基于无细胞体系的生物合成代谢模块设计、构建与快速途径原型

随着代谢工程及合成生物学技术的发展,化学品高效生物合成与绿色制造成为可能。高效生物合成体系的设计与构建是绿色生物制造的核心,其理论体系建立及关键技术突破,将为实现绿色生物制造领域高效生产及资源与环境可持续发展提供有力支撑。本文借助代谢途径模块设计的案例,探讨化合物生物合成过程中潜在通用模块设计原则、设计工具,以及基于无细胞蛋白合成体系的代谢模块快速构建及测试的方法,将突破生物合成途径多基因、多模块“设计-构建-测试”(Design-Build-Test cycle,DBT cycle)高效循环迭代的技术瓶颈。结合机器学习方法的应用,将使“设计-构建-测试”向“设计-构建-测试-学习”(Design-Build-Test-Learn cycle,DBTL cycle)进一步延伸,对高效合成模块的“精准-鲁棒性”设计与构建、推动合成生物学科学与技术发展具有重要意义。

热休克蛋白与肾脏疾病

热休克蛋白(HSPs)是原核细胞和真核细胞产生的一组结构和功能上高度保守的蛋白质,协助其他蛋白质维持构象,参与蛋白质的变构、转运及降解,抑制细胞凋亡,并参与免疫调节。在肾脏HSPs不仅参与维持肾脏的生理状态,更是在保护肾组织免受损伤、促进受损组织修复以及某些肾脏疾病的病理生理过程中发挥重要作用。本文主要对HSPs的分类、生物学功能及其在肾脏方面的研究状况作一简要综述。

还原型牛红细胞Cu(I)Zn-SOD的量化计算

运用G94W量子化学程序,在B3YIP/Lan12dz基组的水平上,对于还原型牛红细胞中CU(I)Zn-SOD活性中心进行了从头计算。研究了分子轨道能量,电荷分布,前线轨道的贡献。结果表明:前沿分子轨道能量均为负值,其电子光谱稳定,与金属配位的咪唑环电荷表现正负交替现象。在前线轨道贡献上Cu(I)的占有轨道贡献大,空轨道的贡献小,不易于接受O_O^-自由基的不成对电子。

Anti-CD132 Monoclonal Antibodies Inducing T Cells Apoptosis after Alloantigen Stimulation and Its Possible Clinical Applications

Aim To investigate the mechanism of anti-CD132 monoclonal antibodies (mAbs)inhibiting T cells proliferation in vitro, and their potential values for clinical use. MethodsBALB/c and C57BL/6 mice splenocytes were harvested for two-ways mixed lymphocyte culture (MLC).Anti-CD132 mAbs (final concentration 100 mg·L^(-1)) were added in MLC on day 0 (group 1) or day 3(group 2). Fluorescence activated cell sorting (FACS) was used to measure the proliferation(carboxy-fluorescein dia cetate, succinimidyl ester, CFSE), apoptosis of T cells (PE-CD3,FTTC-annexin-v), and cell cycle (pro-pidium iodide stain) . The expression of survivin in T cellswas detected by immunochemical stai-ning. Re-sults Multi-generation CFSE-labeled splenocytes werefound dividing and their fluorescent strength decreased in MLC. There was no noticeable change influorescent intensity in group 1 and group 2. On day 3, apoptosis induced by anti-CD132 mAbs wasdetected in part of T cells, but was not detected in the former two days in group 1. In group 2, thenumber of cells in M phase (activated T cells) decreased and apoptot-ic cells increased on day 4.The phenomena were not observed in control group (P < 0.01). Expression of survivin in T cells wasdetected in control group but not in groups 1 and 2. Conclusion Blockade of CD132 signaling pathwayinhibits T cell proliferation in vitro by means of inducing activated alloreactive T cell apoptosisbut not the resting T cells. Anti-CD132 mAbs may be candidates for clinical applications.

