西洛他唑抗动脉粥样硬化的作用机制

目的观察西洛他唑对兔动脉粥样硬化斑块组织的影响,进一步探讨西洛他唑抗动脉粥样硬化作用及其可能机制。方法将30只新西兰雄性白兔随机分为正常对照组、高脂饮食组和西洛他唑组。酶法检测血脂,免疫沉淀法测定血清C反应蛋白水平,免疫组织化学检测基质金属蛋白酶9和核因子κB的表达,病理形态学分析各组主动脉内膜厚度和斑块面积,逆转录聚合酶链反应检测血管组织单核细胞趋化蛋白1mRNA的表达。结果实验第12周末,西洛他唑组总胆固醇、甘油三酯和低密度脂蛋白胆固醇水平低于高脂饮食组,但高于正常对照组(P均<0.01);西洛他唑组主动脉内膜厚度和斑块面积低于高脂饮食组,但大于正常对照组(P均<0.01);西洛他唑组核因子κB、单核细胞趋化蛋白1和基质金属蛋白酶9的表达弱于高脂饮食组,但强于正常对照组(P均<0.01)。结论西洛他唑有抗动脉粥样硬化作用,其作用机制可能与下调核因子κB、单核细胞趋化蛋白1及基质金属蛋白酶9的表达有关。     

颈动脉粥样硬化斑块内MCP-1表达的研究

目的观察颈动脉粥样硬化(AS)斑块内单核细胞趋化蛋白-1(MCP-1)的mRNA和抗原的表达情况。方法分别应用原位杂交和免疫组织化学法测定颈AS斑块中MCP-1mRNA和抗原表达。结果相对于正常动脉内膜,颈AS病变内膜MCP-1阳性细胞数明显增多(P<0.05)。其中颈AS严重病变肩区阳性细胞数最多,以单核-巨噬细胞为主。结论MCP-1与颈AS发生密切相关。     

Monocytes in active multiple sclerosis: intact regulation of HLA-DR density in vitro despite decreased HLA-DR density in vivo

HLA-DR expression on circulating monocytes varies as a function of disease activity in patients with multiple sclerosis (MS), a putative immunopathological demyelinating disorder. Specifically, monocytes isolated from subjects with active MS exhibit reduced HLA-DR antigen density, and immunoregulatory aberrations such as impaired T lymphocyte-mediated suppression correlate strongly with this quantitative defect. To address the mechanism underlying this phenomenon, we compared in vitro regulation of HLA-DR by interferon beta (IFN beta), interferon gamma (IFN gamma), and lipopolysaccharide (LPS) in monocytes from patients with stable and active MS and normal individuals. Interferon-gamma and LPS enhanced monocyte expression of HLA-DR equally in both MS patient groups, suggesting that underexpression of HLA-DR in active MS was not explained by impaired in vivo monocyte responsiveness. Furthermore, interferon regulation of HLA-DR in normals and stable MS subjects was indistinguishable, indicating that aberrant interferon-mediated regulation of class II major histocompatibility complex (MHC) on circulating monocytes does not appear to be a characteristic of the MS disease state.     

Bacterial infection (BI)-INDEX: an improved and simplified rapid flow cytometric bacterial infection marker

The aim of this study was to develop a rapid and simple flow cytometric bacterial infection marker. In this prospective comparative study, quantitative flow cytometric analysis of CD10, CD35, CD66b, CD282, and MHC Class I molecules on human neutrophils, monocytes, and B-lymphocytes from 141 hospitalized febrile patients with suspected infection and from 50 healthy controls was performed. We developed a flow cytometric marker of local and systemic bacterial infections, designated “bacterial infection (BI)-INDEX”, incorporating the quantitative analysis of CD10, CD35, MHCI, CD66b, and CD282 on neutrophils, monocytes, and B-lymphocytes, which displayed 90% sensitivity and 96% specificity in distinguishing between microbiologically confirmed bacterial (n = 31) and viral infections (n = 27) within a 1-h time-frame. We propose that our novel rapid BI-INDEX test will be useful in assisting physicians to ascertain whether antibiotic treatment is required, thus limiting unnecessary antimicrobial usage.     

稳定表达基质细胞衍生因子1α的内皮细胞系构建及其与单核细胞的粘附作用

目的构建稳定表达基质细胞衍生因子1α的ECV304细胞系,研究基质细胞衍生因子1α在单核细胞/内皮细胞粘附中的作用。方法将大鼠基质细胞衍生因子1α基因聚合酶链反应产物及质粒载体pcDNA3.1用EcoRⅠ进行酶切,去磷酸化,置于连接反应体系中进行连接,将连接产物氯化钙法转化DH5α菌。氨苄青霉素筛选并扩增阳性菌落,经酶切鉴定插入方向正确后,再用G418筛选、逆转录聚合酶链反应检测基质细胞衍生因子1α浓度,建立稳定转染ECV304细胞系。在6孔板上种植转染基质细胞衍生因子1α基因的ECV304细胞,加THP-1细胞37℃孵育30min,磷酸缓冲液轻洗3遍,去除未粘附细胞,倒置显微镜下计数上、下、左、右、中5个视野,取其平均值得到每个视野粘附单核细胞数。结果经逆转录聚合酶链反应检测证实,稳定表达基质细胞衍生因子1α的ECV304细胞系构建成功,细胞计数表明,转染ECV304细胞系粘附THP-1细胞数是转染空质粒的十几倍,CXCR4单抗基质细胞衍生因子1α多抗均显著减少其粘附力(P<0.01)。结论内源性基质细胞衍生因子1α能促进ECV304细胞与单核细胞的粘附。     

