CD40-deficient mice generated by recombination-activating gene-2-deficient blastocyst complementation.

To study the role of B-cell antigen CD40 in immune responses, mouse embryonic stem (ES) cells in which both copies of the gene encoding CD40 had been disrupted by homologous recombination were injected in RAG-2 (recombination-activating gene-2)-deficient blastocysts to generate chimeras in which all mature lymphocytes are derived from the CD40-deficient ES cells. T- and B-cell number and phenotype were normal in the CD40-/- chimeras. However, B cells failed to proliferate and undergo isotype switching in vitro in response to soluble CD40 ligand (sCD40L) with interleukin 4 (IL-4) but responded normally to lipopolysaccharide (LPS) with IL-4. CD40-/- chimeras completely failed to mount an antigen-specific antibody response or to develop germinal centers following immunization with the T cell-dependent (TD) antigen keyhole limpet hemocyanin. In contrast, CD40-/- mutant mice responded normally to the T cell-independent (TI) antigens, 2,4,6-trinitrophenyl (TNP)-LPS and TNP-Ficoll. The most noticeable alteration in the serum immunoglobulin levels of young (6-8 weeks old) CD40-/- animals was absence of IgE and severe decrease of IgG1 and IgG2a. These results confirm the essential role of CD40- CD40L interactions in the antibody response to TD antigens and in isotype switching.     

Temporary amelioration of bilirubin conjugation defect in Gunn rats by transplanting conditionally immortalized hepatocytes

BACKGROUND: Conditionally immortalized hepatocytes (CIH) have been used in hepatocyte transplantation as an alternative to primary hepatocytes to cope with the shortage of donor organs. However, CIH are known to undergo apoptosis at body temperature and survive in vivo for a short period. In the present study, we investigated whether CIH function or not and how long their function is maintained in vivo. METHODS: Various CIH cell lines that were established with temperature-sensitive Simian virus 40 large T antigen were transplanted into the spleen of Gunn rats, which are defective in bilirubin uridine diphosphate glucuronoside transferase (BUGT). Then, we measured biological changes over 3 months. RESULTS: Serum bilirubin of the syngeneic CIH recipients decreased by 30%, which was maintained for 8 weeks. Thereafter, it began to rise to basal levels. The recipients of allogeneic CIH showed a minor reduction of bilirubin, although this was not statistically significant. However, there was no significant change in the bilirubin level in recipients of BUGT-defective congeneic CIH throughout the study period. Bilirubin monoglucuronides in the bile were not detected in the recipients of BUGT-defective CIH. However, they appeared in recipients of non-defective CIH and made up approximately 41% of total bile pigments. CONCLUSIONS: Conditionally immortalized hepatocytes expressed hepatocyte function in vivo as well as in vitro, but the function lasted for a couple of months. According to our previous study, the limited functional duration may be related to the inevitable occurrence of apoptosis of these cells at body temperature. These data suggest that CIH can be used in hepatocyte transplantation only for temporary hepatic support.     

Hepatitis B virus infection in microcarrier-attached immortalized human hepatocytes cultured in molecularporous membrane bags: a model for long-term episomal replication of HBV

Studies on the pathobiology of chronic (long-term) hepatitis B virus (HBV) infection and in vitro drug testing have been hampered by the lack of appropriate systems for culturing susceptible cells chronically infected with HBV. Most of the in vitro studies of HBV replication have been performed with HBV genome-transduced cell lines. In this system, viral production is mainly the result of chromosomal replication. In an invitro infection system, owing to medium refreshment (which leads to the removal of infectious particles necessary for the perpetuation of infection) and to trypsinization for cell passages, it is difficult, if not impossible, to maintain chronic HBV infection, despite the use of susceptible cells. To circumvent these unfavourable factors for chronic HBV infection in vitro, we cultured microcarrier-attached immortalized human hepatocytes, infected with HBV, in molecularporous (MW 12000–14000) membrane (dialysis) bags for a duration of 2 months. HBV covalently-closed-circular (ccc) DNA, HBV precore/core and X mRNAs were detected in the cells cultured in this system following infection until the end of the experiment (day 58), while in classical culture conditions (monolayer), markers of HBV replication were also detected. Production of hepatitis B surface antigen (HBsAg) and HBV DNA was detected and their levels in culture medium (collected at the end of experiments from the molecularporous membrane bags) were increased 2.86- and 3.28-fold respectively. Using Southern blot analysis, HBV replicative intermediates could also be demonstrated throughout the experiments. However, integrated HBV DNA was not present. In contrast, HBV ccc DNA, HBV precore/core and X mRNAs, and replicative intermediates were not demonstrable in FTO 2B rat hepatoma cells infected in the same manner in parallel experiments. This in vitro infection system, using susceptible, immortalized human hepatocytes, therefore provides a new tool for studying the long-term effect of HBV infection, mainly involving episomal replication in hepatocytes, and for drug testing.     

