10 ng·mL^-1 Follistatin处理组的MyoD基因表达量显著下降(P＜0.05),PCNA基因表达量显著升高(P＜0.05),Myf5基因表达量显著升高,TGF-β和Smad2基因表达显著升高(P＜0.05),且Smad3基因表达量极限著升高(P＜0.01);Western blotting检测蛋白表达水平结果表明,与对照组相比,TGF-β、Smad2 和Smad3磷酸化水平也显著升高。【结论】10 ng·mL^-1 Follistatin能显著促进鸭骨骼肌卫星细胞增殖,这一过程可通过TGF-β/Smad信号通路实现。使用最佳Follistatin处理浓度能够显著促进鸭骨骼肌卫星细胞增殖,该研究为鸭骨骼肌生长发育调控机理研究奠定分子基础。     

Type, density, and location of immune cells within human colorectal tumors predict clinical outcome

The role of the adaptive immune response in controlling the growth and recurrence of human tumors has been controversial. We characterized the tumor-infiltrating immune cells in large cohorts of human colorectal cancers by gene expression profiling and in situ immunohistochemical staining. Collectively, the immunological data (the type, density, and location of immune cells within the tumor samples) were found to be a better predictor of patient survival than the histopathological methods currently used to stage colorectal cancer. The results were validated in two additional patient populations. These data support the hypothesis that the adaptive immune response influences the behavior of human tumors. In situ analysis of tumor-infiltrating immune cells may therefore be a valuable prognostic tool in the treatment of colorectal cancer and possibly other malignancies.     

Normal development of mice deficient in beta 2M, MHC class I proteins, and CD8+ T cells

Major histocompatibility class I proteins display viral and self antigens to potentially responsive cells and are important for the maturation of T cells; beta 2-microglobulin (beta 2M) is required for their normal expression. Mouse chimeras derived from embryonic stem cells with a disrupted beta 2M gene transmitted the inactivated gene to their progeny. Animals homozygous for the mutated beta 2M gene were obtained at expected frequencies after further breeding. The homozygotes appeared normal, although no class I antigens could be detected on their cells and the animals are grossly deficient in CD4- CD8+ T cells, which normally mediate cytotoxic T cell function.     

Location of gene for beta subunit of human T-cell receptor at band 7q35, a region prone to rearrangements in T cells

The T-cell receptor is formed by two chains, alpha and beta, for which specific clones were recently obtained. In this report the gene for the beta chain of the human T-cell receptor was located on the long arm of chromosome 7, band q35, by means of in situ hybridization. This chromosome region in T cells is unusually prone to develop breaks in vivo, perhaps reflecting instability generated by somatic rearrangement of T-cell receptor genes during normal differentiation in this cell lineage.     

cDNA expression cloning of the IL-1 receptor, a member of the immunoglobulin superfamily

Interleukin-1 alpha and -1 beta (IL-1 alpha and IL-1 beta) are cytokines that participate in the regulation of immune responses, inflammatory reactions, and hematopoiesis. A direct expression strategy was used to clone the receptor for IL-1 from mouse T cells. The product of the cloned complementary DNA binds both IL-1 alpha and IL-1 beta in a manner indistinguishable from that of the native T cell IL-1 receptor. The extracellular, IL-1 binding portion of the receptor is 319 amino acids in length and is composed of three immunoglobulin-like domains. The cytoplasmic portion of the receptor is 217 amino acids long.     

Isotypic exclusion of gamma delta T cell receptors in transgenic mice bearing a rearranged beta-chain gene

The rearrangement of T cell antigen receptor beta- and gamma-chain gene segments was studied in transgenic mice that bear a functional beta-chain gene. Virtually all CD3-positive T cells derived from transgenic mice express beta chains containing the transgene-encoded V beta 8.2 variable region on their surfaces and do not express endogenous beta-chain variable regions. Expression of endogenous V beta genes is inhibited at the level of somatic recombination during thymic ontogeny. Furthermore, rearrangements of the TCR gamma-chain genes are also markedly inhibited in these transgenic animals. Hence expression of the TCR beta transgene has led to allelic exclusion of alpha beta receptors and isotypic exclusion of gamma delta T cell receptors.