AIMS To investigate the alterations of RBC immunoadherence function in patients with various hepatitis B. METHODS RBCC3bRR,RBCICRR and serum CIC levels were measured in 42 patients with acute and chronic hepatitis B a...AIMS To investigate the alterations of RBC immunoadherence function in patients with various hepatitis B. METHODS RBCC3bRR,RBCICRR and serum CIC levels were measured in 42 patients with acute and chronic hepatitis B at ac- tive and convalescence stages. RESULTS RBCC3bRRs at the active/acute stage of various hepatitis were decreased.They were 13,54%±5,23% in AH, 7.61%±4.12% in AFH,and 16.18%±6.10% in CH, respectively,all of which were lower than those in normal persons (18.12%±3.91% ).At the quiescent/recovery stage of various hepatitis,the RBCC3bRRs were increased significantly.The changes of RBCICRR and serum CIC level were contrary to those of RBCC3bRR. CONCLUSIONS RBC immunoadherence function is decreased in acute and chronic hepatitis.The decrease is in direct proportion to the severity of the diseases.展开更多
Objective: To study the protective and therapeutic antitumor immunity against hepatocellular carcinoma (HCC) with the fixed-tumor vaccine.Methods: A tumor vaccine consisting of fixed tumor cells or fixed tumor fragmen...Objective: To study the protective and therapeutic antitumor immunity against hepatocellular carcinoma (HCC) with the fixed-tumor vaccine.Methods: A tumor vaccine consisting of fixed tumor cells or fixed tumor fragments combined with sustained-releasers of cytokines and a non-toxic adjuvant was developed. C57BL/6J mice were immunized intra-dermally with the vaccine on day 0 and 7, followed by intrahepatic challenge with live Hepa 1–6 cells.Results: All of 15 nonimmunized control mice developed the hepatoma. Protection of mice immunized with fixed Hepa 1–6 cells and both of IL-2/GM-CSF microspheres or further mixed with TiterMax Gold reached 80% and 87%, respectively. Mass growth of the established tumors, vaccinated twice at 5 mm in diameter, the tumor of control animals continued to grow. However, 7–10 days after the second injection of the tumor vaccine, the tumor growth was suppressed in 9 of 10 mice and then markedly reduced. Complete tumor regression was observed in 60% (6/10) of mice. Splenocytes from the control mice were not able to lyse target Hepa 1–6 cells and other tumor cells. In contrast splenocytes from the vaccinated mice exhibited a 41% lytic activity against the Hepa 1–6 cells tested at an effector/target (E/T) ratio of 5, whereas they did not exhibited such activity against the melanoma cells (B16-F1), Lewis lung carcinoma cells (LLC), renal carcinoma cells (Renca), and bladder carcinoma cells (MBT-2). The cytotoxic activity was inhibited by the treatment with anti-CD3, anti-CD8, and anti-MHC-class I monoclonal antibodies but not with anti-CD4 and anti-MHC-class II antibodies. In the Phase-I clinical trial, vaccination of HCC patients with the autologous vaccine is a well-tolerated treatment and induces fixed tumor fragment-specific immunity.Conclusion: Fixed HCC vaccination elicited protective and therapeutic antitumor immunity against HCC. The tumor vaccine elicited antigen specific CTL response lysis of the target HCC was mediated by the typical MHC-class I restricted CD8+ T cells. Key words cancer vaccine - cytotoxic T lymphocyte - immunotherapy - hepatoma展开更多