HLA-B27 modulates intracellular survival of Salmonella enteritidis in human monocytic cells

Human major histocompatibility complex class I allele HLA-B27 is associated with a group of diseases called spondyloarthropathies. In reactive arthritis (ReA), the disease is triggered by certain infections, e.g. gastroenteritis caused by Salmonella. The host/microbe interaction is abnormal in susceptible individuals leading to inefficient elimination of arthritis-triggering bacteria, fragments of them, or both, after the initial infection. Using transfected human monocytic U937 cell lines, we demonstrate that the expression of the HLA-B27 antigen does not influence the uptake of S. enteritidis into U937 cells in vitro. Interestingly, HLA-B27 remarkably impairs the elimination of S. enteritidis within the HLA-B27 transfected U937 cells. The impaired elimination of ReA-triggering microbes by HLA-B27⁺ monocytes may offer an explanation for the persistence of ReA-triggering microbes in susceptible HLA-B27⁺ individuals. This modulation of the host/microbe interaction by HLA-B27 may have an important role in the pathogenesis of ReA.     

Expression and function of NKRP1A molecule on human monocytes and dendritic cells

In this study, we analyzed the expression and function of the lymphocyte surface lectin NKRP1A on peripheral blood monocytes (Mo) or Mo and dendritic cells (DC) derived from thymic and bone marrow precursors. De novo expression of NKRP1A and CD14 molecules was detected upon culture of CD2^? CD3^? CD14^? CD16^? CD1a^? NKRP1A^? immature thymic precursors for 7 days in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF). Under these culture conditions, by day 21, a fraction of cells had lost CD14 and acquired both CD80 (B7.1) and CD86 (B7.2) molecules. These cells displayed a DC-like morphology and were surface NKRP1A positive. CD34⁺ NKRP1A^? CD14^? precursors, isolated from bone marrow and cultured in the presence of GM-CSF, also expressed both NKRP1A and CD14: these antigens were newly expressed on about one third of cells which had lost the CD34 precursor marker. In addition, NKRP1A was constitutively present on resting CD14⁺ peripheral blood Mo. When these cells were cultured in the presence of GM-CSF, the resulting DC population retained the expression of NKRP1A and acquired CD80, while they lost the CD14 antigen. Functional analysis revealed that the engagement of NKRP1A molecule leads to a strong intracellular calcium ([Ca²⁺]_i) increase both in resting peripheral blood Mo and in vitro-derived DC. [Ca²⁺]_i increase was mainly due to extracellular calcium influx, as it was completely abrogated by the addition of EGTA. More importantly, the engagement of the NKRP1A molecule induced interleukin (IL)-1β and IL-12 production by resting Mo and DC, respectively. Altogether these data indicate that NKRP1A lectin is present at the surface of Mo and DC and may play a relevant role in the activation and function of both cell types.     

Effect of filtration leukocytapheresis therapy: modulation of white blood cell enzyme activities in patients with rheumatoid arthritis

We treated 12 patients with rheumatoid arthritis by filtration leukocytapheresis (FLCP) and evaluated its effect on leukocyte enzyme activities. We calculated the number of leukocytes removed and assessed the clinical response. We also evaluated the cellular enzyme activities of elastase and dipeptidylpeptidase IV (DPP IV). Out of 12 patients, 7 patients achieved 20% improvement for 4 weeks following FLCP. The FLCP treatment resulted in removal of 96% of granulocytes, 98% of monocytes, and 61% of lymphocytes. Granulocytes and monocytes with high elastase activity were effectively removed by FLCP. The elastase activity of granulocytes was increased 4 weeks after the last FLCP only in responders. On the other hand, the DPP IV activity of lymphocytes was low at 4 weeks after the last FLCP in responders. Modulation of leukocyte enzyme activities is one of the main effects of FLCP therapy and alteration of granulocytes, monocytes, and lymphocytes.     

Additive inhibition of dendritic cell allostimulatory capacity by alcohol and hepatitis C is not restored by DC maturation and involves abnormal IL-10 and IL-2 induction

Background: Excessive alcohol use results in impaired immunity, and it is associated with increased incidence and progression of chronic hepatitis C virus (HCV) infection. Here we investigated the effects of HCV infection and alcohol on myeloid dendritic cells (DC) that are critical in antiviral immunity. Methods: Immature and mature DCs were generated from monocytes of chronic HCV infected patients (HCV‐DC) and controls (N‐DC) with IL‐4 plus granulocyte‐macrophage colony stimulating factor (GM‐CSF) in the presence or absence of alcohol (25 mM). DC allostimulatory capacity was tested in mixed lymphocyte reaction (MLR) and cytokine production by ELISA. Results: Allostimulatory capacity of HCV‐DCs was reduced compared to N‐DCs and it was further inhibited by alcohol treatment (p < 0.01). MLR was also decreased with alcohol‐treated N‐DCs. DC phenotypic markers and apoptosis were comparable between HCV‐DCs and N‐DCs irrespective of alcohol treatment. However, HCV‐DCs and alcohol‐treated N‐DCs exhibited elevated IL‐10 and reduced IL‐12 production. Reduced MLR with HCV‐DCs and its further inhibition by alcohol coexisted with decreasing IL‐2 levels (p < 0.017). DC maturation partially improved but failed to fully restore the reduced allostimulatory function of either alcohol‐treated or alcohol‐naïve HCV‐DCs (p < 0.018). Conclusions: Alcohol and HCV independently and together inhibit DC allostimulatory capacity, increase IL‐10, reduce IL‐12 and IL‐2 production that cannot be normalized by DC maturation. HCV and alcohol interact to modulate innate and adaptive immune responses via dendritic cells.