Establishment of mice model with human viral hepatitis B

AIM:To establish a mice model of hepatitis B by using HBV-transgenic mice, and to transfer HBV-specific cytotoxic T lymphoo/tes (CTL) induced from syngeneic BALB/c mice immunized by a eukaryotic expression vector containing HBV complete genome DNA.METHODS: HBV DNA was obtained from digested pBR3222HBV and ligated with the vector pcDNA3. Recombinant pcDNA3-HBV was identified by restriction endonuclease assay and transfected into human hepatoma cell line HepG2 with lipofectin. ELISA was used to detect the expression of HBsAg in culture supernatant, and RT-PCR to determine the existence of HBV PreS1 mRNA.BALB/c mice were immunized with pcDNA3-HBV or pcDNA3 by intramuscular injection.ELISA was used to detect the expression of HBsAb in serum. MTT assay was used to measure non-specific or specific proliferation ability and specific killing activity of spleen lymphocytes. Lymphocytes from immunized mice were transferred into HBV-transgenic mice (2.5×10^7 per mouse).Forty-eight hours later,the level of serum protein and transaminase was detected with biochemical method,liver and kidney were sectioned and stained by HE to observe the pathological changes.RESULTS: By enzyme digestion with Eco RI, Xho I and Hind Ⅲ,the recombinant pcDNA3-HBV was verified to contain a single copy of HBV genome,which was inserted in the positive direction.HepG2 cells transfected with the recombinant could stably express PreS1 mRNA and HBsAg.After immunized by pcDNA3-HBV for 4 weeks,HBsAb was detected in the serum of BALB/c mice. The potential of spleen lymphoo/tes for both non-specific and specific proliferation and the specific killing activity against target cells were enhanced. The transgenic mice in model group had no significant changes in the level of serum protein but had an obvious increase of ALT and AST. The liver had obvious pathological changes, while the kidney had no evident damage.CONCLUSION:A eukaryotic expression vector pcDNA3-HBV containing HBV complete genome is constructed successfully. HepG2 cells transfected with the recombinant can express PreS1 mRNA and HBsAg stably.Specific cellular immune response can be induced in mice immunized by pcDNA3-HBV.A mice model of acute hepatitis with HBV has been established.     

Susceptibility of chimeric mice with livers repopulated by serially subcultured human hepatocytes to hepatitis B virus

We previously identified a small population of replicative hepatocytes in long-term cultures of human adult parenchymal hepatocytes (PHs) at a frequency of 0.01%-0.09%. These hepatocytes were able to grow continuously through serial subcultures as colony-forming parenchymal hepatocytes (CFPHs). In the present study, we generated gene expression profiles for cultured CFPHs and found that they expressed cytokeratin 19, CD90 (Thy-1), and CD44, but not mature hepatocyte markers such as tryptophan-2,3-dioxygenase (TO) and glucose-6-phosphatase (G6P), confirming that these cells are hepatic progenitor-like cells. The cultured CFPHs were resistant to infection with human hepatitis B virus (HBV). To examine the growth and differentiation capacity of the cells in vivo, serially subcultured CFPHs were transplanted into the progeny of a cross between albumin promoter/enhancer-driven urokinase plasminogen activator-transgenic mice and severe combined immunodeficient (SCID) mice. The cells were engrafted into the liver and were able to grow for at least 10 weeks, ultimately reaching a maximum occupancy rate of 27%. The CFPHs in the host liver expressed differentiation markers such as TO, G6P, and cytochrome P450 subtypes and could be infected with HBV. CFPH-chimeric mice with a relatively high replacement rate exhibited viremia and had high serum levels of hepatitis B surface antigen. CONCLUSION: Serially subcultured human hepatic progenitor-like cells from postnatal livers successfully repopulated injured livers and exhibited several phenotypes of mature hepatocytes, including susceptibility to HBV. In vitro-expanded CFPHs can be used to characterize the differentiation state of human hepatic progenitor-like cells.     