Objective To genetically correct a disease-causing point mutation in human induced pluripotent stem cells （iPSCs） derived from a hemophilia B patient. Methods First, the disease-causing mutation was detected by ...Objective To genetically correct a disease-causing point mutation in human induced pluripotent stem cells （iPSCs） derived from a hemophilia B patient. Methods First, the disease-causing mutation was detected by sequencing the encoding area of human coagulation factor IX （F IX） gene. Genomic DNA was extracted from the iPSCs, and the primers were designed to amplify the eight exons of F IX. Next, the point mutation in those iPSCs was genetically corrected using CRISPR/Cas9 technology in the presence of a 129-nucleotide homologous repair template that contained two synonymous mutations. Then, top 8 potential off-target sites were subsequently analyzed using Sanger sequencing. Finally, the corrected clones were differentiated into hepatocyte-like cells, and the secretion of F IX was validated by immunocytochemistry and ELISA assay.Results The cell line bore a missense mutation in the 6th coding exon （c.676 C〉T） of F IX gene. Correction of the point mutation was achieved via CRISPR/Cas9 technology in situ with a high efficacy at about 22% （10/45） and no off-target effects detected in the corrected iPSC clones. F IX secretion, which was further visualized by immunocytochemistry and quantified by ELISA in vitro, reached about 6 ng/ml on day 21 of differentiation procedure. Conclusions Mutations in human disease-specific iPSCs could be precisely corrected by CRISPR/Cas9 technology, and corrected cells still maintained hepatic differentiation capability. Our findings might throw a light on iPSC-based personalized therapies in the clinical application, especially for hemophilia B.展开更多
A case of a 53-year-old female patient with reactive lymphoid hyperplasia （RLH）, clinically designated as pseudolymphoma of the liver is described in this article. The patient was admitted to our hospital for furthe...A case of a 53-year-old female patient with reactive lymphoid hyperplasia （RLH）, clinically designated as pseudolymphoma of the liver is described in this article. The patient was admitted to our hospital for further evaluation of hepatic tumors incidentally discovered at another hospital. Various diagnostic methods, including ultrasonography （US）, computerized tomography （CT）, magnetic resonance imaging （MRI） and hepatic angiography displayed three small lesions in the liver with outstanding findings consistent with hepatocellular carcinoma （HCC）. Surgical resection was performed and the three lesions were microscopically diagnosed as RLH of the liver. The lesions comprised a massive infiltration of lymphoid cells with follicles and hyalinized inter- follicular spaces. Immunohistochemical examination revealed that infiltrating lymphocytes had no prominent nuclear atypia and polyclonality. RLH of the liver is a very rare condition and only twelve cases have been reported in the English literature. Majority of the reported cases were middle-aged women and about half of them had some immunologic abnormalities such as autoimmune thyroiditis, Sjogren＇s syndrome, primary immunodeficiency, primary biliary cirrhosis. Since they are olden clinically misdiagnosed as HCC, surgery is the choice of treatment for these patients. Although their pathology resembles malignant lymphoma, the clinical course is completely benign. The authors propose that RLH of the liver can be discriminated from HCC by its clinical features.展开更多
AIM： To identify a multi serum protein pattern as well as single protein markers using surface-enhanced laser desorption/ionisation time-of-flight mass spectrometry （SELDI-TOF-MS） for detection and differentiation ...AIM： To identify a multi serum protein pattern as well as single protein markers using surface-enhanced laser desorption/ionisation time-of-flight mass spectrometry （SELDI-TOF-MS） for detection and differentiation of liver fibrosis （F1-F2）, liver cirrhosis （F4） and hepatocellular carcinoma （HCC） in patients with chronic hepatitis C virus （HCV）. METHODS： Serum samples of 39 patients with F1/F2 fibrosis, 44 patients with F4 fibrosis, 34 patients with HCC were applied to CM10 arrays and analyzed using the