The instability of membrane markers expressed by human monocytes and macrophages in culture

Surface markers were tested on freshly isolated human monocytes and following their in vitro maturation to macrophages. The markers tested were HLA-DR antigens, receptors for the Fc of IgG and complement as well as membrane markers defined by monoclonal antibodies. The results revealed a dynamic expression of some of the markers on monocytes which was influenced by several variables. The expression of the markers was modulated by the presence of different sera, by treatment with lymphokines and interferon and following the in vitro maturation of monocytes to macrophages. The most unstable marker was found to be the HLA-DR, which was modulated by all these variables. The 63D3 was affected by different sera and culture supernatant, as well as following the maturation of monocytes to macrophages, but not by lymphokines and interferon. One of the markers, the Mac 120, was found to be relatively stable and did not change significantly following the maturation of monocytes to macrophages. The Fc and complement receptors were also stable in their expression under these conditions, but were probably partially blocked in the presence of human serum. These results indicated that at least some of the heterogeneity related to the monocyte population was probably not due to the occurrence of stable subsets of cells, but rather to reversible changes in marker expression.     

A gene signature of inhibitory MHC receptors identifies a BDCA3(+) subset of IL-10-induced dendritic cells with reduced allostimulatory capacity in vitro

Dendritic cells (DC) are crucial gatekeepers in regulating immunity. Whereas mature immunostimulatory myeloid DC (DC(ims)) potently promote immune responses, IL-10-induced myeloid DC (DC-IL-10) counteract T cell activation. To elucidate the molecular repertoire by which DC-IL-10 secure reduced T cell activation, comparative gene expression profiling was done using Affymetrix U133A microarrays. Among the genes overexpressed in DC-IL-10, eight immunoreceptor tyrosine-based inhibitory motif (ITIM)-containing inhibitory molecules (ILT2, ILT3, ILT4, ILT5, DCIR, PILRA, FcgammaRIIB, SLAM) were found. Phenotypic analysis of DC-IL-10 defined an ILT(high) DC subset further characterized by expression of CD14, TLR2, DC-SIGN, and CD123 and the lack of lymphocyte, monocyte/macrophage, and plasmacytoid DC markers such as CD3, CD8, and CD68. A unique feature of the ILT(high) DC subset was expression of the novel DC marker BDCA3, which was not detected on monocytes, immature DC, DC(ims), or ILT(low) DC-IL-10. While the allogeneic T cell proliferation induced by the entire DC-IL-10 population was approximately 30% of that stimulated by DC(ims), allogeneic MLR responses driven by the ILT(high) DC subset were reduced to 8% of the allostimulatory capacity of DC(ims), although secretion of the inhibitory cytokine IL-10 and other Th1/Th2-associated cytokines was similar in these cells.     

Pyrosequencing for detection of mutations in the connexin 26 (GJB2) and mitochondrial 12S RNA (MTRNR1) genes associated with hereditary hearing loss

Hereditary hearing loss (HHL) is one of the most common congenital disorders and is highly heterogeneous. Mutations in the connexin 26 (CX26) gene (GJB2) account for about 20% of all cases of childhood deafness, and approach 50% in documented recessive cases of non-syndromic hearing loss. In addition, a single mitochondrial DNA mutation, mt1555A>G, in the 12S rRNA gene (MTRNR1), is associated with familial cases of progressive deafness. Effective screening of populations for HHL necessitates rapid assessment of several of these potential mutation sites. Pyrosequencing links a DNA synthesis protocol for determining sequence to an enzyme cascade that generates light whenever pyrophosphate is released during primer strand elongation. We assessed the ability of Pyrosequencing to detect common mutations causing HHL. Detection of the most common CX26 mutations in individuals of Caucasian (35delG), Ashkenazi (167delT), and Asian (235delC, V37I) descent was confirmed by Pyrosequencing. A total of 41 different mutations in the CX26 gene and the mitochondrial mt1555A>G mutation were confirmed. Genotyping of up to six different adjacent mutations was achieved, including simultaneous detection of 35delG and 167delT. Accurate and reproducible results were achieved taking advantage of assay flexibility and experimental conditions easily optimized for a high degree of standardization and cost-effectiveness. The standardized sample preparation steps, including target amplification by PCR and preparation of single-stranded template combined with automated sequence reaction and automated genotype scoring, positions this approach as a potentially high throughput platform for SNP/mutation genotyping in a clinical laboratory setting. .     