Effect of interferon-γ and tumor necrosis factor-α on hepatitis B virus following lamivudine treatment

AIM： To evaluate anti-hepatitis B virus （HBV） activity and cytotoxicity of interferon-α, （IFN-3,） and tumor ne- crosis factor-α （TNF-α） following lamivudine treatment of HepG2.2.15 cells. METHODS： HepG2.2.15 cells were treated with 2 pmol/L lamivudine for 16 d （lamivudine group）, cultured for 10 d, followed by 5 ng/ml TNF-α and 1000 U/mL IFN-γ, for 6 d （cytokine group）, or treated with 2 ~tmol/L lami- vudine for 10 d followed by 5 ng/mL TNF-α and 1000 U/mL IFN-γ, for 6 d （sequential group）, or cultured without additions for 16 d （control group）. Intracellular DNA was extracted from 3 ×10^ HepG2.2.15 cells from each group. The extracted DNA was further purified with mung bean nuclease to remove HBV relaxed circu- lar DNA that may have remained. Both HBV covalently closed circular DNA （cccDNA） and HBV DNA were exam- ined with real-time polymerase chain reaction. The titers of hepatitis B surface antigen （HBsAg） and hepatitis B e antigen （HBeAg） were quantified with enzyme-linkedimmunosorbent assay. Cell viability was measured with the cell counting kit-8 assay. RESULTS： Compared to lamivudine alone （22.63%±0.12%）, both sequential （51.50% ± 0.17%, P = 0.034） and cytokine treatment （49.66% ± 0.06%, P = 0.041） showed a stronger inhibition of HBV cccDNA; the dif- ference between the.sequential and cytokine groups was not statistically significant （51.50% ± 0.17% vs 49.66% ± 0.06%, P = 0.88）. The sequential group showed less inhibition of HBV DNA replication than the lamivudine group （67.47% ±0.02% vs 82.48% ± 0.05%, P = 0.014）; the difference between the sequen- tial and cytokine groups was not statistically significant （67.47% ± 0.02% vs 57.45% ± 0.07%, P = 0.071）. The levels of HBsAg and HBeAg were significantly de- creased in the sequential treatment group compared to the other groups [HBsAg： 3.48 ± 0.04 （control）, 3.09 ± 0.08 （lamivudine）, 2.55± 0.13 （cytokine）, 2.32 ± 0.08 （sequential）, P = 0.042 for each between-group comparison; HBeAg 3.48 ± 0.01 （control）, 3.08 ± 0.08 （lamivudine）, 2.57 ± 0.15 （cytokine）, 2.34 ± 0.12 （se- quential）, P = 0.048 for each between-group compari- son]. Cell viability in the cytokine group was reduced to 58.03% ± 8.03% compared with control cells （58.03% ± 8.03% vs 100%, P = 0.000）. Lamivudine pretreat- ment significantly reduced IFN-γ, ± TNF-αmediated toxicity of HepG2.2.15 cells [85.82% =1= 5.43% （sequen- tial） vs 58.03% ± 8.03% （cytokine）, P = 0.002]. CONCLUSION： Sequential treatment overcame the lower ability of lamivudine alone to inhibit cccDNA and precluded the aggressive cytotoxicity involving IFN-y and TNF-α by decreasing the viral load.

Relationship of human leukocyte antigen class II genes with the susceptibility to hepatitis B virus infection and the response to interferon in HBV-infected patients