SELDI-TOF ProteinChip System （PBS-Ⅱc; Ciphergen Biosystems） after anion-exchange fractionation. All patients had chronic hepatitis C and histologically confirmed fibrosis stage/HCC. Data were analyzed for protein patterns by multivariate statistical techniques and artificial neural networks. RESULTS： A 4 peptide/protein multimarker panel （7486, 12843, 44293 and 53598 Da） correctly identified HCCs with a sensitivity of 100% and specificity of 85% in a two way-comparison of HCV-cirrhosis versus HCV-HCC training samples （AUROC 0.943）. Sensitivity and specificity for identification of HCC were 68% and 80% for random test samples. Cirrhotic patients could be discriminated against patients with F1 or F2 fibrosis using a 5 peptide/protein multimarker pattern （2873, 6646, 7775, 10525 and 67867 Da） with a specificity of 100% and a sensitivity of 85% in training samples （AUROC 0.976） and a sensitivity and specificity of 80% and 67% for random test samples. Combination of the biomarker classifiers with APR/score and alfa-fetopotein （AFP） improved the diagnostic performance. The 6646 Da marker protein for liver fibrosis was identified as apolipoprotein C-I. CONCLUSION： SELDI-TOF-MS technology combined with protein pattern analysis seems a valuable approach for the identification of liver cirrhosis and hepatocellular carcinoma in patients with chronic hepatitis C. Host probably a combination of different serum markers will help to identify liver cirrhosis and early-stage hepatocellular carcinomas in the future.展开更多
Lymphoepitelioma is a particular form of undifferentiat-ed carcinoma, characterized by a prominent lymphoid stroma, originally described in the nasopharynx. Lym-phoid stroma-rich carcinomas arising in other organs hav...Lymphoepitelioma is a particular form of undifferentiat-ed carcinoma, characterized by a prominent lymphoid stroma, originally described in the nasopharynx. Lym-phoid stroma-rich carcinomas arising in other organs have been termed lymphoepithelioma-like carcinoma (LELC). In the liver, primary LELCs are very rare, and the majority has been identified as cholangiocarcino-mas. Here a rare case of lymphoepithelioma-like hepa-tocellular carcinoma (HCC) is described. A 47-year old woman presented with abdominal pain. Ultrasonogra-phy revealed a liver nodule, 2.2 cm in diameter, local-ized in the right lobe, adjacent to the gallbladder. Viral markers for hepatic B virus (HBV), hepatic C virus (HCV) and Epstein-Barr virus (EBV) were negative. The nod-ule was hypoechogenic. The patient underwent sur-gery, with resection of the nodule. Histology showed hepatocellular carcinoma, characterized by a promi-nent lymphoid infiltrate. At immunocytochemistry, tumor cells were reactive for Hep Par1 and glypican 3. Immunophenotyping of tumor infiltrating lymphocytes evidenced the predominance of CD8+ cytotoxic sup-pressor T cells. The postoperative clinical outcome was favorable and the patient was recurrence-free 15 mo after resection. This case, to the best of our knowl-edge, is the first reported non EBV and non cirrhosis-associated lymphoepithelioma-like hepatocellular carci-noma. The association between the lack of EBV infec-tion, the absence of cirrhosis, a "cytotoxic profile" of the inflammatory infiltrate and a good prognosis could identify a variant of lymphoepithelioma-like HCC with a favorable clinical outcome.展开更多