长期恩替卡韦经治慢性乙型肝炎患者低病毒血症的相关影响因素

目的探讨长期接受恩替卡韦治疗的慢性乙型肝炎(CHB)患者仍维持低水平病毒复制的相关影响因素。方法选取2018年11月—2020年6月于徐州医科大学附属医院门诊接受恩替卡韦抗病毒治疗至少1年的CHB患者,根据至观察期结束患者HBV DNA载量分为低病毒血症(LLV)组和持续病毒学应答(SVR)组。观察患者人口学特征参数和实验室检测指标。计量资料两组间比较采用独立样本t检验或Mann-Whitney U检验;计数资料两组间比较采用χ2检验。采用多因素logistic回归分析长期恩替卡韦经治患者出现LLV的影响因素。结果共纳入560例CHB患者,其中LLV组204例,SVR组356例。两组患者比较,年龄(Z=-3.530,P<0.001)、性别(χ2=4.270,P=0.039)、是否存在肝硬化(χ2=53.879,P<0.001)、服药依从性(χ2=5.326,P=0.021)、HBeAg阳性率(χ2=90.681,P<0.001)、治疗前基线HBV DNA载量(Z=-8.337,P<0.001)、基线HBsAg定量(Z=-10.472,P<0.001)以及用药类型(χ2=7.558,P=0.006)差异均有统计学意义。多因素logistic回归分析显示,治疗前基线HBeAg状态(OR=3.381,95%CI:1.985~5.756,P<0.001)、HBV DNA载量(OR=1.223,95%CI:1.050~1.424,P=0.010)和HBsAg定量(OR=2.448,95%CI:1.743~3.438,P<0.001)是长期恩替卡韦抗病毒治疗出现LLV的危险因素。结论在临床实践中,基线高载量HBV DNA水平、高HBsAg定量和HBeAg阳性的CHB患者即使长期坚持服用恩替卡韦抗病毒治疗,也存在较高的LLV风险。因此,对于此类人群应予以重视,需动态监测HBsAg定量、HBV DNA载量、HBeAg状态。     

Early dynamics of hepatitis B virus in chimeric mice carrying human hepatocytes monoinfected or coinfected with genotype G

Of the 8 genotypes of HBV (genotypes A-H), genotype G is unique in that it has an insertion in the core gene and two stop codons in the precore region preventing the synthesis of hepatitis B e antigen. Most individuals with genotype G are coinfected with other genotypes, typically genotype A. Mice with severe combined immunodeficiency disease carrying human hepatocytes were infected with HBV particles propagated in Huh7 cells in culture. Mice monoinfected with genotype G did not raise detectable HBV DNA in serum, although products of the core gene emerged 4 to 8 weeks after inoculation. When they were superinfected with genotype A at week 10, however, HBV DNA of genotype A developed, which was replaced almost completely by that of genotype G within 10 weeks. Such a rapid takeover was also observed in mice initially infected with genotype A or C and superinfected with genotype G. Similar viral dynamics occurred in mice simultaneously coinfected with genotypes G and A. Takeover was markedly enhanced in mice inoculated with a serum passage containing genotype G with a trace of genotype A. Coinfection of mice with genotypes G and A induced abundant cellular steatosis along with increased fibrosis in the liver, which was not detected in mice monoinfected with genotype A or G. CONCLUSION: Genotype G can monoinfect chimeric mice at very low levels, and its replication increases maredly when coinfected with other genotypes. Coinfection with genotype G could enhance fibrosis under immunocompromised states.     

