MHC-chromosome dosage effects: evidence for increased expression of Ia glycoprotein and alteration of B cell subpopulations in neonatal aneuploid chickens

Quantitative variation in the expression of MHC-encoded class II (Ia) glycoproteins has been associated with stages of lymphocyte development and a number of disease conditions. We have used an avian MHC dosage model to study the regulation of Ia expression and the effects of quantitative variation in membrane Ia on B-cell development. Lymphocyte membrane expression of Ia glycoprotein molecules and the frequency of small-versus-large lymphocytes were examined in trisomic line chickens containing either two (disomic), three (trisomic), or four (tetrasomic) copies of the microchromosome encoding the MHC. This was accomplished by quantitative laser flow cytometry analysis of bursa-resident B lymphocytes from neonatal trisomic line chickens. The aneuploids (trisomics and tetrasomics) expressed more cell surface Ia than did normal disomic birds. Furthermore, the aneuploids exhibited a greater frequency of small B lymphocytes as compared to disomic chickens. Dual parameter analysis of Ia. quantity and cell size was undertaken to study B lymphocyte subpopulations in these birds. It was observed that the aneuploids had altered frequencies of two distinct subpopulations of cells: (1) an increased percentage of small cells which express high levels of Ia antigen and (2) a decreased percentage of large cells which express medium levels of Ia antigen. These findings support the view that MHC class II genes are regulated and expressed in a dosage-dependent manner. Therefore, increases in the number of MHC copies per cell result in the increased expression of Ia glycoprotein on bursa-resident B cells. The stepwise increase in membrane Ia on trisomic and tetrasomic B cells is correlated, and perhaps casually linked, with progressive degrees of alteration of developing B cell subpopulations in the bursa of aneuploid chicks. These events may ultimately alter the humoral immunity of the aneuploid animals.     

Expression of human B-cell specific receptor FCRL1 in normal individuals and in patients with autoimmune diseases

The comparative analysis of expression level of FCRL1 gene encoding human B-cell surface receptor in healthy individuals and patients with autoimmune diseases was carried out. For the expression estimation we used results of DNA dot-hybridization on the membranes, containing cDNA samples from subpopulations of blood cells of patients with autoimmune diseases. The quantitative estimation of hybridization signals showed that expression level of FCRL1 gene in peripheral blood B-lymphocytes was significantly higher in patients with a multiple sclerosis, lupus anticoagulans, Takayasu's arteritis and also in von Willebrand disease than in healthy individuals. FCRL1-specific monoclonal and polyclonal antibodies were raised. They were proven to detect FCRL1 in Western blotting, immunohistochemistry and flow cytometry. It was found that FCRL1 is expressed on the surface of CD19+ mature B-cells. In tonsil FCRL1-positive cells were located in crypt area: in mantle zone of secondary lymphoid follicles and among cells of lymphoepithelium. FCRL1-positive cells were also found in B-cell follicles of the spleen.     

Cell-cell contact of human T cells with fibroblasts changes lymphocytic mRNA expression: increased mRNA expression of interleukin-17 and interleukin-17 receptor

Using random arbitrarily primed-reverse transcribed-PCR and sequence analysis, we investigated changes in lymphocytic molecules after cell-cell contact with fibroblasts. An mRNA species which was upregulated in Jurkat T cells by cell-cell contact with MRHF cells (a human foreskin fibroblast line) was identified as coding for the human interleukin-17 receptor. This finding was confirmed by quantitative RT-PCR for the HUT78 and Jurkat T cell lines, for peripheral blood lymphocytes, and for tonsillar T cells. Furthermore, the interleukin-17 mRNA, coding for a proinflammatory cytokine, was also upregulated in peripheral blood lymphocytes and tonsillar T cells after cell-cell contact with fibroblasts. Supernatants obtained from cell-cell contact-stimulated peripheral blood lymphocytes enhance the production of interleukin-6 and interleukin-8 by fibroblast-like synoviocytes and this effect could be blocked by interleukin-17 antibodies. Changes in the mRNA levels of Jurkat T cells induced by cell-cell contact with adherent cells were also found for M-type pyruvate kinase, for tropomyosin TM30 and for the p54nrb gene product.     

Relative ability of different bovine leukocyte populations to support active replication of rinderpest virus.

