肝癌基因谱动态表达及HSPgp96异常的临床价值

目的:探讨肝细胞癌变过程中基因表达谱的动态变化, 观察热休克蛋白(HSP)gp96表达及其在肝癌早期诊断及判断预后方面的临床价值. 方法:以2-乙酰氨基芴(2-FAA, 0.05%)喂饲SD大鼠诱发肝癌发生, 观察肝病理组织学(HE染色)改变, 用全基因组芯片分析肝基因表达谱的动态变化, 以巢式逆转录酶链反应扩增gp96基因片段.结果: SD大鼠在喂饲2-FAA后, 肝细胞在诱癌早期发生颗粒样变性, 中期出现不典型增生, 后期见大量癌巢结节. 肝癌发生过程中大鼠肝基因表达谱明显改变, 与对照大鼠比较, 癌变大鼠gp96上调3倍; 诱癌变过程中, 肝组织gp96 mRNA表达明显增加, 变性组, 癌前组和癌变组的肝组织中gp96基因表达均明显高于正常对照组(90.9%, 100%, 100% vs 16.67%, P<0.05). 结论:肝细胞癌变过程中基因表达谱明显改变, 其中gp96 mRNA呈过度表达, 其分析有利于肝癌的诊断和预后判断.     

Abnormal expression of HSP gp96 associated with HBV replication in human hepatocellular carcinoma

BACKGROUND: Heat shock protein (HSP) gp96 is a member of the HSP90 family and presumably overexpresses as a result of stimulation by mutated or abnormal proteins. Its abnormal expression correlates with carcinogenesis, progression and prognosis of hepatocellular carcinoma (HCC). In this study, we investigated the pathological characteristics of liver gp96 expression and its relationship with hepatitis B virus (HBV) replication in HCC patients. METHODS: Tumor specimens were prospectively collected from 30 HCC patients undergoing liver resection. Total RNAs were extracted from HCC or their noncancerous tissues. The distribution of gp96 expression in hepatocytes was investigated by streptavidin peroxidase (S-P) immunohistochemistry and tissue HBV-DNA was detected by the in situ molecular hybridization technique. The association of gp96 expression with HBV replication, and the histopathological characteristics of HCC were analyzed. RESULTS: The gp96 was strongly expressed in HCC (73.3%, 22 of 30) and weakly (46.7%, 14 of 30) in non-cancerous tissues. The gp96 expression in HCC tissues was correlated with degree of tumor differentiation and tumor size, but not with tumor number (P>0.05). Immunohistochemical analysis showed that 17 of 19 HCC patients with HBVDNA -positive were strongly expressed for gp96, whereas only 5 of 11 patients with HBV-DNA-negative were positive for gp96. A significant difference was found between the two groups (89.5% vs. 45.5%, P<0.05). CONCLUSIONS: The abnormal expressions of HSP gp96 in HCC tissues are associated with HBV replication. This finding indicates that HBV infection plays an important role in the development of HCC.     

热休克蛋白gp96在原发性肝癌组织中的检测及其意义

热休克蛋白gp96的发现为肿瘤和病毒性肝炎等传染性疾病的免疫治疗开辟了新的途径。用免疫组织化学方法对gp96存原发性肝癌组织中的表达进行检测，探讨gp96在抗肝癌免疫反应中的作用。     

热休克蛋白gp96在慢性乙型肝炎组织中的检测及其意义

对慢性乙型肝炎进行gp96检测 ,探讨gp96表达状况与肝炎病变进展过程的相关性及其在抗乙型肝炎病毒免疫反应中的作用。采用免疫组织化学 (IHC)方法 ,对 77例慢性乙型肝炎病例的活检肝组织标本进行gp96检测 ,其中轻度慢性肝炎者 37例 ,中度 2 0例 ,重度 2 0例。以 16例非肝炎肝组织作为本实验对照。gp96在对照组正常肝组织和慢性乙型肝炎中的平均阳性细胞率分别为 0 5 3%和 15 94 % ,两组间具有非常显著性差异。在慢性肝炎轻、中、重三组 ,阳性细胞平均率分别为 6 14 %、2 1 39%及 2 9 18% ,统计学检验表明组与组之间有显著差异 ,gp96表达率随着炎症程度的加重而上升。提示gp96与机体抗乙肝病毒的免疫应答程度有一定的相关性     