HLA-DRB1*07与慢性乙肝患者Th1/Th2因子表达水平的相关性

目的 :探讨广东地区汉族慢性乙型肝炎患者HLA DRB1 0 7与Th1 Th2因子表达水平的相关性。方法 :收集 12 0例广东地区汉族慢性乙肝患者新鲜抗凝血各 8ml,通过序列特异性引物套式PCR(PCR SSP)方法进行HLA DRB1 0 7检测 ,并同时用双抗体夹心法检测患者治疗前后外周血CD4 +T细胞分泌IFN γ、IL 2、IL 10和IL 4的水平。结果 :12 0例慢性乙肝患者HLA DRB1 0 7携带者 31例 ,携带率为 2 5 8% ,明显高于广东地区汉族人群的平均携带率 7 84 % ;HLA DRB1 0 7阳性患者IFN γ平均表达水平为 (1132 0 4± 75 36 )pg ml,IL 2平均表达水平为 (1184 0 6± 81 4 2 )pg ml,IL 4平均表达水平为 (876 79± 4 7 5 3)pg ml,IL 10平均表达水平为 (817 4 8± 2 4 4 0 )pg ml ;HLA DRB1 0 7阴性患者IFN γ平均表达水平为 (12 32 10± 198 13)pg ml,IL 2平均表达水平为 (12 0 8 17± 116 12 )pg ml,IL 4平均表达水平为 (6 81 99± 6 1 5 9)pg ml,IL 10平均表达水平为 (6 38 84± 76 17)pg ml。HLA DRB1 0 7阳性患者IL 4、IL 10表达水平高于阴性患者 (P <0 0 5 ) ,而IFN γ、IL 2表达水平与阴性患者差异无统计学意义 (P >0 0 5 )。结论 :HLA DRB1 0 7(+)慢性乙肝患者Th2因子表达水平高于HLA DRB1 0 7(- )慢性乙肝患者。     

Bac-to-Bac杆状病毒表达系统表达鼠IL-4受体拮抗体蛋白的研究

目的 利用Bac-to-Bac杆状病毒表达系统表达鼠IL-4受体拮抗体 (mIL-4RA)蛋白.方法 PCR方法扩增小鼠IL-4 C118截断型基因,定向克隆入转移质粒pFastBacHTB中,转化感受态DH10Bac细胞,在DH10Bac细胞内重组pFastBacHTB与杆粒发生转座.筛选阳性克隆,提取重组杆粒,转染sf9昆虫细胞株,获取完整重组杆状病毒.反复感染sf9细胞,扩增病毒同时表达目的 蛋白;用ELISA方法进行蛋白鉴定并初步定量.结果 经核苷酸序列测定及PCR方法,鉴定成功获得含mIL-4RA基因的重组杆粒;通过杆粒转染后sf9细胞所表现出来的细胞病变,推断成功转染并获得重组杆状病毒;最后ELISA方法初步定量sf9细胞培养上清中mIL-4RA蛋白表达量为(1.15±0.12) ng/mL.结论 本研究利用Bac-to-Bac杆状病毒表达系统成功表达mIL-4RA蛋白,为进一步研究其生物学活性及功能奠定了实验基础,同时亦为其他蛋白质的真核表达提供了方法学的参考.     

Effective induction of immune tolerance by portal venous infusion with IL-10 gene-modified immature dendritic cells leading to prolongation of allograft survival

Dendritic cells (DC) not only initiate T cell responses, but are also involved in the induction of tolerance. The functional properties of DC are strictly dependent on their state of maturation. It has been shown that immature DC can induce immune tolerance and prolong allograft survival. Interleukin-10 (IL-10) is an important immunosuppressive cytokine which inhibits maturation and function of DC. In order to improve the tolerogenicity of DC, we and others showed that adenovirus vectors can effectively mediate IL-10 genetic modification of DC, and IL-10 genetic modification can inhibit MHC II, B7.2, and CD40 expression, IL-12 secretion and the T cell stimulatory capacity of DC. The primary aim of this study is to examine the in vivo effects of this approach on allograft survival in a murine cardiac allograft transplantation model. To our surprise, we observed that infusion of immature DC genetically modified to express IL-10 (DC-IL-10) via the tail vein could not prolong allograft survival in the recipients, but shortened their survival. More interestingly, portal venous infusion of DC-IL-10 markedly prolonged allograft survival. The diverse effects of DC-IL-10 infusion through different routes may be due to the different immune responses to alloantigens in recipients that received DC-IL-10 via either the portal or the tail vein. Decreased cytotoxicity, polarization of Th2 response, poor T cell stimulating activity of liver DC and enhanced incidence of donor DC in the recipients may contribute to the more efficient prolongation of allograft survival observed after portal venous infusion of DC-IL-10. These results suggest that portal venous infusion may be an effective approach for immature DC to induce immune tolerance or hyporesponsiveness against donor antigens, and prolong allograft survival.Abbreviations APC Antigen-presenting cells - CTL Cytotoxic T lymphocytes - DC Dendritic cells - DC-IL-10 IL-10 gene-modified immature dendritic cells - iDC Immature dendritic cells - IL-10 Interleukin-10 - MLR Mixed leukocyte reaction - MOI Multiplicity of infection     

