腺病毒载体在小鼠肝脏的分布及其表达规律研究

目的研究腺病毒载体介导的β-半乳糖苷酶(LacZ)报告基因在小鼠肝脏的分布及表达情况,评价腺病毒载体作为基因治疗转导系统的有效性及安全性,为进一步的目的基因导入及表达奠定基础。方法将Balb/cJ小鼠随机分为正常组及暴发性肝炎组,给小鼠经尾静脉注射2×108PFU腺病毒,收集24h、48h、72h、96h、120h和第7天小鼠血清,检测谷丙转氨酶和总胆红素,同时取肝脏、肺脏、心脏、肾脏标本进行X-gal染色,以观察腺病毒载体在各脏器的表达情况。取MHV-3诱导的暴发性肝炎组小鼠24h、48h、72h、96h肝脏,检测腺病毒载体在肝脏的表达情况。结果腺病毒载体主要在小鼠肝脏表达,并于72小时表达最高,在正常小鼠及暴发性肝炎小鼠肝脏中表达效率分别约55.7%和25.7%,之后逐渐消减;正常组小鼠注射腺病毒载体后,肝功能无明显变化。结论腺病毒载体介导的报告基因可在肝脏高效表达且未见明显的肝损害,可作为基因治疗中安全有效的基因传递系统用于治疗肝脏疾病。     

MHV-3诱导暴发性肝炎小鼠肝组织KCNJ15的表达变化

目的研究病毒感染所致的急性肝衰竭模型中离子通道基因KCNJ15表达水平,探讨该疾病模型中KCNJ15的变化及其与疾病进程的关系。方法利用MHV-3病毒诱导的Balb/cJ小鼠暴发型肝炎模型,采用定量PCR和免疫组化技术分别从基因及蛋白水平检测肝脏KCNJ15表达水平,并应用流式细胞术检测肝脏淋巴细胞亚群KCNJ15表达。结果随着MHV-3感染时间的延长,肝组织中KCNJ15表达水平逐渐增高,以感染后72小时最为显著。在肝脏CD4+T细胞,KCNJ15蛋白表达水平于48小时显著升高,达到25.17±7.68%,与0h(3.92±1.33%)相比差异具有统计学意义(P<0.001),随后于72小时回落;肝脏表达KCNJ15的CD8+T细胞比例变化与CD4+T细胞趋势一致,于48h达到37.08±8.73%,与0h(6.98±3.48%)相比有显著差异(P<0.001);而表达KCNJ15的肝脏NK细胞比例则从7.72±1.34%上升到感染后24小时的峰值19.80±4.25%(P<0.001),随后回落。结论肝组织及肝脏淋巴细胞过表达KCNJ15可能参与了MHV-3诱导的暴发性肝炎小鼠免疫诱导的肝脏损伤过程。     

Foxp3+ regulatory T cells protect the liver from immune damage and compromise virus control during acute experimental hepatitis B virus infection in mice

The strength of antiviral T cell responses correlates with clearance of hepatitis B virus (HBV) infection, but the immunological mechanisms mitigating or suppressing HBV-specific T cells are still poorly understood. In this study, we examined the role of CD4(+) Foxp3(+) regulatory T cells (Tregs) in a mouse model of acute HBV infection. We initiated HBV infection via an adenoviral vector transferring a 1.3-fold overlength HBV genome (AdHBV) into transgenic DEREG mice, where Tregs can be transiently but selectively depleted by injection of diphtheria toxin. The effect of Treg depletion on the outcome of HBV infection was characterized by detailed virological, immunological, and histopathological analysis. Numbers of Tregs increase in the liver rapidly after initiation of HBV replication. Initial depletion of Tregs revealed their complex regulatory function during acute infection. Tregs mitigated immunomediated liver damage by down-regulating the antiviral activity of effector T cells by limiting cytokine production and cytotoxicity, but did not influence development of HBV-specific CD8 T cells or development of memory T cells. Furthermore, Tregs controlled the recruitment of innate immune cells such as macrophages and dendritic cells to the infected liver. As a consequence, Tregs significantly delayed clearance of HBV from blood and infected hepatocytes. Conclusion: Tregs limit immunomediated liver damage early after an acute infection of the liver, thereby contributing to conservation of tissue integrity and organ function at the cost of prolonging virus clearance. (HEPATOLOGY 2012;56:873-883).     

Characterization of splenic cells during the early phase of infection with neuropathogenic mouse hepatitis virus

The highly neuropathogenic cl-2 and less virulent srr7 viruses isolated from the neurotropic JHM strain of the mouse hepatitis virus exhibit super acute spread of virus (SAS), a term applied when rapid viral spread from an organ or part of the initially infected site to another non-adjacent organ or part is detected within 12 h after infection. Herein, we used a cytospin procedure to confirm SAS in splenic cells derived from mice whose brains were infected with these viruses. The cytospin procedure enabled effective preservation of the cells on glass slides. With this method, we could characterize extremely low populations of infected cells in the spleen (less than 0.1%) at 12 h post-inoculation with srr7. We observed that all kinds of splenic cells examined were infected, including B220(+)Ly-6C(+) plasmacytoid dendritic cells. The population of viral antigen-positive splenic cells was only slightly higher in cl-2 infection than in srr7 infection, but the cells showing viral production were present in numbers significantly higher in cl-2 infection compared with srr7 infection.     