AIM： To study the relationship of human leukocyte antigen （HLA）-DRB1 and -DQB1 alleles with the genetic susceptibility to HBV infection and the response to interferon （IFN） in HBV-infected patients. METHODS： Low-resolution DNA typing kit was used to determine HLA-DR-1 and -DQB1 genes in 72 patients with chronic hepatitis B （CHB） and HLA-DRB1 in 200 healthy people ready to donate their bone marrow in Shanghai. Among CHB patients, 35 were treated with IFNα-1b for 24 wk. RESULTS： The frequencies of HLA-DRBI＊06, DRBI＊08 and DRB1＊16 alleles in 72 patients were higher than in 200 healthy people （2.08% vs0%, OR = 3.837, P= 0.018; 11.11% vs5.50%, OR = 2.148, P= 0.034; and 6.94% vs 3.00%, OR = 0.625, P = 0.049, respectively）; whereas that of DRBI＊07 allele was lower （2.78% vs 7.75%, OR = 0.340, P= 0.046）. The frequency of HLA-DRBI＊ 14 allele was higher in 11 responders to IFN compared with 24 non-responders （18.18% vs2.08%, OR = 10.444, P = 0.031）, whereas that of DQBI＊07 allele was inverse （9.09% vs37.50%, OR = 0.167, P= 0.021）. CONCLUSION： The polymorphism of HLA class II may influence the susceptibility to HBV infection and the response to IFN in studied CHB patients. Compared with other HLA-DRB1 alleles, HLA-DRBI＊06, DRBI＊08, and DRB1＊16 may be associated with chronicity of HBV infection, HLA-DRBI＊07 with protection against HBV infection, and HLA-DRB1＊14 allele may be associated with a high rate of the response of CHB patients to IFN treatment. Compared with other HLA-DQB1 alleles, HLA-DQBI＊07 may be associated with low response rate to IFN. 2005 The WJG Press and Elsevier Inc. All rights reserved

Herbal compound 861 regulates mRNA expression of collagen synthesis- and degradation-related genes in human hepatic stellate cells

AIM： To identify the role of herbal compound 861 （Cpd 861） in the regulation of mRNA expression of collagen synthesis- and degradation-related genes in human hepatic stellate cells （HSCs）. METHODS： mRNA levels of collagen types I and III, matrix metalloproteinase 1 （MMP-1）, matrix metalloproteinase 2 （MMP-2）, membrane type-1 matrix metalloproteinase （MT1-MMP）, tissue inhibitor of metalloproteinase 1 （TIMP-1）, and transforming growth factor β1 （TGF-β1） in cultured-activated HSCs treated with Cpd 861 or interferon-γ, （IFN-γ,） were determined by real-time PCR. RESULTS： Both Cpd 861 and IFN-γ reduced the mRNA levels of collagen type Ⅲ, MMP-2 and TGF-β1. Moreover, Cpd 861 significantly enhanced the MMP-1 mRNA levels while down-regulated the TIMP-1 mRNA expression, increasing the ratio of MMP-1 to TIMP-1 to （6.3 ＋ 0.3）- fold compared to the control group. CONCLUSION： The anti-fibrosis function of Cpd 861 may be mediated by both decreased interstitial collagen sythesis by inhibiting the transcription of collagen type Ⅲ and TGF-β1 and increased degradation of these collagens by up-regulating MMP-1 and down-regulating TIMP-1 mRNA levels.

Expression of a soluble form of CTLA4 on macrophage and its biological activity

Interaction between cytotoxic T lymphocyte-asso-ciated antigen-4 (CTLA4, CD152) and B7 molecules (B7-1and B7-2) is of importance in the cellular events of lym-phocyte, including antigen-specific T-cell activation andinduction of autoreactive T-cell. We describe here the firstintroduction of a murine soluble CTLA4 gene, CTLA4Ig,to Mm1 cells, a macrophagic cell line. CTLA4Ig waJssuccessfully expressed on Mm1 cells and the expressedCTLA4Ig was found to be functionally active in their bind-ing to B7 molecules by flow cytometry and immunofluo-rescence studies- The biological activity of CTLA4Ig fromthe transfected Mm1 cells was studied and showed in-hibitory activity on mixed lymphocyte culture. A highCTLA4Ig producing macrophagic cell line was obtained.As Mm1 cells were regarded as difficult for gene transfec-tion and there had so far been no report on expression ofCTLA4Ig gene on Mm1 cells, these results suggested thatthe CTLA4Ig expressing Mm1 cells could be useful forExpression of CTLA4 on Mml and its biological activityanalysis of CTLA4 and B7 molecule interaction in bothmacrophage and T-cell.

