穿孔素与艾滋病病毒感染

在HIV感染中,CTL是控制HIV感染细胞免疫应答中重要的效应细胞,主要通过穿孔素介导的颗粒胞吐途径,特异性清除HIV感染细胞,在HIV原发感染期对于快速消除病毒血症起关键作用,但是CTL仍不能彻底清除HIV。近年来有关穿孔素与HIV感染的相关性研究日益受到国内外学者的关注,此文将对穿孔素介导细胞毒效应的分子机制、穿孔素在HIV感染中作用的研究进展作一简要综述。     

大鼠心脏移植急性排斥反应过程中穿孔素及颗粒蛋白酶B mRNA的表达

目的通过动态监测大鼠异体异位心脏移植外周血（PB）中穿孔素（pedorin，P）和颗粒酶B（granryme B，OB）的表达水平，判断其与急性排斥反应的相关性。方法实验组（n=16），供受体分别为Wistar和SD大鼠；对照组（n=17），供受体均为SD大鼠，两组术后均未接受抗排斥治疗。于术前及术后不同的时间采血，同时进行活组织病理检查，应用荧光定量RT-PCR（FQ-PCR）的方法，测定P及GBmRNA的表达水平，并与同期的病理检查结果相对照。结果实验组于移植后24hP及GBmRNA的表达上调，术后72h的表达明显增加（P〈0．001），术后3—5d达峰值，7—11d维持高水平，其变化早于病理改变；而对照组P及GBmRNA的表达及病理改变，在同期的观察过程中均没有明显的变化（P〉0．05）。结论同种异体大鼠心脏移植的急性排斥反应过程中，外周血淋巴细胞的穿孔素与颗粒酶BmRNA的表达水平术后呈现递增性，早于病理改变，可以作为急性排斥反应的监测指标。     

定量RT—PCR检测穿孔素mRNA方法的建立与临床初步应用

目的　建立检测穿孔素mRNA的定量RT PCR方法。方法　用限制性内切酶制备竞争性模板 ,建立定量RT PCR方法 ,检测正常人及部分肿瘤病人外周血的穿孔素mRNA水平。结果　 6例正常人的外周血穿孔素mRNA含量为 0 5 1± 0 40pmol/ml,消化道肿瘤患者的穿孔素mRNA水平与正常相比 ,有显著差异 (P <0 .0 1) ,乳腺肿瘤及白血病患者的穿孔素mRNA水平与正常人相比 ,没有差异。结论　穿孔素mRNA水平的降低可能是肿瘤发生的一个重要原因 ;除穿孔素途径以外 ,还存在别的抗肿瘤的效应机制。     

扩张型心肌病活检心肌穿孔素表达及其与心肌病变的关系

目的　研究扩张型心肌病 (DCM)患者心内膜活检心肌中穿孔素 (PFP)表达水平及与心肌病变的关系。方法　 19例DCM患者均行心内膜心肌活检留取心肌 ,10例意外死亡之非心脏病人心肌作为正常对照。利用原位杂交方法检测心肌浸润细胞PFPmRNA表达水平并与苏木精和伊红(HE)染色心肌病理积分进行比较。结果　与正常对照心肌相比 ,DCM患者心肌中有炎性细胞浸润 ,每高倍镜视野大于 5个白细胞的浸润灶明显增多。 19例DCM中 ,15例可见PFPmRNA阳性的浸润细胞 ,每高倍镜视野平均 1 91± 0 72个细胞。而 10例正常对照中未见PFPmRNA阳性的细胞。PFPmRNA表达水平与心肌病理积分正相关 ,r =0 82 ,P <0 0 5。结论　PFPmRNA表达水平与心肌病变的程度密切相关 ,提示PFP介导的细胞毒作用可能为DCM中心肌损害的重要机制之一     

人外周血CD56+NK细胞亚群表型和生物学特征

目的 探讨人CD56^＋NK细胞亚群、表型和生物学特征。方法 分离正常人外周血单个核细胞,利用多种抗体进行细胞表面和细胞内细胞毒效应分子（颗粒酶B和穿孔素）染色,通过流式细胞仪,在单个细胞水平上分析CD56^＋NK细胞亚群的表型及其生物学特征。结果 根据CD56分子表达密度的不同,将人外周血中NK细胞分为CD56^bright出和CD56^dim两大亚群。CD56^bright和CD56^dim细胞均表达CD95,CD56^bright细胞亚群中,CD95^bright和CD95^dim表达百分率分别为21．4％和77．0％;CD56^dim细胞亚群中,CD95^bright和CD95^dim表达百分率分别为23．1％和75．6％。与CD56^bright出细胞亚群相比,CD56^bright细胞表达CD8、颗粒酶B和穿孔素细胞的阳性率较高,分别为9．1％、6．2％和8．O％。在CD56^bright和CD56^bright亚群中,CD95^brightCD8^＋亚群高表达颗粒酶B和穿孔素。结论 人外周血CD56^＋NK细胞由不同表型和生物学特征的细胞亚群组成,CD56^dim”和CD56^dimCD8^＋NK细胞亚群在杀伤破坏靶细胞中发挥重要作用。     