Bovine peripheral blood mononuclear cells (PBMC) were infected with the pathogenic Saudi isolate of rinderpest virus (RPV) in order to identify the cell subpopulation(s) susceptible to active replication of this virus. Flow cytometry analysis, using a monoclonal antibody recognizing the H glycoprotein of RPV, showed that monocytes were the main subpopulation in which the virus replicated, whereas <2% of lymphocytes expressed viral antigen. The activation of PBMC with concanavalin A before infection resulted in an increase in the capacity of lymphocytes to support RPV replication; >90% of CD4+ and CD8+ T lymphocytes expressed viral antigen at 3 days postinfection, although < or = 40% of gamma/delta T cells were productively infected. B-lymphocyte activation with pokeweed mitogen also resulted in increased replication of this virus in these cells, involving up to 40% of B lymphocytes. An enhancement of lymphocyte susceptibility to infection and active replication by RPV was observed upon coculture of RPV-infected PBMC on bovine endothelial cells. Such enhancement was most marked with the B-cell and CD4+ T-cell subpopulations. Contact between lymphocytes and extracellular matrix components did not alter the capacity of RPV to replicate in lymphocytes. This intercellular contact with endothelial cells increased the viability of certain lymphocyte subpopulations, but it alone could not explain the increased sensitivity to RPV. Intercellular signalling, which resulted in interleukin-2 receptor upregulation, probably played a role. In summary, monocytes are the main target for active, productive infection by RPV. Similar replication in lymphocytes depends on their activation state and on contact with accessory cells such as endothelial cells. These characteristics have important implications for virus traffic in vivo and the pathogenesis of this disease.     

Excessive production of IFN-gamma in patients with systemic lupus erythematosus and its contribution to induction of B lymphocyte stimulator/B cell-activating factor/TNF ligand superfamily-13B

Expression and immunological significance of IFN-gamma, a pivotal cytokine in murine lupus, have not been clearly demonstrated in human systemic lupus erythematosus (SLE). In the present study we investigated the expression of IFN-gamma in peripheral blood T cells from patients with SLE and its role in the production of the soluble B lymphocyte stimulator (sBLyS). Peripheral blood T cells from patients with SLE expressed significantly larger amounts of IFN-gamma in response to stimulation with anti-CD3 mAb plus anti-CD28 mAb than those from normal controls as shown by three analytical methods, including ELISA, flow cytometry, and quantitative RT-PCR. The ratio of IFN-gamma-producing T cells to effector memory T cells in CD3(+)CD4(+) and CD3(+)CD8(+) populations in patients with SLE was significantly higher than that of normal controls. The T-box expressed in T cells (T-bet) mRNA/GATA-binding protein-3 (GATA-3) mRNA ratio was significantly higher in patients with SLE than in normal controls. T cell culture supernatants from patients with SLE contained significantly higher sBLyS-inducing activity than normal controls; this was almost completely inhibited by the addition of anti-human IFN-gamma mAb. Percentages of BLyS-expressing peripheral blood monocytes in patients with SLE were significantly higher than those of normal controls. Monocytes from patients with SLE produced significantly larger amounts of sBLyS in response to IFN-gamma than those from normal controls. Taken together, these data strongly indicate that the overexpression of IFN-gamma in peripheral blood T cells contributes to the immunopathogenesis of SLE via the induction of sBLyS by monocytes/macrophages, which would promote B cell activation and maturation.     

Antigen reactive memory T cells are defined by Ta1

Ta1 is a 105,000 dalton protein that is weakly expressed on a small fraction of resting human peripheral blood T cells but strongly expressed in vitro on T cell clones and a substantial proportion of activated T cells. Unlike receptors for growth factors such as IL 2, the Ta1 antigen is present on T cell lines and clones irrespective of cell cycle. The function of Ta1 was investigated after separation of T lymphocytes into Ta1-enriched and Ta1-depleted subpopulations that were obtained from normal human subjects. Although Ta1-enriched T cells constitute only 10 to 15% of the E rosette-positive lymphocyte population, most, if not all, of the anamnestic response to the recall antigens tetanus toxoid and mumps reside in the Ta1+ population. Both Ta1-enriched and -depleted cells responded equally well to the mitogen PHA. The autologous mixed lymphocyte response was also greater in the Ta1-enriched subpopulation but not to the degree seen with soluble antigen. Increased proliferation was not due simple to increased inducer cell function within the Ta1+ subpopulations because both Ta1- and Ta1+ cells induced similar amounts of Ig synthesis in the presence of PWM. Additionally, increasing numbers of Ta1- cells did not suppress the enhanced proliferative responses of Ta1+ cells, and thus Ta1- cells do not appear to be functioning as suppressor cells. The Ta1 antigen appears to be a marker for previously activated T cells in peripheral blood, and this subpopulation appears to include T memory cells.     

