Dendritic cells pulsed with hsp70-peptide complexes derived from human hepatocellular carcinoma induce specific anti-tumor immune responses

AIM： To investigate the anti-tumor effect of dendritic cells （DCs） pulsed with hsp70-peptide complexes derived from human hepatocellular carcinoma （HCC） cells on human T cells. METHODS： Hsp70-peptide complexes were purified from human HCC cells with column chromatography using ADP-agarose and DEAE-Sepharose. DCs were derived from peripheral blood mononuclear cells of healthy donors in the presence of human GM-CSF and IL-4. The anti-tumor effect of DCs pulsed with hsp70-peptide complexes on human T-cell was assayed by CTL and enzyme-linked immunospot （ELISPOT） tests. RESULTS： Hsp70-peptide complexes derived from human HCC cells activated phenotypic and functional maturation of DCs. The matured DCs stimulated a high level of autologous T-cell proliferation and type Ⅰ cytokine secretion, and induced HCC-specific cytotoxic T lymphocytes （CTLs）, which specifically killed HCC cells by a MHC class Ⅰ restricted mechanism. CONCLUSION： Hsp70-peptide complexes derived from human HCC cells can serve as a potent tumor antigen source for pulsing DCs, the pulsed DCs are very effective in activating specific T-cell responses against HCC cells. 2005 The WJG Press and Elsevier Inc. All rights reserved

STUDY ON THE ANTI-TUMOR EFFICACY INDUCED BY HEAT SHOCK PROTEIN 70-PEPTIDE COMPLEXES DERIVED FROM TUMOR CELLS

OBJECTIVE: To study the efficacy and explore the mechanism of the anti-tumor immunity elicited by heat shock protein 70-peptide complexes (HSP70-PC) derived from tumor cells. METHODS: Cells culture, flow cytometric analysis, affinity chromatography for protein purification, SDS-PAGE, Western-blotting and animal experiment were used. RESULTS: HSP70-PC immunization rendered protective effect to both naive tumorl-bearing mice. All of the naive mice obtained complete resistance to Hcaf cell attack; 40% of the tumor-bearing mice survived for over 90 days, whereas the mice of control group died within 2 weeks (P

Enhancing the treatment effects of tumor cell purified autogenous heat shock protein 70-peptide complexes on HER-3-overexpressing breast cancer

Objective The aim of this study was to enhance the treatment effect of tumor purified autogenous heat shock protein 70-peptide complexes(HSP70-PCs)on HER-3-overexpressing breast cancer.Methods In this study,we first studied the expression of HER-3 in breast cancer tissues and its relationship with patient characteristics.We then purified HSP70-PCs from primary breast cancer cells with different HER-2 and HER-3 expression profiles and determined the cytotoxicity of autogenous dendritic cells(DCs)and CD8+T cells induced by these complexes.Third,recombinant human HSP70-HER-3 protein complexes were used to inhibit the autogenous HSP70-PCs purified from HER-3-overexpressing breast cancer cells,and the resulting immunological response was examined.Results The results show that HSP70-PCs can be combined with recombinant HSP70-HER-3 protein complexes to induce stronger immunological responses than autogenous HSP70-PCs alone and that these treatments induce autogenous CD8+T cell killing of HER-3-positive breast cancer cells.Conclusion These findings provide a new direction for HSP70-DC-based immunotherapy for patients with HER-3-overexpressing breast cancer.

异种HSP_(70)及固相抗体法体外扩增人γδT细胞的比较

目的 :比较异种HSP70 及固相抗体法体外扩增人γδT细胞。方法 :分别用固相抗体法、HSP70BCG 及小鼠榄香烯复合瘤苗热休克蛋白 70 肽复合物 (HSP70m 肽复合物 )体外扩增人PBMCs的γδT细胞 ,流式细胞仪检测细胞表型及亚型分布 ,RT PCR检测各亚型基因的取用。MTT检测γδT细胞的细胞毒作用。结果 :培养 14天后 ,抗体法扩增的PMBCs总数增加 30～ 4 0倍 ,γδT细胞的比率由 3.2 %提高到 86 .7% ;HSP70BCG 组PBMCs总数增加 7～ 8倍 ,γδT细胞的比率由 3.2 %提高到 71.32 % ;HSP70m 肽复合物组PBMCs总数增加 4～ 5倍 ,γδT细胞的比率由 3.2 %提高到 2 7.2 6 %。抗体及HSP70BCG 扩增的γδT细胞以表达Vγ9 Vδ2亚型为主 ,且Vδ1、Vδ2、Vδ3三种基因均得到取用 ,对Daudi具较高的杀伤活性。结论 :抗体及HSP70BCG 均能扩增出高纯度的γδT细胞 ,且具有完整的受体库 ,扩增的γδT细胞具细胞毒活性。

Expression and Purification of Goose HSP70 and Compound Formation with Virus Polypeptide