HCMV-controlling NKG2C+ NK cells originate from novel circulating inflammatory precursors

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Angiocentric lymphoma with granulomatous panniculitis in the skin expressing natural killer cell and large granular T-cell phenotypes

We investigated three patients suffering from angiocentric lymphoma with granulomatous panniculitis in the skin. All three patients presented with multiple purple subcutaneous nodules. Immunohistologically, the lymphoma cells in all three patients expressed CD2 (T-11), CD56 (neural cell adhesion molecule), and Mik-1 (interleukin-2 receptor). CD3s (CD3, Leu-4)-positive lymphoma cells were found in two patients. A pore-forming protein (perforin) was detected in the lymphoma cells of all three patients. Perforin-possessing lymphoid cells were focally scattered in 2 of 15 patients with CD56-negative cutaneous lymphomas who served as controls. By the Southern blot method, one patient showed a rearranged T-cell receptor (TcR) gene in the biopsied specimen, and the other two patients had germline configurations of TcRs and immunoglobulin heavy chain genes. One patient had serum anti-human T-cell lymphotropic virus (HTLV)-I antibody, but showed no integration of its proviral DNA. Ultrastructurally, membrane-bound azurophilic granules were detected in the atypical lymphoid cells of all three patients. Angiocentric lymphoma with panniculitis in three patients showed the characteristics of natural killer and large granular T-cells. The histological features might be due to the characteristics of the neoplastic cells with azurophilic granules and perforin.This work was supported in part by Grants in Aid from the Fukuoka Cancer Society, Japan     

The roles of perforin- and Fas-dependent cytotoxicity in protection against cytopathic and noncytopathic viruses

In vitro, T cell-dependent cytotoxicity is mediated by two distinct mechanisms, one being perforin-, the other Fas-dependent. The contribution of both of these mechanisms to clearance of viral infections was investigated in mice for the noncytopathic lymphocytic choriomeningitis virus (LCMV) and the cytopathic vaccinia, vesicular stomatitis (VSV) and Semliki forest (SFV) viruses. Clearance of an acute LCMV infection was mediated by the perforin-dependent mechanism without measurable involvement of the Fas-dependent pathway. For the resolution of vaccinia virus infection and for resistance against VSV and SFV, however, neither of the two pathways was required. These data suggest that perforin-dependent cytotoxicity mediated by T cells is crucial for protection against noncytopathic viruses, whereas infections with cytopathic viruses are controlled by nonlytic T cell-dependent soluble mediators such as cytokines (IFN-γ against vaccinia virus) and neutralizing antibodies (against VSV and SFV).     

Myelin oligodendrocyte glycoprotein-induced autoimmune encephalomyelitis is chronic/relapsing in perforin knockout mice,but monophasic in Fas- and Fas ligand-deficient lpr and gld mice

The expression and action of Fas/Fas ligand (FasL) in multiple sclerosis has been postulated as a major pathway leading to inflammatory demyelination. To formally test this hypothesis, C57BL/6-lpr and -gld mice, which due to gene mutation express Fas and FasL in an inactive form, were immunized with myelin oligodendrocyte glycoprotein peptide_35–55. Whereas in wild-type C57BL/6 mice, experimental autoimmune encephalomyelitis (EAE), was chronic/relapsing, EAE in lpr and gld mice was characterized by a lower incidence of disease and a monophasic course. This contrasts with C57BL/6 perforin knockout mice, which showed the most severe form of EAE of all mouse strains tested, the course being chronic relapsing. The difference noted cannot be attributed to an involvement of FasL in oligodendrocyte damage since oligodendrocytes are insensitive to FasL-mediated cytotoxicity in vitro, and since in the acute phase of EAE gld mice also show CD4⁺T cell infiltrates with associated demyelination in brain and spinal cord. Unlike oligodendrocytes, astrocytes were killed by FasL in vitro. It remains to be established whether this latter finding explains the different disease course of lpr and gld mice compared to wild-type and perforin knockout mice.