We report a case of fatal liver failure due to reactivation of lamivudine-resistant HBV. A 53-year-old man was followed since 1998 for HBV-related chronic hepatitis. Serum HBV-DNA was 150 MEq/mL (branched DNA signal a...We report a case of fatal liver failure due to reactivation of lamivudine-resistant HBV. A 53-year-old man was followed since 1998 for HBV-related chronic hepatitis. Serum HBV-DNA was 150 MEq/mL (branched DNA signal amplification assay) and ALT levels fluctuated between 50-200 IU/L with no clinical signs of liver cirrhosis. Lamivudine (100 mg/d) was started in May 2001 and serum HBV-DNA subsequently decreased below undetectable levels. In May 2002, serum HBV-DNA had increased to 410 MEq/mL, along with ALT flare (226 IU/L). The YMDD motif in the DNA polymerase gene had been replaced by YIDD. Lamivudine was continued and ALT spontaneously decreased to the former levels. On Oct 3 the patient presenting with general fatigue, nausea and jaundice was admitted to our hospital. The laboratory data revealed HBV reactivation and liver failure (ALT: 1828 IU/L, total bilirubin: 10 mg/dL, and prothrombin INR: 3.24). For religious reasons, the patient and his family refused blood transfusion, plasma exchange and liver transplantation. The patient died 10 d after admission. The autopsy revealed remarkable liver atrophy.展开更多
文摘目的:研究负载肝癌抗原的肝癌树突状细胞(dendritic cell,DC)瘤苗诱导的抗肿瘤作用。方法:于体外用rhGM-CSF和rhIL-4从肝癌患者外周血诱导DC,并用肝癌细胞抗原冲击致敏,制成负载肝癌抗原的肝癌DC瘤苗。肝癌DC瘤苗活化自体T淋巴细胞分化为细胞毒性T淋巴细胞(cytotoxic of T-lymphocytes,CTL),采用流式细胞术检测CTL分型;乳酸脱氢酸(LDH)4 h释放法检测CTL对肝癌细胞的特异性杀伤作用。MTT法检测肝癌DC瘤苗及其活化CTL培养上清对肿瘤细胞的抑制作用;ELISA法检测肝癌DC瘤苗活化的CTL培养上清中IL-12、IFN-γ和TNF-α水平。结果:经肝癌DC瘤苗活化后的T淋巴细胞群中,CD56+细胞数量显著减少,CD4+T和CD8+T细胞数量增加,其中以CD8+T细胞增加较明显;细胞毒实验显示,该CTL对自体肝癌细胞具有较强的杀伤作用,而对与致敏DC的肝癌抗原无关的CT26结肠腺癌细胞无明显杀伤作用;单纯肝癌DC瘤苗或其激活的CTL培养上清也表现出较强的抑瘤作用,而且这种抑瘤作用具有广谱性,可抑制多种肿瘤细胞的生长;在CTL培养上清中可检测到IL-12、TNF-α和IFN-γ的含量。结论:肝癌DC瘤苗不仅诱导了对肝癌细胞的特异性CTL杀伤作用,而且可激活CD4+T和CD8+T细胞分泌IL-12、TNF-α和IFN-γ等细胞因子,以非杀伤方式直接或间接地抑制肿瘤细胞生长,发挥非特异性的抗肿瘤作用。
文摘AIMS To investigate the alterations of RBC immunoadherence function in patients with various hepatitis B. METHODS RBCC3bRR,RBCICRR and serum CIC levels were measured in 42 patients with acute and chronic hepatitis B at ac- tive and convalescence stages. RESULTS RBCC3bRRs at the active/acute stage of various hepatitis were decreased.They were 13,54%±5,23% in AH, 7.61%±4.12% in AFH,and 16.18%±6.10% in CH, respectively,all of which were lower than those in normal persons (18.12%±3.91% ).At the quiescent/recovery stage of various hepatitis,the RBCC3bRRs were increased significantly.The changes of RBCICRR and serum CIC level were contrary to those of RBCC3bRR. CONCLUSIONS RBC immunoadherence function is decreased in acute and chronic hepatitis.The decrease is in direct proportion to the severity of the diseases.
文摘Objective: To study the protective and therapeutic antitumor immunity against hepatocellular carcinoma (HCC) with the fixed-tumor vaccine.Methods: A tumor vaccine consisting of fixed tumor cells or fixed tumor fragments combined with sustained-releasers of cytokines and a non-toxic adjuvant was developed. C57BL/6J mice were immunized intra-dermally with the vaccine on day 0 and 7, followed by intrahepatic challenge with live Hepa 1–6 cells.Results: All of 15 nonimmunized control mice developed the hepatoma. Protection of mice immunized with fixed Hepa 1–6 cells and both of IL-2/GM-CSF microspheres or further mixed with TiterMax Gold reached 80% and 87%, respectively. Mass growth of the established tumors, vaccinated twice at 5 mm in diameter, the tumor of control animals continued to grow. However, 7–10 days after the second injection of the tumor vaccine, the tumor growth was suppressed in 9 of 10 mice and then markedly reduced. Complete tumor regression was observed in 60% (6/10) of mice. Splenocytes from the control mice were not able to lyse target Hepa 1–6 cells and other tumor cells. In contrast splenocytes from the vaccinated mice exhibited a 41% lytic activity against the Hepa 1–6 cells tested at an effector/target (E/T) ratio of 5, whereas they did not exhibited such activity against the melanoma cells (B16-F1), Lewis lung carcinoma cells (LLC), renal carcinoma cells (Renca), and bladder carcinoma cells (MBT-2). The cytotoxic activity was inhibited by the treatment