Significance of hepatitis B viremia levels determined by a quantitative polymerase chain reaction assay in patients with hepatitis B e antigen-negative chronic hepatitis B virus infection

OBJECTIVES: In hepatitis B e antigen (HBeAg)-negative chronic hepatitis B virus (HBV) infection, the clinical relevance of low viremia levels remains unclear. We evaluated the clinical significance of a single baseline serum HBV DNA measurement by a quantitative polymerase chain reaction (PCR) assay in this setting. METHODS: In total, 196 patients with HBeAg-negative chronic HBV infection (62 inactive carriers, 134 with chronic hepatitis B) were studied. ALT activity was normal at baseline in 25/134 HBeAg-negative chronic hepatitis B patients (18.7%), whereas it remained normal throughout follow-up in all inactive carriers. RESULTS: HBV DNA was <30,000 copies/ml in 14 (10.5%) and <100,000 copies/ml in 17 (12.9%) HBeAg-negative chronic hepatitis B patients, whereas it was <30,000 copies/ml in all inactive carriers (undetectable in 14). In particular, HBV DNA levels were <100,000 copies/ml in eight (32%) and <30,000 copies/ml in five (20%) of the 25 patients with HBeAg-negative chronic hepatitis B and normal baseline ALT values. HBV DNA levels with a cut-off at 30,000 or 100,000 copies/ml could correctly classify 92.9% or 91.3% of patients with HBeAg-negative chronic HBV infection, whereas ALT or IgM anti-HBc (IgM class antibody to HBV core antigen) index > 0.200 could correctly classify only 87.2% and 82.1% of patients, respectively. A combined HBV DNA and IgM anti-HBc index performed better by correctly classifying 94.4% of cases. CONCLUSIONS: Serum HBV DNA levels evaluated by sensitive quantitative PCR assays can be used for differentiation between HBeAg-negative chronic hepatitis B and inactive hepatitis B surface antigen carrier state, but the cut-off level should be set at approximately 30,000 copies/ml and certainly lower than the recently suggested level of 100,000 copies/ml.     

乙型肝炎病毒C基因逆转录病毒载体的构建及在永生化人外周血B细胞中的表达

目的　体外研究乙型肝炎病毒Ｃ（ＨＢＶ／Ｃ） 基因变异的生物学意义。方法　应用逆转录病毒载体ｐＸＴ１ 构建ＨＢＶ／Ｃ 基因表达载体,并转染永生化人外周血Ｂ 细胞使之稳定表达ＨＢｃＡｇ。结果　重组质粒ｐＸＴ１ＨＢＶ／Ｃ 经聚合酶链反应（ＰＣＲ） 和ＢｇｌⅡ及ＸｈｏⅠ双酶切鉴定均阳性,转染后的永生化人外周血Ｂ 细胞ＰＣＲ 鉴定含目的ＤＮＡ,流式细胞仪分析显示,约４７ ．４ ％ 的细胞膜上表达了ＨＢｃＡｇ。结论　ＨＢＶ／Ｃ基因逆转录病毒表达载体有较高的转染效率,目的基因能在宿主细胞中稳定表达,有利于系列研究ＨＢＶ／Ｃ基因变异的生物学意义。     

商陆抗病毒蛋白在急性人乙型肝炎病毒感染小鼠体内的抑制作用

目的 探讨商陆抗病毒蛋白(PAP)在急性HBV感染小鼠模型体内抗HBV的效果.方法 通过尾静脉注射具有复制能力的HBV真核表达质粒来建立小鼠急性HBV感染模型.根据注射后第1天小鼠血清HBsAg和HBeAg水平,从35只小鼠中选出24只进行配对,分为PAP治疗组(腹腔注射0.25 mg/kg PAP)和对照组.于PAP注射前、注射后第1、3、5、7天,时间分辨免疫荧光分析法检测小鼠血清HBsAg、HBeAg和抗-HBc水平,荧光定量PCR检测小鼠血清HBV DNA水平.注射后第7天HE染色检测肝组织病理变化,免疫组织化学检测肝组织HBsAg、HBeAg表达.采用配对t检验进行统计学分析.结果 与对照组相比,在PAP腹腔注射后第1、3、5天对HBsAg的抑制率分别为23%(t=116.3,P<0.05)、47%(t=38.2,P<0.05)、68%(t=23.7,P<0.05),对HBeAg的抑制率分别为36%(t=34.2,P<0.05)、55%(t=61.6,P<0.05)、83%(t=98.8,P<0.05),对HBV DNA的抑制率分别为70.7%(t=6.6,P<0.05)、86.9%(t=5.9,P<0.05)、95.2%(t=36.6,P<0.05)、95.3%(t=19.7,P<0.05).结论 0.25 mg/kg剂量的PAP在急性HBV感染小鼠模型体内对血清和肝组织的HBsAg、HBeAg的表达及HBV DNA的复制均有明显的抑制效果.     