Interaction between heat-shock protein 73 and HLA-DRB1 alleles associated or not with rheumatoid arthritis

OBJECTIVE: HLA-DRB1 alleles whose third hypervariable region contains a QKRAA/QRRAA/RRRAA motif are associated with rheumatoid arthritis (RA) through unknown mechanisms. We previously demonstrated that the QKRAA motif was also expressed on the Escherichia coli 40-kd heat-shock protein (HSP) DnaJ. The QKRAA motif helps DnaJ bind its partner chaperone, the E coli 70-kd HSP DnaK. Furthermore, we observed that in lymphoblastoid cells, Hsp73, the constitutive 70-kd HSP, associates with HLA-DRB1*0401 (an allele with a QKRAA motif) and targets it to lysosomes. In this study, we sought to classify different HLA-DRB1 alleles according to their ability to bind Hsp73. METHODS: To evaluate how well different HLA-DRB1 alleles could bind Hsp73, we developed a quantitative precipitation assay and a direct binding assay. RESULTS: Quantitative precipitation assay from total cellular proteins and from lysosomal extracts demonstrated that RA-associated HLA-DRB1 alleles bound Hsp73 better than did HLA-DRB1 alleles that were not associated with RA. HLA-DRB1*0401 was the best Hsp73 binder. These findings were confirmed by direct binding assay between purified proteins. CONCLUSION: HLA-DRB1*0401 was the best Hsp73 binder among the 8 different HLA-DRB1 alleles that were tested.     

Endoplasmic reticulum chaperone gp96 is essential for infection with vesicular stomatitis virus

The envelope glycoprotein of vesicular stomatitis virus (VSV-G) enables viral entry into hosts as distant as insects and vertebrates. Because of its ability to support infection of most, if not all, human cell types VSV-G is used in viral vectors for gene therapy. However, neither the receptor nor any specific host factor for VSV-G has been identified. Here we demonstrate that infection with VSV and innate immunity via Toll-like receptors (TLRs) require a shared component, the endoplasmic reticulum chaperone gp96. Cells without gp96 or with catalytically inactive gp96 do not bind VSV-G. The ubiquitous expression of gp96 is therefore essential for the remarkably broad tropism of VSV-G. Cells deficient in gp96 also lack functional TLRs, which suggests that pathogen-driven pressure for TLR-mediated immunity maintains the broad host range of VSV-G by positively selecting for the ubiquitous expression of gp96.     

Heat-shock protein gp96/grp94 is an essential chaperone for the platelet glycoprotein Ib-IX-V complex

The platelet glycoprotein Ib-IX-V complex (GPIb-IX-IV) is the receptor for VWF and is responsible for VWF-mediated platelet activation and aggregation. Loss of the GPIb-IX-V complex is pathogenic for Bernard-soulier Syndrome (BSS), which is characterized by macrothrombocytopenia and impaired platelet function. It remains unclear how the GPIb-IX-V complex is assembled and whether there is a role for a specific molecular chaperone in the process. In the present study, we report that the assembly of the GPIb-IX-V complex depends critically on a molecular chaperone in the endoplasmic reticulum (ER): gp96 (also known as grp94 and HSP90b1). gp96/grp94 deletion in the murine hematopoietic system results in thrombocytopenia, prolonged bleeding time, and giant platelets that are clinically indistinguishable from human BSS. Loss of gp96/grp94 in vivo and in vitro leads to the concomitant reduction in GPIb-IX complex expression due to ER-associated degradation. We further demonstrate that gp96/grp94 binds selectively to the GPIX subunit, but not to gpIbα or gpIbβ. Therefore, we identify the platelet GPIX subunit of the GPIb-IX-V complex as an obligate and novel client of gp96/grp94.     