Proposal for a Reasonable Model of the Visual System： Principles of Clinical Neurosociology

This paper maintains that when photons enter the pupil and reach the Inner Limiting Membrane （ILM）, they are reflected onto a point or centroid of the vitreous body, which could be the lens, to be transmitted by the Muiller cells to the Retinal Pigment Epithelium （RPE） hexagonal cells, where an almost complete image is formed in each of them, overlapping with the adjacent images, to be carried subsequently by each of the ganglion cell axons to a place where the single image we are aware of is formed. This process calls for a high degree of control and coordination, which must be effected by the horizontal, amacrine, and interplexiform cells, gap junctions and the feedback provided from the V1 area to the Lateral Geniculate Nucleus （LGN）. But, as the ILM covers the optic disc but not the fovea, the latter must produce the blind spot and the rays reflected radially from the centroid must have the same centre as the Muller cells in order to be able to channel them to the RPE cells.

Human leukocyte antigen class II DQB1*0301, DRB1*1101 alleles and spontaneous clearance of hepatitis C virus infection: A meta-analysis

AIM： To assess the associations of human leukocyte antigen （HI_A） class Ⅱ DQB1＊0301 and/or DRB1＊1101 allele with spontaneous hepatitis C virus （HCV） clearance by meta-analysis of individual dataset from all studies published till date. METHODS： To clarify the impact of HLA class Ⅱ polymorphisms on viral clearance, we performed a metaanalysis of the published data from 11 studies comparing the frequencies of DQB1＊0301 and DRB1＊1101 alleles in individuals with spontaneous resolution to those with persistent infection. As we identified the heterogeneity between studies, summary statistical data were calculated based on a random-effect model. RESULTS： Meta-analyses yielded summary estimatesodds ratio （OR） of 2.36 [95%CI （1.62, 3.43）, P〈0.00001] and 2.02 [95%CI （1.56, 2.62）, P〈0.00001] for the effects of DQB1＊0301 and DRB1＊1101 alleles on spontaneous clearance of HCV, respectively. CONCLUSION： These results support the hypothesis that specific HLA class Ⅱ alleles might influence the susceptibility or resistance to persistent HCV infection. Both DQB1＊0301 and DRB1＊1101 are protective alleles and present HCV epitopes more effectively to CD4^＋T lymphocytes than others, and subjects with these two alleles are at a lower risk of developing chronic HCV infection. Large, multi-ethnic confirmatory and welldesigned studies are needed to determine the host genetic determinants of HCV infection.

Development of HBsAg-Binding Aptamers that bind HepG2.2.15 cells via HBV surface antigen

Hepatitis B virus surface antigen （HBsAg）, a specific antigen on the membrane of Hepatitis B virus （HBV）-infected cells, provides a perfect target for therapeutic drugs. The development of reagents with high affinity and specificity to the HBsAg is of great significance to the early-stage diagnosis and treatment of HBV infection. Herein, we report the selection of RNA aptamers that can specifically bind to HBsAg protein and HBsAg-positive hepatocytes. One high affinity aptamer, HBs-A22, was isolated from an initial 115 met library of -1.1 ×10^15 random-sequence RNA molecules using the SELEX procedure. The selected aptamer HBs-A22 bound specifically to hepatoma cell line HepG2.2.15 that expresses HBsAg but did not bind to HBsAg-devoid HepG2 cells. This is the first reported RNA aptamer which could bind to a HBV specific antigen. This newly isolated aptamer could be modified to deliver imaging, diagnostic, and therapeutic agents targeted at HBV-infected cells.