with anti-CD3, anti-CD8, and anti-MHC-class I monoclonal antibodies but not with anti-CD4 and anti-MHC-class II antibodies. In the Phase-I clinical trial, vaccination of HCC patients with the autologous vaccine is a well-tolerated treatment and induces fixed tumor fragment-specific immunity.Conclusion: Fixed HCC vaccination elicited protective and therapeutic antitumor immunity against HCC. The tumor vaccine elicited antigen specific CTL response lysis of the target HCC was mediated by the typical MHC-class I restricted CD8+ T cells. Key words cancer vaccine - cytotoxic T lymphocyte - immunotherapy - hepatoma
基金Supported by the National Science and Technology Major Project(2011ZX09102-010-04)
文摘Objective To genetically correct a disease-causing point mutation in human induced pluripotent stem cells （iPSCs） derived from a hemophilia B patient. Methods First, the disease-causing mutation was detected by sequencing the encoding area of human coagulation factor IX （F IX） gene. Genomic DNA was extracted from the iPSCs, and the primers were designed to amplify the eight exons of F IX. Next, the point mutation in those iPSCs was genetically corrected using CRISPR/Cas9 technology in the presence of a 129-nucleotide homologous repair template that contained two synonymous mutations. Then, top 8 potential off-target sites were subsequently analyzed using Sanger sequencing. Finally, the corrected clones were differentiated into hepatocyte-like cells, and the secretion of F IX was validated by immunocytochemistry and ELISA assay.Results The cell line bore a missense mutation in the 6th coding exon （c.676 C〉T） of F IX gene. Correction of the point mutation was achieved via CRISPR/Cas9 technology in situ with a high efficacy at about 22% （10/45） and no off-target effects detected in the corrected iPSC clones. F IX secretion, which was further visualized by immunocytochemistry and quantified by ELISA in vitro, reached about 6 ng/ml on day 21 of differentiation procedure. Conclusions Mutations in human disease-specific iPSCs could be precisely corrected by CRISPR/Cas9 technology, and corrected cells still maintained hepatic differentiation capability. Our findings might throw a light on iPSC-based personalized therapies in the clinical application, especially for hemophilia B.
文摘A case of a 53-year-old female patient with reactive lymphoid hyperplasia （RLH）, clinically designated as pseudolymphoma of the liver is described in this article. The patient was admitted to our hospital for further evaluation of hepatic tumors incidentally discovered at another hospital. Various diagnostic methods, including ultrasonography （US）, computerized tomography （CT）, magnetic resonance imaging （MRI） and hepatic angiography displayed three small lesions in the liver with outstanding findings consistent with hepatocellular carcinoma （HCC）. Surgical resection was performed and the three lesions were microscopically diagnosed as RLH of the liver. The lesions comprised a massive infiltration of lymphoid cells with follicles and hyalinized inter- follicular spaces. Immunohistochemical examination revealed that infiltrating lymphocytes had no prominent nuclear atypia and polyclonality. RLH of the liver is a very rare condition and only twelve cases have been reported in the English literature. Majority of the reported cases were middle-aged women and about half of them had some immunologic abnormalities such as autoimmune thyroiditis, Sjogren＇s syndrome, primary immunodeficiency, primary biliary cirrhosis. Since they are olden clinically misdiagnosed as HCC, surgery is the choice of treatment for these patients. Although their pathology resembles malignant lymphoma, the clinical course is completely benign. The authors propose that RLH of the liver can be discriminated from HCC by its clinical features.