Acute phase HBV-specific T cell responses associated with HBV persistence after HBV/HCV coinfection

To characterize acute-phase hepatitis B virus (HBV)-specific T cell responses associated with self-limited and persistent HBV infections, we compared a patient with acute HBV/HCV coinfection, who was able to control HCV but developed chronic hepatitis B, with patients who resolved acute HBV infection spontaneously. Acute-phase CD4 responses were efficient in self-limited infections but undetectable in the coinfected patient with HBV persistence. CD8 responses were multispecific irrespective of the outcome of infection, but the CD8 repertoire associated with HBV persistence lacked the most dominant specificities detectable in self-limited infections. In conclusion, insufficient CD4 help and defective CD8 repertoire may play a role at the early stages of infection in influencing HBV persistence.     

Enhancement of humoral immune responses to HBsAg by heat shock protein gp96 and its N-terminal fragment in mice

AIM: Most studies on the immune effect of gp96 were focused on its enhancement of CTLs. It is interesting to know whether gp96 could influence the humoral immune response, and whether the recombinant N-terminal fragment of gp96 could substitute native gp96 to stimulate the immune system. METHODS: gp96 isolated from livers of normal mice and its N-terminal fragment (amino acid 22-355) expressed in E coli were used for immunization of BALb/c mice. Eight groups of mice received one of the following regiments subcutaneously in 100 μL phosphate buffered saline (PBS) at an interval of 3 wk. Group 1: PBS only; group 2: gp96 only; group 3: N-terminal fragment only; group 4: HBsAg only; group 5: HBsAg+gp96; group 6: HBsAg+N-terminal fragment; group 7: HBsAg+incomplete Freud's adjuvant; group 8: HBsAg+N-terminal fragment (95℃ heated for 30 min). Serum anti-HBsAg antibody levels were assayed by ELISA. CTL responses in splenocytes were analyzed by ELISPOT after the last vaccination. RESULTS: The average titer of serum anti-HBsAg antibody in the mice immunized with HBsAg together with gp96 or its N-terminal fragment were much higher than those immunized with HBsAg alone detected by ELISA. The cellular immune response of the mice immunized with HBsAg together with gp96 or its N-terminal fragment was not different with those immunized with HBsAg alone measured by ELISPOT assay. CONCLUSION: gp96 or its N-terminal fragment greatly improved humoral immune response induced by HBsAg, but failed to enhance the CTL response, which demonstrated the potential of using gp96 or its N-terminal fragment as a possible adjuvant to augment humoral immune response against HBV infection.     

Essential role of CD91 in re-presentation of gp96-chaperoned peptides

Heat shock proteins (HSPs) such as gp96 are released from cells as a result of necrotic cell death. The ability of endogenous HSP-peptide complexes to elicit antigen-specific T cells requires representation of the chaperoned peptides by antigen-presenting cells. Re-presentation requires the uptake of HSP-peptide complexes through a receptor, suggested to be the low-density lipoprotein receptor-related protein or CD91. We have used short interfering RNA for CD91 to show that, as antigen-presenting cells lose expression of CD91, their re-presenting ability undergoes a corresponding and dramatic decline. Furthermore, as the cells recover from extinction of CD91 expression, they regain the ability to re-present peptides. The ability of cells to bind alpha(2) macroglobulin, a previously known CD91 ligand, or HSP gp96, and their ability to process peptides chaperoned by alpha(2) macroglobulin, undergo identical variations. These results have been obtained from studies in vitro and from an assay that measures re-presentation in vivo. In additional studies in vivo, protective tumor immunity elicited by tumor-derived gp96-peptide complexes is shown to be abrogated by anti-CD91 antisera. These studies show that CD91 is essential for re-presentation of gp96-chaperoned peptides by MHC molecules and have an important bearing on the mechanism of immunogenicity of necrotic cells.     