丹参多酚酸盐对急诊PCI患者术后氧化应激、心功能、血管内皮功能的影响

目的探究丹参多酚酸盐对急诊PCI患者术后氧化应激、心功能、血管内皮功能的影响。方法选择衡水市人民医院2017年8月—2018年7月收治的150例急性心肌梗死(AMI)患者,均采用急诊PCI治疗,对照组术前予以阿司匹林肠溶缓释片、硫酸氢氯吡格雷片口服,术后予以心肌梗死二级预防干预;观察组于术后予以注射用丹参多酚酸盐治疗,均连续治疗14 d。对比2组治疗前后血清丙二醛(MDA)、吞噬细胞还原型辅酶Ⅲ氧化酶(NADPH氧化酶)p22phox活性及心功能、血管内皮功能及药物不良反应发生情况,并随访6个月,对比2组临床预后。结果观察组治疗后的血清MDA、NADPH氧化酶p22phox活性均明显低于对照组(P均<0.05)。观察组治疗后血清肌酸激酶(CK)水平明显低于对照组(P<0.05),左室射血分数(LVEF)、一氧化氮(NO)水平均明显高于对照组(P均<0.05)。2组均未发生严重药物不良反应。术后随访6个月,观察组术后心血管事件率、术后再狭窄率均低于对照组(P均<0.05)。结论对急诊PCI患者术后辅以丹参多酚酸盐治疗有助于减轻氧化应激反应及改善血管内皮功能和心功能,可降低术后心血管事件率、术后再狭窄率,且用药安全可靠。

A comparative review of HLA associations with hepatitis Band C viral infections across global populations

Hepatitis B （HBV） and hepatitis C （HCV） viral infection or co-infection leads to risk of development of chronic infection, cirrhosis and hepatocellular carcinoma （HCC）. Immigration and globalization have added to the challenges of public health concerns regarding chronic HBV and HCV infections worldwide. The aim of this study is to review existing global literature across ethnic populations on HBV and HCV related human leukocyte antigen （HLA） associations in relation to susceptibility, viral persistence and treatment. Extensive literature search was conducted to explore the HLA associations in HBV and HCV infections reported across global populations over the past decade to understand the knowledge status, weaknesses and strengths of this information in different ethnic populations. HLA DR13 is consistently associated with HBV clearance globally. HLADRB1＊11/＊12 alleles and DQB1＊0301 are associated with HBV persistence but with HCV clearance worldwide. Consistent association of DRB1＊03 and ＊07 is observed with HCV susceptibility and non-responsiveness to HBV vaccination across the population. HLA DR13 is protective for vertical HBV and HCV transmission in Chinese and Italian neonates, but different alleles are associated with their susceptibility in these populations. HLA class I molecule interactions with Killer cell immunoglobulin like receptors （KIR） of natural killer （NK） cells modulate HCV infection outcome via regulating immune regulatory cells and molecules. HLA associations with HBV vaccination, interferon therapy in HBV and HCV, and with extra hepatic manifestations of viral hepatitis are also discussed. Systematic studies in compliance with global regulatory standards are required to identify the HLA specific viral epitope, stage specific T cell populations interacting with different HLA alleles during disease progression and viral clearance of chronic HBV or HCV infections among different ethnic populations. These studies would facilitate stage specific therapeutic strategies for clearance of HBV and HCV infections or co-infections across global populations and aid in identification of HBV-HCV combined vaccine. HLA associations of chronic HBV or HCV development with confounding host factors including alcohol, drug abuse, insulin resistance, age and gender are lacking and warrant detailed investigation across global populations.

Clinical significance of loss of heterozygosity for M6P/IGF2R in patients with primary hepatocellular carcinoma

AIM:To investigate the relationship between loss of heterozygosity (LOH) for mannose 6-phosphate/insulin- like growth factor 2 receptor (M6P/IGF2R) and the outcomes for primary HCC patients treated with partial hepatectomy. METHODS: The LOH for M6P/IGF2R in primary HCC patients was assessed using six different gene-specific nucleotide polymorphisms. The patients studied were enrolled to undergo partial hepatectomy. RESULTS: M6P/IGF2R was found to be polymorphic in 73.3% (22/30) of the patients, and of these patients, 50.0% (11/22) had tumors showing LOH in M6P/IGF2R. Loss of heterozygosity in M6P/IGF2R was associated with significant reductions in the two year overall survival rate (24.9% vs 65.5%; P = 0.04) and the disease-free survival rate (17.8% vs 59.3%; P = 0.03). CONCLUSION: These results show M6P/IGF2R LOH predicts poor clinical outcomes in surgically resected primary HCC patients.