PESV 对K562 细胞BCR/ABL 融合基因及凋亡调控因子Bcl-2、Bad 表达的影响

目的:探讨PESV对K562细胞BCR/ABL融合基因及凋亡调控因子bcl-2和bad表达的影响.方法:将体外培养K562细胞,经PESV处理不同时间后,流式细胞术检测细胞凋亡率,荧光定量RT-PCR检测BCR/ABL、Bcl-2、Bad mRNA水平变化.结果:与对照组相比,PESV处理后K562细胞,凋亡率增加,BCR/ABL融合基因表达降低,抗凋亡相关基因Bcl-2 mRNA表达降低,促凋亡基因Bad mRNA表达增加.结论:PESV能降低降低K562细胞BCR/ABL融合基因的表达,可能通过调节Bcl-2和Bad表达,抑制K562细胞增殖,促进其凋亡.     

Cannabinoids inhibit insulin secretion and cytosolic Ca2+ oscillation in islet beta-cells via CB1 receptors

Obesity is the main risk factor for the development of metabolic syndrome. Endogenous cannabinoids act on the cannabinoid type 1 (CB1) receptor, a GPCR, and stimulate appetite via central and peripheral actions, while blockade of CB1 receptor reduces body weight in humans. In this study, we aimed to explore a role of the peripheral endocannabinoid system in insulin secretion, which could be important in the metabolic effects of the cannabinoid-CB1 system. We found that mRNA for CB1 receptor, but not CB2 receptor, was expressed in mouse pancreatic islets using RT-PCR. Immunohistochemical study revealed that CB1 receptor was expressed in beta-cells. Furthermore, anandamide and a CB1 agonist, arachidonylcyclopropylamide (ACPA), inhibited glucose-induced insulin secretion from mouse pancreatic islets. Both anandamide and ACPA inhibited glucose-induced cytosolic Ca(2+) oscillation in mouse pancreatic beta-cells. These results demonstrate a novel peripheral action of cannabinoids to inhibit insulin secretion via CB1 receptors.     

Regulation of BSF-2/IL-6 production by human mononuclear cells. Macrophage-dependent synthesis of BSF-2/IL-6 by T cells

Regulation of BSF-2/IL-6 production in peripheral mononuclear cells (MNC) was studied. BSF-2 mRNA expression in mitogen-stimulated MNC showed a biphasic response, the first peak around 4 h and the second peak around 48 h. This was caused by different kinetics of BSF-2 mRNA expression in distinct subpopulations of MNC; M phi expressed BSF-2 mRNA at 5 h in the absence of any stimulation, and mitogen-stimulated T cells and B cells expressed BSF-2 mRNA 48 h after stimulation. Immunohistochemical staining of the cells with anti-BSF-2 antibody demonstrated that macrophages, T cells and B cells could produce BSF-2. T cells in peripheral MNC produced BSF-2 in the presence of M phi. The requirement of macrophage for BSF-2 production in T cells could be replaced by TPA but not by IL-1 or BSF-2.     

Phenotypic and Immunomodulatory Properties of Equine Cord Blood-Derived Mesenchymal Stromal Cells

Multipotent mesenchymal stromal cells (MSC) have attracted interest for their cytotherapeutic potential, partly due to their immunomodulatory abilities. The aim of this study was to test the robustness of our equine cord blood (CB) MSC isolation protocol, to characterize the CB-MSC before and after cryopreservation, and to evaluate their immunosuppressive phenotype. We hypothesized that MSC can be consistently isolated from equine CB, have unique and reproducible marker expression and in vitro suppress lymphoproliferation. Preliminary investigation of constitutive cytoplasmic Toll-like receptor (TLR) 3 and 4 expression was also preformed due to their possible association with anti- or pro-inflammatory MSC phenotypes, respectively. Surface markers were assessed for antigen and mRNA expression by flow cytometry and quantitative polymerase chain reaction (qPCR). Immunomodulatory properties were evaluated in mixed lymphocyte reaction assays, and TLR3 and TLR4 expression were measured by qPCR and immunocytochemistry (ICC). CB-MSC were isolated from each off nine cord blood samples. CB-MSC highly expressed CD29, CD44, CD90, and lacked or had low expression of major histocompatibility complex (MHC) class I, MHC-II, CD4, CD8, CD11a/18 and CD73 before and after cryopreservation. CB-MSC suppressed in vitro lymphoproliferation and constitutively expressed TLR4. Our findings confirmed CB as a reliable MSC source, provides an association of surface marker phenotype and mRNA expression and suggest anti-inflammatory properties of CB-MSC. The relationship between TLRs and lymphocyte function warrants further investigation.     