三步法提取纯化肺腺癌组织中HSPgp96多肽复合物

目的建立从人肺癌组织中提取,为肺癌肿瘤免疫治疗的临床应用提供实验依据纯化热休克蛋白（HSP heatshock protein）gp96多肽复合物的方法 ;并通过体外实验对其在肺癌肿瘤免疫中的作用进行初步研究。方法运用三步蛋白纯化法从肺腺癌（PAa）组织中提取、纯化HSPgp96肽复合物,SDSPAGE、Westernblot鉴定HSPgp96多肽复合物。提取的gp96-多肽复合物与树突状细胞共孵育培养3～10天后,第10天加入到由人外周血中分离的淋巴细胞,继续培养3～7天刺激细胞毒T淋巴细胞的产生;以gp96-多肽复合物致敏淋巴细胞作为阳性对照组;以未加入任何抗原成分的DC为阴性对照。收集被致敏淋巴细胞的上清,采用IFN-γELISA检测试剂盒分析淋巴细胞所释放的IFN-γ量作为CTL反应的指标。结果应用该方法提取的蛋白质经SDSPAGE和Westernblot特异性鉴定,证实是HSPgp96多肽复合物;蛋白产率为每克PAa肺癌组织可纯化HSPgp96肽复合物约50μg。肿瘤免疫学实验发现,以gp96-多肽复合物作为肿瘤抗原负载DC,以及单独以gp96-多肽复合物作为肿瘤抗原,分别致敏淋巴细胞后均可以诱导产生CTL反应;且gp96/DC为疫苗所释放的IFN-γ量高于单独以gp96为疫苗所释放的IFN-γ量,有显著性差异（P〈0.01）。结论三步蛋白纯化法不仅操作简便、重复性高,而且提取的HSPgp96肽复合物具有高获率、高纯度、高免疫效应等优点。gp96-多肽复合物能诱导出肿瘤特异性CTL反应,而将其负载DC后能激发起更强的CTL,显示出gp96-多肽复合物/DC疫苗抗肿瘤作用的广泛前景。     

Inhibition of adjuvant-induced arthritis by DNA vaccination with the 70-kd or the 90-kd human heat-shock protein: immune cross-regulation with the 60-kd heat-shock protein

OBJECTIVE: Adjuvant arthritis can be induced in Lewis rats by immunization with Mycobacterium tuberculosis (Mt). The mycobacterial 65-kd heat-shock protein (Hsp65) is targeted by arthritogenic T cells. However, Hsp65 and the mycobacterial 71-kd heat-shock protein are also recognized by T cells that can down-regulate adjuvant-induced arthritis (AIA). We have recently demonstrated that vaccination with human Hsp60 DNA inhibits AIA. The present study was undertaken to analyze the role of the T cell responses to self HSP molecules other than Hsp60 in the control of AIA. METHODS: Lewis rats were immunized with DNA vaccines coding for human Hsp70 or Hsp90 (Hsp70 plasmid [pHsp70] or pHsp90), and AIA was induced. The T cell response to Mt, Hsp60, Hsp70, and Hsp90 (proliferation and cytokine release) was studied, and the T cell response to Hsp60 was mapped with overlapping peptides. RESULTS: The Hsp70 or Hsp90 DNA vaccines shifted the arthritogenic T cell response from a Th1 to a Th2/3 phenotype and inhibited AIA. We detected immune crosstalk between Hsp70/90 and Hsp60: both the Hsp70 and Hsp90 DNA vaccines induced Hsp60-specific T cell responses. Similarly, DNA vaccination with Hsp60 induced Hsp70-specific T cell immunity. Epitope mapping studies revealed that Hsp60-specific T cells induced by pHsp70 vaccination reacted with known regulatory Hsp60 epitopes. CONCLUSION: T cell immunity to Hsp70 and to Hsp90, like Hsp60-specific immunity, can modulate the arthritogenic response in AIA. In addition, our results suggest that the regulatory mechanisms induced by Hsp60, Hsp70, and Hsp90 are reinforced by an immune network that connects their reactivities.     