Establishment of an orthotopic transplantation tumor model of hepatocellular carcinoma in mice

AIM:To improve the outcome of orthotopic transplantation in a mouse model,we used an absorbable gelatin sponge(AGS) in nude mice to establish an orthotopic implantation tumor model.METHODS:MHCC-97L hepatocellular carcinoma(HCC)cells stably expressing the luciferase gene were injected into the subcutaneous region of nude mice.One week later,the ectopic tumors were harvested and transplanted into the left liver lobe of nude mice.The AGS was used to establish the nude mouse orthotopic implantation tumor model.The tumor suppressor gene,paired box gene 5(PAX5),which is a tumor suppressor in HCC,was transfected into HCC cells to validate the model.Tumor growth was measured by bioluminescence imaging technology.Semi-quantitative reverse transcription polymerase chain reaction(RT-PCR) and histopathology were used to confirm the tumorigenicity of the implanted tumor from the MHCC-97L cell line.RESULTS:We successfully developed an orthotopic transplantation tumor model in nude mice with the use of an AGS.The success rate of tumor transplantation was improved from 60% in the control group to 100% in the experimental group using AGS.The detection of fluorescent signals showed that tumors grew in all live nude mice.The mice were divided into 3 groups:AGS-,AGS+/PAX5-and AGS+/PAX5 +.Tumor size was significantly smaller in PAX5 transfected nude mice compared to control mice(P < 0.0001).These fluorescent signal results were consistent with observations made during surgery.Pathologic examination further confirmed that the tissues from the ectopic tumor were HCC.Results from RT-PCR proved that the HCC originated from MHCC-97L cells.CONCLUSION:Using an AGS is a convenient and efficient way of establishing an indirect orthotopic liver transplantation tumor model with a high success rate.

DNA TYPING SYSTEM FOR HLA-A2 ALLELES BY POLYMERASE CHAIN REACTION WITH SEQUENCE- SPECIFIC PRIMERS

Objective. To establish a PCR- SSP method for discriminating as many HLA- A* 02 alleles, which could easily be introduced into a routine laboratory. Methods. In this study we typed HLA- A* 02 polymorphisms by a sequence- specific primer (SSP) method, which involved round 1 and round 2 PCR reactions to detect 17 HLA- A* 02 alleles (they are HLA- A* 0201- 0217 alleles) covering exon 2 and exon 3. Results. We have found that DNA sample concentration and purity were the most important variables in determining the quality of the results. For identifying correct band size, the size marker used was important. We noticed that different PCR machines performed differently. By this method, we detected 20 HLA- A* 02 positive genomic DNA samples and found 4 kinds of HLA- A* 02 alleles. They were HLA- A* 0201, 0203, 0206 and 0210. Conclusion. The HLA- A* 02 PCR- SSP method was proven to be a reliable and easily applicable typing method. Our results suggest that the SSP described here provides an optimal HLA- A* 02 typing technique that may be useful in selecting donor- recipient pairs in bone marrow transplantation between unrelated individuals.