Cell-surface bound pertussis toxin induces polyclonal T cell responses with high levels of interferon-gamma in the absence of interleukin-12

Pertussis toxin (PTx), an exotoxin produced by Bordetella pertussis, has long been used as a mucosal adjuvant. We examined the T cell stimulatory properties of PTx in order to dissect its mechanisms of adjuvanticity. PTx or the B-oligomer of PTx (PTxB) failed to activate purified murine CD4+ or CD8+ T cells, as measured by a lack of proliferation or expression of early T cell activation markers. However, these T cells proliferated extensively in response to the toxin in the presence of syngeneic DC, and proliferation was accompanied by a high level of IFN-gamma production in the absence of IL-12. Interestingly, such responses were independent of signals mediated by MHC-TCR interaction. Both PTx and PTxB were found to bind stably to the surface of DC, and increased the adherence of DC to surrounding cells. These data suggest that polyclonal T cell responses mediated by the toxin are likely to be caused by the toxin bound on the surface of APC, either cross-linking cell surface molecules on T cells, or directly stimulating T cells together with the co-stimulatory molecules expressed on APC. B. pertussis may use this toxin as a mechanism to evade a specific immune response.     

Calcium homeostasis and glucose uptake of murine myotubes exposed to insulin,caffeine and 4‐chloro‐m‐cresol

The modulation of glucose uptake by cytosolic calcium and the role of insulin on calcium homeostasis in insulin‐target cells are incompletely understood and results are contradictory. To address this issue, we used the C2C12 murine skeletal muscle cell line model and examined the influence of caffeine and 4‐chloro‐m‐cresol, two ryanodine receptor agonists known to mobilize intracellular calcium stores and increase cytosolic free calcium concentration. We followed ⁴⁵calcium efflux, a validated indicator of cytosolic calcium concentration, and 3‐O‐methyl‐[1–³H]‐d ‐glucose uptake in parallel. We also determined if insulin incubation affected ⁴⁵calcium influx rate. A 30‐min treatment by 1 μm insulin highly significantly increased ⁴⁵calcium efflux by 8.5% (P = 0.0014), despite a significant reduction of ⁴⁵Ca²⁺ influx already measurable after 20 and 30 min of insulin stimulation (?16.6%, P = 0.0119 and ?21.3%, P = 0.0047, respectively). Caffeine (1–20 mm ) and 4‐chloro‐m‐cresol (0.05–10 mm ) concentration‐dependently increased ⁴⁵calcium efflux, the latter being more potent and efficacious. These agents, in a concentration‐dependent manner, inhibited both basal and, more potently, insulin‐stimulated glucose uptake. This resulted in a negative correlation of glucose uptake and ⁴⁵calcium efflux (r > 0.95, P < 0.001). This effect was ～5 times greater for caffeine than for 4‐chloro‐m‐cresol, suggesting a calcium‐independent part of the glucose uptake inhibition by caffeine. In our in vitro model of cultured muscle cells, insulin appears to prevent calcium overload by both stimulating efflux and inhibiting cell storage. This effect, taken together with the observed inhibitory, inverse relationship between ⁴⁵calcium efflux and glucose uptake, contributes to describing the complex insulin–calcium interplay involved in target cells.     

TGF-β1 Null Mutation Leads to CD154 Upregulated Expression in Affected Tissues

The TGF-1(–/–) mouse is a murine model for systemic autoimmune disease. The aim of this study is to elucidate the immunological mechanism that leads to multifocal tissue inflammation and autoantibody production in TGF-1(–/–) mice. Heart, lung, liver, and salivary gland from TGF-1(–/–) were assessed for CD154 expression by RT-PCR and immunohistochemistry. Compared to wild-type littermates, CD154 expression was elevated in all tissues studied. Furthermore, IL-12 mRNA was expressed in the salivary gland and heart of TGF-1(–/–) mice and not in wild-type littermates. This suggests that the CD154 pathway is activated in these tissues. This shows that TGF-1 regulates CD154 expression leading to spontaneous IL-12 production and autoimmunity.