Stimulation of human T cell colony growth by a lymphocyte colony enhancement factor derived from lymphocyte subpopulations

Blood mononuclear cells (MNC) develop into T cell colonies when the cells are sensitized with PHA and seeded in a two-layer soft agar system. Conditioned medium (CM) derived from MNC enhanced lymphocyte colony formation when it was added to the culture system. CFU-TL appear to be stimulated into colony formation by molecules secreted by lymphocyte subpopulations contained in the seeded cells. In this study, human peripheral blood MNC were fractionated by a battery of techniques into adherent, E+, CD4+, CD8+, B and null cells. CM was prepared from each of the subpopulations and its effects on T cell colony growth assayed. All the lymphocyte subpopulations were found to generate lymphocyte colony enhancement factor (LCEF). After several purification procedures, CM prepared from CD4 and CD8+, displayed LCEF activity corresponding to proteins of molecular weight 30-40 and 100-140 kD.     

siRNA沉默t-bet基因对CD4、CD8T淋巴细胞亚群IFN-γ产生的影响

利用化学合成针对人t-bet基因的小干扰RNA（siRNA）,转染体外培养的人外周血单个核细胞,利用流式细胞仪分选纯化CD4^＋、CD8^＋T淋巴细胞,半定量RT-PCR检测CD4^＋、CD8^＋T淋巴细胞中t-betmRNA的表达变化,流式细胞术检测转染前后IFN-γ产生的变化情况。探讨转录因子t-bet对人CD4^＋、CD8^＋T淋巴细胞亚群IFN-γ产生的调控作用。与对照组相比,转染后的人CD4^＋、CD8^＋T淋巴细胞t-betmRNA表达水平明显下降;转染siRNA后,CD4＋T淋巴细胞中IFN-γ＋细胞比例为（18.46±6.86）%,与对照组（50.20±5.91）%比较有显著性差异（P〈0.01）;CD8＋T淋巴细胞IFN-γ＋细胞比例为（74.18±9.33）%,和对照组（76.51±6.49）%比较差异不明显（P〉0.05）。体外转录合成的siRNA可有效降低人CD4^＋、CD8^＋T淋巴细胞t-bet的基因表达;转录因子t-bet对人不同淋巴细胞亚群IFN-γ的产生所起作用不同。     

人脐带间充质干细胞的分离培养与鉴定简

目的：建立并优化人脐带间充质干细胞分离纯化方法,并对其表面标志与多向分化潜能进行鉴定。方法：收集健康足月产胎儿脐带组织,采用组织块贴壁法进行原代培养,流式细胞仪对其表面标志进行检测,通过向成骨成脂分化对其多向分化潜能进行鉴定,RT-PCR对其干细胞特性基因Oct4、Nanog、Sox2、Nestin进行检测。结果：采用组织块贴壁法可在2周左右获得大量间充质干细胞,培养的细胞经流式细胞仪检测,高表达CD29、CD44、CD105、CD106,低表达CD34、CD45;经成骨成脂诱导2周后可分化为成骨细胞和成脂细胞,RT-PCR检测发现原代细胞表达Oct4、Nanog、Sox2、Nestin基因。结论：人脐带间充质干细胞可在体外扩增培养,具有多向分化潜能,可作为组织工程种子细胞来源。     

腺病毒介导的人抑瘤素M基因对A375人黑色素瘤细胞的抑制作用

目的:构建抑瘤素M(OSM)重组腺病毒载体,研究其对人黑色素瘤细胞A375的抑制作用。方法:以PEGZ-OSM重组质粒为模板,通过PCR技术扩增出OSM片段,采用腺病毒载体的基因重组和体外包装技术获得表达与人OSM氨基酸序列相同的重组腺病毒子Ad-OSM,感染A375细胞,用荧光显微镜、RT-PCR、Western blot法检测OSM在A375细胞中的转录和表达;荧光显微镜观察A375细胞的形态学改变;MTT法和流式细胞术(FCM)检测Ad-OSM对A375细胞的生长抑制和细胞周期的抑制效应;半定量RT-PCR法检测OSM基因表达对A375细胞中的Bax、Bcl-2基因表达的影响。结果:基因测序和PCR分析结果显示,成功构建了Ad-OSM腺病毒表达载体;RT-PCR和Western blot法检测到OSM基因在A375细胞中的转录和表达;OSM基因的表达对A375细胞增殖有明显抑制作用,并可诱导细胞凋亡,OSM基因可通过上调细胞中Bax和下调Bcl-2基因表达诱导细胞凋亡。结论:成功构建了Ad-OSM腺病毒表达载体,感染OSM基因可明显抑制A375人黑色素瘤细胞的生长,诱导其凋亡,该现象可能是通过改变Bax、Bcl-2基因表达水平来发挥抗肿瘤作用。