甘蔗Rieske Fe/S蛋白前体基因ScPetC的克隆及表达分析

细胞色素b6f复合体还原型铁硫蛋白前体(Cytochrome b6f complex Rieske Fe/S precursor protein,PetC)是由细胞核PetC基因编码的蛋白,其成熟蛋白参与构成细胞色素b6f复合体,是电子传递的重要元件。基于前期构建的受高粱花叶病毒(Sorghum mosaic virus,SrMV)侵染的甘蔗转录组数据库,从主栽甘蔗品种‘新台糖22号’叶片中成功克隆到该基因,并命名为ScPetC(GenBank登录号为MH333037.1)。生物信息学分析发现,ScPetC基因cDNA全长824bp,包含了一个678bp的开放阅读框,编码226个氨基酸。ScPetC属于PRK13473超家族,其C末端具有典型的Rieske保守结构域;蛋白理论等电点为8.19,属于稳定的、亲水性蛋白;二级结构多为无规则卷曲,三级结构预测其比其他植物PetC多出一段α螺旋结构。在本氏烟(Nicotiana benthamiana)叶片瞬时表达中,ScPetC定位于叶绿体、细胞质和细胞膜。尽管前人研究表明,ScPetC的表达量会受SrMV侵染的影响,不同于半夏(Pinellia ternata)PetC与大豆花叶病毒(Soybean mosaic virus,SMV)P1蛋白之间的互作,ScPetC不能与SrMV-P1蛋白互作,但能与甘蔗黄叶病毒(Sugarcane yellow leaf virus,SCYLV)P0蛋白互作。实时荧光定量PCR分析表明,ScPetC基因在甘蔗不同组织中均有表达,但在叶片中的表达量最高。甘蔗受脱落酸胁迫3 h时,ScPetC表达量显著上调,但是随着处理时间的延长,表达受到抑制;在茉莉酸甲酯、水杨酸、氯化铜、氯化镉和氯化钠胁迫下,ScPetC表达量均显著下调。本研究通过对ScPetC生物学功能、环境外源激素与重金属等胁迫下的表达模式及其与甘蔗病原病毒蛋白互作的初步探究,增加了对甘蔗PetC的了解,也为深入研究其在甘蔗受黄叶病毒侵染中的作用奠定基础。

Antifibrotic effect of aloe vera in viral infection-induced hepatic periportal fibrosis

AIM:To investigate the anti-oxidative and anti-fibrotic effects of aloe vera in patients with liver fibrosis.METHODS:Aloe vera high molecular weight fractions(AHM) were processed by patented hyper-dry system in combination of freeze-dry technique with microwave and far infrared-ray radiation.Fifteen healthy volunteers as the control group and 40 patients were included.The patients were randomly subdivided into two equal groups:the conventional group was treated with placebo(starch),and AHM group was treated with 0.15 gm/d AHM,both for 12 consecutive weeks.The patients were investigated before and after treatment.Serum activity of aspartate aminotransferase(AST),alanine aminotransferase(ALT),alkaline phosphatase(ALP),hyaluronic acid(HA),transforming growth factor-β(TGF-β) and matrixmetalloproteinase-2(MMP-2) were determined.The reduced glutathione(GSH) and malondialdehyde(MDA) levels in liver were assayed and the expression of hepatic α-smooth muscle actin(α-SMA) was identified by immunohistochemistry.RESULTS:At the start of the study,the hematoxylin and eosin staining revealed fibro-proliferated bile ductules,thick fibrous septa and dense inflammatory cellular infiltration in the patients before treatment.The use of AHM for 12 wk significantly ameliorated the fibrosis,inhibited the inflammation,and resulted in minimal infiltration and minimal fibrosis compared to the conventional group.The enzyme activities of the liver(ALT,AST and ALP) were attenuated after treatment in both groups,and the decrease in the AHM group was more significant as compared with the conventional group.Similar to the AST,the MDA levels were significantly higher before treatment,and were attenuated after treatment in both groups.In contrast,the hepatic glutathione content in the patients were decreased significantly in the AHM group compared to the controls.The serum levels of the fibrosis markers(HA,TGF-β and MMP-2) were also reduced significantly after treatment.The expression of α-SMA was modified in patients before and after treatment as compared with the normal controls.In the conventional group,there was only thin and incomplete parenchymal α-SMA positive septum joining the thickened centrilobular veins,while in the AHM group,few α-SMA positive cells were present in sinusoid and lobule after treatment.CONCLUSION:Oral supplementation with AHM could be helpful in alleviating the fibrosis and inflammation of hepatic fibrosis patients.