A novel system for efficient gene transfer into primary human hepatocytes via cell-permeable hepatitis B virus-like particle

Protein transduction domains (PTDs) have been used to deliver a variety of biologically active cargo across cellular membranes. However the potential of PTDs to mediate transport of nanoparticular structures into the cytoplasm bypassing the endosomal compartment remains unclear. Cell-permeable virus-like particles (VLPs) harboring a marker gene based on hepatitis B virus nucleocaspids were established. Cell permeability was achieved by fusion with translocation motif (TLM)-PTD. Electron and confocal microscopy revealed that these VLPs translocate as complete particles across the plasma membrane and transverse the cytoplasm toward the nucleus. Inhibition of endocytosis did not affect translocation of these VLPs into the cytoplasm. Based on these particles, a gene transfer system was developed. To this end the particles were loaded with DNA-encoding small hepatitis B virus surface antigen (SHBs) or green fluorescence protein (eGFP) that served as marker genes. Although the DNA-packaging efficiency was very low, applying the appropriate number of VLPs to primary human hepatocytes a gene transfer efficiency of approximately 95% was observed. In conclusion, the TLM-PTD has the potential to mediate efficient transfer of assembled particles and its cargo, nucleic acids, into primary human hepatocytes. This provides the basis for development of novel transducible therapeutic or diagnostic particles.     

Repopulation of mouse liver with human hepatocytes and in vivo infection with hepatitis B virus

Mice containing livers repopulated with human hepatocytes would provide excellent in vivo models for studies on human liver diseases and hepatotropic viruses, for which no permissive cell lines exist. Here, we report partial repopulation of the liver of immunodeficient urokinase-type plasminogen activator (uPA)/recombinant activation gene-2 (RAG-2) mice with normal human hepatocytes isolated from the adult liver. In the transplanted mice, the production of human albumin was demonstrated, indicating that human hepatocytes remained functional in the mouse liver for at least 2 months after transplantation. Inoculation of transplanted mice with human hepatitis B virus (HBV) led to the establishment of productive HBV infection. According to human-specific genomic DNA analysis and immunostaining of cryostat liver sections, human hepatocytes were estimated to constitute up to 15% of the uPA/RAG-2 mouse liver. This is proof that normal human hepatocytes can integrate into the mouse hepatic parenchyma, undergo multiple cell divisions, and remain permissive for a human hepatotropic virus in a xenogenic liver. This system will provide new opportunities for studies on etiology and therapy of viral and nonviral human liver diseases, as well as on hepatocyte biology and hepatocellular transplantation.     

Expression of beta 2-microglobulin on hepatocytes in acute and chronic type B hepatitis

beta 2-Microglobulin display was examined in 131 liver biopsies from patients with acute and chronic type B hepatitis, using an indirect immunoperoxidase method. Enhanced expression of beta 2-microglobulin on hepatocyte membranes was observed in patients with acute hepatitis, chronic active hepatitis with moderate to severe activity and cirrhosis, when compared with normal liver. In acute hepatitis, beta 2-microglobulin-positive hepatocytes were mainly observed in perivenular areas in association with bridging necrosis. In chronic hepatitis, beta 2-microglobulin-positive hepatocytes were observed mainly in periportal zones and in some areas of lobular activity. Diffuse-enhanced display of beta 2-microglobulin on hepatocytes was observed in 5 of 6 patients treated with lymphoblastoid interferon as part of a trial of antiviral therapy. The mechanism by which beta 2-microglobulin display is enhanced on hepatocytes in patients not treated with interferon is uncertain. However, display of beta 2-microglobulin on hepatocytes probably reflects display of HLA-A, B and C antigens and may influence the course of hepatitis B virus infection by increasing susceptibility of the affected cells to T cell-mediated immune attack.     