基金Supported by a research grant of the Jurgen Manchot Stiftung
文摘AIM： To identify a multi serum protein pattern as well as single protein markers using surface-enhanced laser desorption/ionisation time-of-flight mass spectrometry （SELDI-TOF-MS） for detection and differentiation of liver fibrosis （F1-F2）, liver cirrhosis （F4） and hepatocellular carcinoma （HCC） in patients with chronic hepatitis C virus （HCV）. METHODS： Serum samples of 39 patients with F1/F2 fibrosis, 44 patients with F4 fibrosis, 34 patients with HCC were applied to CM10 arrays and analyzed using the SELDI-TOF ProteinChip System （PBS-Ⅱc; Ciphergen Biosystems） after anion-exchange fractionation. All patients had chronic hepatitis C and histologically confirmed fibrosis stage/HCC. Data were analyzed for protein patterns by multivariate statistical techniques and artificial neural networks. RESULTS： A 4 peptide/protein multimarker panel （7486, 12843, 44293 and 53598 Da） correctly identified HCCs with a sensitivity of 100% and specificity of 85% in a two way-comparison of HCV-cirrhosis versus HCV-HCC training samples （AUROC 0.943）. Sensitivity and specificity for identification of HCC were 68% and 80% for random test samples. Cirrhotic patients could be discriminated against patients with F1 or F2 fibrosis using a 5 peptide/protein multimarker pattern （2873, 6646, 7775, 10525 and 67867 Da） with a specificity of 100% and a sensitivity of 85% in training samples （AUROC 0.976） and a sensitivity and specificity of 80% and 67% for random test samples. Combination of the biomarker classifiers with APR/score and alfa-fetopotein （AFP） improved the diagnostic performance. The 6646 Da marker protein for liver fibrosis was identified as apolipoprotein C-I. CONCLUSION： SELDI-TOF-MS technology combined with protein pattern analysis seems a valuable approach for the identification of liver cirrhosis and hepatocellular carcinoma in patients with chronic hepatitis C. Host probably a combination of different serum markers will help to identify liver cirrhosis and early-stage hepatocellular carcinomas in the future.
文摘Lymphoepitelioma is a particular form of undifferentiat-ed carcinoma, characterized by a prominent lymphoid stroma, originally described in the nasopharynx. Lym-phoid stroma-rich carcinomas arising in other organs have been termed lymphoepithelioma-like carcinoma (LELC). In the liver, primary LELCs are very rare, and the majority has been identified as cholangiocarcino-mas. Here a rare case of lymphoepithelioma-like hepa-tocellular carcinoma (HCC) is described. A 47-year old woman presented with abdominal pain. Ultrasonogra-phy revealed a liver nodule, 2.2 cm in diameter, local-ized in the right lobe, adjacent to the gallbladder. Viral markers for hepatic B virus (HBV), hepatic C virus (HCV) and Epstein-Barr virus (EBV) were negative. The nod-ule was hypoechogenic. The patient underwent sur-gery, with resection of the nodule. Histology showed hepatocellular carcinoma, characterized by a promi-nent lymphoid infiltrate. At immunocytochemistry, tumor cells were reactive for Hep Par1 and glypican 3. Immunophenotyping of tumor infiltrating lymphocytes evidenced the predominance of CD8+ cytotoxic sup-pressor T cells. The postoperative clinical outcome was favorable and the patient was recurrence-free 15 mo after resection. This case, to the best of our knowl-edge, is the first reported non EBV and non cirrhosis-associated lymphoepithelioma-like hepatocellular carci-noma. The association between the lack of EBV infec-tion, the absence of cirrhosis, a "cytotoxic profile" of the inflammatory infiltrate and a good prognosis could identify a variant of lymphoepithelioma-like HCC with a favorable clinical outcome.
文摘We report a case of fatal liver failure due to reactivation of lamivudine-resistant HBV. A 53-year-old man was followed since 1998 for HBV-related chronic hepatitis. Serum HBV-DNA was 150 MEq/mL (branched DNA signal amplification assay) and ALT levels fluctuated between 50-200 IU/L with no clinical signs of liver cirrhosis. Lamivudine (100 mg/d) was started in May 2001 and serum HBV-DNA subsequently decreased below undetectable levels. In May 2002, serum HBV-DNA had increased to 410 MEq/mL, along with ALT flare (226 IU/L). The YMDD motif in the DNA polymerase gene had been replaced by YIDD. Lamivudine was continued and ALT spontaneously decreased to the former levels. On Oct 3 the patient presenting with general fatigue, nausea and jaundice was admitted to our hospital. The laboratory data revealed HBV reactivation and liver failure (ALT: 1828 IU/L, total bilirubin: 10 mg/dL, and prothrombin INR: 3.24). For religious reasons, the patient and his family refused blood transfusion, plasma exchange and liver transplantation. The patient died 10 d after admission. The autopsy revealed remarkable liver atrophy.