Study of human B7 homolog 1 expression in patients with hepatitis B virus infection

AIM: To further investigate the role of human B7 homolog 1 (B7-H1) in the mechanism of persistent hepatitis B virus (HBV) infection. METHODS: Peripheral and intra-hepatic B7-H1 expression were compared by flow cytometry and immunochemical staining between two 2 distinct groups, one being chronic HBV tolerance patients (CHB-T) and the other being acute hepatitis B patients (AHB). B7-H1 mRNA expression level was also compared by real time polymerase chain reaction between CHB-T and AHB patients. The location of intra-hepatic B7-H1 and CD40 expression were analyzed by immunofluorescence. The levels of B7-H1 and CD40 expression on cultured myeloid dendritic cells (mDCs) with or without hepatitis B surface antigen (HBsAg) treatment were analyzed dynamically by flow cytometry. Intracellular interferon-γ (IFN-γ) staining and the stimulatory capacity of mDC of cultured mDC with or without HBsAg treatment were also compared by flow cytometry. RESULTS: Peripheral B7-H1 expression on mDCs was increased significantly in AHB compared to CHB-T patients (P < 0.05). In the liver tissues from CHB-T patients, B7-H1 positive cells were almost absent despite a persistently elevated serum HBsAg load. In contrast, there were indeed increased B7-H1-positive cells in situ in the liver tissue from AHB. In vitro analysis showed the parallel upregulation of B7-H1 and CD40 on CD11c+ mDCs after the onset of stimulation. Addition of recombinant hepatitis B surface antigen (rHBsAg) significantly decreased CD40 expression (P < 0.05 at 16 h, 20 h and 24 h time points). B7-H1 expression was also inhibited by rHBsAg, and the inhibition rate of CD40 was greater than that of B7-H1. This preferential inhibition of CD40 expression on mDCs by rHBsAg resulted in the dysfunction of mDCs and T cells in the mixed leucocyte reaction (MLR) system. With rHBsAg pretreatment, in a carboxyfluorescein diacetate succinimidyl ester (CFSE) labeled MLR system at a ratio of 1:5 responder cell-stimulator cell (R/S), the CFSE dim percentage of T cells decreased from 85.1% to 25.4% and decreased from 30.3% to 12.0% at 1:10 R/S. IFN-γ production by CD8+ T cells, in the MLR system, was reduced significantly by HBsAg pretreatment. At ratios of 1:5 R/S, the percentage of IFN-γ and CD8 dual positive T cells decreased from 55.2% ± 5.3% to 15.1% ±3.1% (P < 0.001), and decreased from 35.0% ± 5.1% to 7.3% ± 2.7% at ratios of 1:10 R/S (P < 0.001). CONCLUSION: B7-H1 is not a signature of immune dysfunction, but an inflammation marker. HBsAg regulate immune response by tipping the balance between B7-H1 and CD40.

Successful en bloc resection of primary hepatocellular carcinoma directly invading the stomach and pancreas

Multivisceral surgical resection for cure was successfully performed in a 70-year-old man suffering from a primary hepatocellular carcinoma(HCC)associated with direct invasion to the stomach and pancreas.The patient presented with gastric outlet obstruction,upper abdominal pain and a history of chronic liver disease due to hepatitis B virus(HBV)infection.Upper gastro-intestinal(GI)endoscopy revealed an infiltrating tumor protruding through the gastric wall and obliterating the lumen.Computer tomograghy(CT)and magnetic resonance imaging(MRI)scan demonstrated a 15-cm tumor in the left lateral segment of the liver with invasion to the stomach and pancreas.Alpha-foetoprotein (AFP)levels and liver function tests were normal.The patient underwent an en bloc left hepatectomy,total gastrectomy,distal pancreatectomy with splenectomy and radical lymphadenectomy.Pathology revealed a poorly differentiated,giant cell HCC involving the stom-ach and pancreas.Disease-free margins of resection were achieved.The patient's postoperative course was uneventful.Sixteen months after surgery,he has norecurrence or distal metastasis.Direct invasion of HCC into the GI tract is rarely encountered.Complete surgical resection should be considered in selected patients with an appropriate hepatic functional reserve.