The experiment was conducted to study the specific expression of HSP70 caused by heat shock, HSP70 purification and the characteristics of coalescence with antigenic peptide in the formation of the complex. Sixty healthy 6-week-old male Wulong geese were selected and randomly divided into three groups. The control group was slaughtered without heat treatment. Treatment group 1 was shocked with an acute heat treatment at （42 ± 1）℃ for 5 h before they were immediately slaughtered. Treatment group 2 was kept for 12 h after the heat treatment under normal conditions in order to recover and was then slaughtered. Cardiac tissue was taken in order to make paraffin sections for the immunohistochemistry experiment and the liver tissue was used to purify HSP70. The geese heart HSP70 expression differences in the three groups were determined and at the same time the experiments of HSP70 purification and appraisal in the liver tissue were carried on. HSP70 purification and synthesis of HBV PreS1 multi-peptides unified the complex, which was determined by bi-specific antibody enzyme-linked immune sandwich assay. The results indicated that widespread HSP70 positive pellets in the cardiac muscle were found under hot shock conditions. HSP70 expression in the treatment group 1 was centered in the karyotheca and its periphery, but in treatment group 2, it was centered in the surrounding cell membrane. The HSP70 purification could be obtained through two sets of purification plans; both the synthesis peptide and the HSP70 purification form the complex under certain conditions. The double antibody sandwich ELISA technique was applied to detect if the complex had been formed. Positive results showed that the complex was formed. The specific expression of HSP70 under heat shock shifted with time, suggesting that HSP70 possibly had some function in cell protection. High-purity HSP70 protein can be obtained under low-pressure chromatography conditions, and in comparison with each other, it was better in the flow of the molecular sieve preliminary separation, ConA-agarose chromatography and the ADP-agarose chromatography. Under certain condition in vitro, the synthetic peptide could combine with HSP70 to form the compound, thereby providing a further experimental foundation for the immunity function of the complex.

小鼠肺腺癌热休克蛋白70-抗原肽复合物的纯化

目的 探索肿瘤热休克蛋白 70抗原肽复合物 (HCA70 )分离、纯化的方法。方法 采用低渗、反复冻融匀浆、ADP -Agarose亲和层析结合DEAE离子交换层析 ,从体外培养的小鼠肺腺癌细胞 (LA - 795)中纯化HAC70 ;通过SDS -PAGE电泳、Westernblot检测、小鼠移植瘤动物实验证实所获得蛋白是否为HAC70。结果 获得蛋白分子量为 70kD左右 ,与抗HSP70特异单抗结合 ,用此蛋白主动免疫的小鼠可抵抗LA795细胞的攻击 ,获得了高纯度的HAC70。结论 采用ADP -Agarose亲和层析结合DEAE离子交换层析可获得高纯度的HAC70。

The complex of Fas-associated factor 1 with Hsp70 stabilizes the adherens junction integrity by suppressing RhoA activation

Fas-associated factor 1 (FAF1) is a scaffolding protein that plays multiple functions, and dysregulation of FAF1 is associated withmany types of diseases such as cancers. FAF1 contains multiple ubiquitin-related domains (UBA, UBL1, UBL2, UAS, and UBX), eachdomain interacting with a specific partner. In particular, the interaction of UBL1 with heat shock protein 70 (Hsp70) is associatedwith tumor formation, although the molecular understanding remains unknown. In this study, the structural analysis revealed thatHis160 of FAF1 is important for its interaction with Hsp70. The association of Hsp70 with FAF1 is required for the interaction withIQGAP1. FAF1 negatively regulates RhoA activation by FAF1–Hsp70 complex formation, which then interacts with IQGAP1. Thesesteps play a key role in maintaining the stability of cell-to-cell junction. We conclude that FAF1 plays a critical role in the structureand function of adherens junction during tissue homeostasis and morphogenesis by suppressing RhoA activation, which induces theactivation of Rho-associated protein kinase, phosphorylation of myosin light chain, formation of actin stress fiber, and disruptionof adherens junction. In addition, depletion of FAF1 increased collective invasion in a 3D spheroid cell culture. These results provideinsightinto how the FAF1–Hsp70 complex acts as a novelregulator ofthe adherens junction integrity. The complex can be a potentialtherapeutic target to inhibit tumorigenesis and metastasis.

Investigation on the effect of peptides mixture from tumor cells inducing anti-tumor specific immune response

The peptides mixture was prepared from tumor cells by freezing-thawing cells, precipitation by heating, followed by acidification of the solution. The activation and proliferation of mouse splenocytes by HSP70-peptide complex, formed by the binding of HSP70 and peptides in vitro, were observed, so was the specific cytotoxicity of the proliferative lymphocytes to tumor cells. The phenotypes of the proliferative lymphocytes were analyzed by a flow cytometer. BALB/c mice inoculated with H22 hepatocarcinoma cells in peritoneal cavity or hind thigh were immunized by injection with HSP70-peptides complex to observe the inhibitory effect of the immunization on tumor and lifetime of tumor-bearing mice. On the other hand, blood samples were collected from the immunized mice to check the functions of liver and kidney. The results showed that the peptides mixture from tumor cells contained tumor-specific antigen peptides which could be presented by HSP70 to activate lymphocytes in vitro, the proliferative lymphocytes were T cells which were specifically cytotoxic to tumor cells, the in vivo growth of both ascitic and solid carcinoma could be suppressed by immunization with HSP70-peptides and the lifetime of tumor-bearing mice was prolonged, the in vivo immunization with HSP70-H22-peptides had no impact on the function of mouse liver and kidney, suggesting that there was no occurrence of autoimmunity in vivo after immunization.