Cell surface expression of an endoplasmic reticulum resident heat shock protein gp96 triggers MyD88-dependent systemic autoimmune diseases

Heat shock proteins have been implicated as endogenous activators for dendritic cells (DCs). Without tissue distress or death, these intracellular molecules are inaccessible to surface receptor(s) on DCs, possibly to avoid uncontrolled DC activation and breakdown of immunologic tolerance. We herein addressed this hypothesis in transgenic mice by enforcing cell surface expression of gp96, a ubiquitous heat shock protein of the endoplasmic reticulum. Although a pan-specific promoter is used for transgene expression, neither the expression level nor the tissue distribution of the endogenous gp96 was altered by this maneuver. However, cell surface gp96 induced significant DC activations and spontaneous lupus-like autoimmune diseases, even though the development/functions of lymphocytic compartments were unaltered. Using a bone marrow chimera approach, we further demonstrated that both DC activation and autoimmunity elicited by cell surface gp96 are dependent on the downstream adaptor protein MyD88 for signaling by Toll/IL-1 receptor family. Our study not only established the proinflammatory property of cell surface gp96 in vivo, but also suggested a chronic stimulation of DCs by gp96 as a pathway to initiate spontaneous autoimmune diseases.     

热休克蛋白gp96、髓样细胞白血病-1和阻抑蛋白在肝硬化和肝癌组织中的表达

目的:分析热休克蛋白gp96、髓样细胞白血病-1(MCL-1)和阻抑蛋白(PHB)在LC和HCC组织中的表达及其临床意义.方法:免疫组化二步法分别检测19例LC,32例HCC和21例对照组肝组织中gp96,MCL-1和PHB的表达,并分析其与肝癌临床病理学特征的关系.结果:gp96在对照组、LC组和HCC组的表达逐渐增强(P＜0.05);HCC无包膜、有坏死和门静脉有癌栓者gp96表达较高;gp96表达与HCC分化程度呈负相关(r=-0.4655,P=0.0073),与TNM分期呈正相关(r=0.5157,P=0.0025).MCL-1和PHB在HCC组织中过表达,HCC有坏死者MCL-1表达高于无坏死者,HCC无包膜者PHB表达高于有包膜者(P＜0.05).结论:gp96,MCL-1和PHB的过表达可能与HCC的发生、发展有关.gp96可能E参与LC的发生、发展及向HCC的恶性转化,有助于判断HCC患者的预后.     

A cellular protein that associates with the transforming protein of Rous sarcoma virus is also a heat-shock protein

A single viral protein (pp60src) mediates neoplastic transformation of cells infected with Rous sarcoma virus. Immunoprecipitation of pp60src has revealed two cellular proteins (Mr 50,000 and 89,000) that appear to associate with pp60src in a specific manner. Neither of the cellular proteins has been well characterized, but it is thought that both may participate in the function of pp60src. Treatment of avian cells with unphysiological temperature or certain chemical agents amplifies the production of several proteins in the manner of the "heat shock" response earlier described for Drosophila. We report here that one of these proteins, with a molecular weight of 89,000 is identical to the 89-kilodalton protein found associated with pp60src. The 89-kilodalton protein is a major constituent of both uninfected and infected cells, even in the absence of inducing agents, but only a small fraction of this protein appears to associate with pp60src in cells transformed by Rous sarcoma virus. The complex containing pp60src and the 89-kilodalton protein can be precipitated by an immune reaction involving pp60src alone. The complexed form of the 89-kilodalton protein did not react directly with antibodies but regained its reactivity subsequent to release from the complex. We conclude that the 89-kilodalton protein is bound to pp60src in a relatively stable complex. We suggest that the 89-kilodalton protein may have overlapping roles in viral oncogenesis and the heat shock response, and that evidence on the function of the protein in either setting may illuminate its function in the other. In addition, it may prove profitable to search for other overlaps between the cellular response to heat shock and the neoplastic transformation of cells by pp60src.     

CIGB-814, an altered peptide ligand derived from human heat-shock protein 60, decreases anti-cyclic citrullinated peptides antibodies in patients with rheumatoid arthritis

Clinical Rheumatology - Rheumatoid arthritis (RA) is a chronic T cell-mediated autoimmune disease. Serum autoantibodies against cyclic citrullinated peptides (anti-CCP) are significant markers for...