Defective propagation of signals generated by sympathetic nerve stimulation in the liver of connexin32-deficient mice.

The gap junctional protein connexin32 is expressed in hepatocytes, exocrine pancreatic cells, Schwann cells, and other cell types. We have inactivated the connexin32 gene by homologous recombination in the mouse genome and have generated homozygous connexin32-deficient mice that were viable and fertile but weighed on the average approximately 17% less than wild-type controls. Electrical stimulation of sympathetic nerves in connexin32-deficient liver triggered a 78% lower amount of glucose mobilization from glycogen stores, when compared with wild-type liver. Thus, connexin32-containing gap junctions are essential in mouse liver for maximal intercellular propagation of the noradrenaline signal from the periportal (upstream) area, where it is received from sympathetic nerve endings, to perivenous (downstream) hepatocytes. In connexin32-defective liver, the amount of connexin26 protein expressed was found to be lower than in wild-type liver, and the total area of gap junction plaques was approximately 1000-fold smaller than in wild-type liver. In contrast to patients with connexin32 defects suffering from X chromosome-linked Charcot-Marie-Tooth disease (CMTX) due to demyelination in Schwann cells of peripheral nerves, connexin32-deficient mice did not show neurological abnormalities when analyzed at 3 months of age. It is possible, however, that they may develop neurodegenerative symptoms at older age.     

Evaluation of liver tissue by polymerase chain reaction for hepatitis B virus in patients with negative viremia

AIM: To assess the clinical significance of Hepatitis B virus (HBV) DNA localization in the liver tissue of patients with positive HBsAg and negative viremia. METHODS: HBV virological parameters of 33 HBsAg positive chronic hepatitis patients, including seromarkers and HBV DNA amplification in both sera and liver biopsies, were evaluated. RESULTS: Ten patients had negative viremia and positive HBV DNA in their liver biopsies. Most of them had HBeAg-negative/HBeAb-positive chronic hepatitis. Their liver biochemical and histopathological profiles were different from the viremic patients. Their disease pattern was designated as "hepatitis B in situ". CONCLUSION: Hepatitis B in situ is a consequential entity which can be missed in clinical practice. It is a new clinical pattern of chronic HBV infection that considers HBV in liver biopsy and adds a new indication for antiviral therapy.     

A new model for rheumatoid arthritis generated by engraftment of rheumatoid synovial tissue and normal human cartilage into scid mice

Objective. A new animal model was used to study the interaction between rheumatoid synovial cells and cartilage and to explore the cellular basis of rheumatoid joint destruction. Methods. Fresh synovial tissue derived from patients with rheumatoid arthritis was implanted with normal human cartilage into SCID mice, either subcutaneously or under the renal capsule, for up to 304 days. The implants were analyzed by light and electron microscopy, as well as by immunohistochemistry and in situ hybridization. Results. Human synovial tissue and cartilage implanted in SCID mice are maintained by the animals for up to 304 days. After 35 days, focal erosions occur at the site of attachment of synovial lining cells to the cartilage. After 105 days, a pannus-like formation, consisting of proliferating synovial fibroblast-like cells invading the cartilage, is observed. The fibroblast nature of these cells was supported by observation of only focal expression of the macrophage markers CD14 and CD68. Cells at the immediate site of cartilage destruction express messenger RNA for cathepsin L, whereas cathepsin D messenger RNA was detected in subsynovial regions away from the site of destruction. The human origin of the tissue involved in cartilage destruction was demonstrated using monoclonal antibodies to HLA-ABC and human type IV collagen. Conclusion. The present approach introduces a novel in vivo model of rheumatoid arthritis for the study of the molecular and cellular mechanisms of rheumatoid joint destruction at sites of synovial attachment to cartilage. In this model, the SCID mouse acts as a useful host for studying the properties of rheumatoid synovium in the absence of circulating human blood components.