Changes in individual plasma amino acids following experimentally induced and fly fever virus infection

Fasting concentrations of 21 individual plasma amino acids were determined in daily (7:30 a.m.) serial samples from eight volunteers infected with sand fly fever virus and compared to values obtained in six separate daily preexposure baseline measurements in each volunteer, as well as to serial measurements in three unexposed control subjects. By 47 hr after inoculation and before the onset of fever or other clinical indications of infection, most individual plasma amino acids were significantly depressed below preexposure values. These changes began before the marked decrement in protein intake during the illness. Such changes in plasma amino acids did not occur in control subjects. Reduction in amino acid concentrations persisted until after the lysis of fever and did not coincide in timing with alterations in white blood counts or serum Zn and Fe values. Urinary total nitrogen, urea, and alpha amino nitrogen were not altered during the course of sand fly fever in these subjects. Although food intake was reduced during sand fly fever, the magnitude of the amino acid depression was far greater than that reported during starvation or protein deprivation in non-infected subjects, and the sequence of changes in plasma valine, alanine, and glycine followed patterns different from those reported during starvation. It may be postulated that unusually large quantities of certain plasma amino acids were taken up by the cells of the liver and other visceral tissues during this infection. Plasma phenylalanine responded in a manner different from that of the other amino acids. It was decreased on day 2 after exposure to the virus but by day 4 and 5 was significantly increased above preinfection values. This resulted in a significant increase in the phenylalaninetyrosine ratio during the febrile phase of sand fly fever.     

Free amino acids in plasma and skeletal muscle of patients with liver cirrhosis

Free amino acids were measured under postabsorptive conditions in plasma and intracellular water of skeletal muscle obtained by needle biopsy in nine healthy controls and 14 subjects suffering from clinically stable liver cirrhosis. The aromatic amino acids phenylalanine and tyrosine in cirrhotics were elevated to the same extent in plasma and in muscle water. Branched-chain amino acids were uniformly reduced in plasma, but in muscle water only valine was significantly lower (222 +/- 92 mumoles per kg intracellular water vs. 368 +/- 82, p less than 0.001), while isoleucine (142 +/- 63 vs. 103 +/- 30), leucine (223 +/- 88 vs. 226 +/- 36) and branched-chain amino acids as a whole (589 +/- 186 vs. 681 +/- 88) were normal or elevated with an increased muscle:plasma ratio (3.12 +/- 2.03 vs. 1.41 +/- 0.37, p less than 0.05 for isoleucine; 3.00 +/- 1.28 vs. 1.85 +/- 0.27, p less than 0.025 for leucine; 2.24 +/- 0.64 vs. 1.69 +/- 0.13, p less than 0.05 for total branched-chain amino acids. Our data show that, in cirrhosis, plasma concentrations of branched-chain amino acids do not reflect their levels in muscle cellular water; only the intracellular pool of valine is severely depleted. This suggests that higher amounts of valine supplementation may be useful in nutritional treatment of liver cirrhosis. The elevated muscle:plasma gradients for branched-chain amino acids may result from abnormalities in their transport through muscle-plasma membrane.(ABSTRACT TRUNCATED AT 250 WORDS)     

Acute and chronic changes in the microcirculation of the liver in inbred strains of mice following infection with mouse hepatitis virus type 3

The acute and chronic effects of mouse hepatitis virus type 3 on the microcirculation of the liver in both semisusceptible C3HeB/FeJ and fully resistant A/J mice were studied. In the C3HeB/FeJ mice, abnormalities of microcirculatory flow were noted as early as 12 hr after infection and by 24 hr, localized avascular foci appeared. Disturbances were characterized by granular blood flow, sinusoidal microthrombi, distortion of sinusoids by edematous hepatocytes and necrotic lesions. Following the acute infection, Day 10, two patterns of chronic disease were observed. Eighty percent of the mice developed chronic granulomatous hepatitis whereas in the remaining 20% a more severe chronic aggressive hepatitis was observed which was characterized by ongoing hepatocellular necrosis and a marked mononuclear cell infiltrate. In both cases, in vivo microcirculatory abnormalities were found predominantly around visible lesions. Onset of the microcirculatory abnormalities was found to be concomitant with a rise in monocyte related procoagulant activity. Procoagulant activity rose acutely and remained elevated throughout the chronic phase but was higher in animals with severe disease. In contrast to the above, normal blood flow and histology were seen in the resistant A/J mice at all times following infection, and procoagulant activity remained at basal levels despite active viral replication as demonstrated by immunofluorescence studies and recovery of infectious virus. These observations suggest a role for monocyte procoagulant activity in the development of microcirculatory abnormalities following mouse hepatitis virus type 3 infection which may be important in the pathogenesis of the disease.     

Immunosuppression with cyclosporine during the incubation period of experimental woodchuck hepatitis virus infection increases the frequency of chronic infection in adult woodchucks.

The immunosuppressive agent cyclosporine was given to adult woodchucks during acute experimental infection with woodchuck hepatitis virus (WHV). All 17 woodchucks given WHV alone or with a vehicle resolved the infection (i.e., zero chronicity), but when cyclosporine was given throughout the incubation and acute phases of infection (0-12 or 14 weeks; n = 12), the rate of chronic infection increased to 92%. When cyclosporine was given only during the incubation period (0-4 weeks; n = 10) or only during the acute phase of infection (2-12 weeks; n = 9), the rates increased to 50% and 55%, respectively. However, when the drug was given after the acute phase (8-18 weeks; n = 9), the chronic infection rate (11%) did not differ from that in untreated and vehicle controls. Immune responses inhibited by cyclosporine are important in resolution of acute WHV infection and occur mainly during the first 8 weeks. Immunosuppression of these responses for even short intervals during incubation (e.g., 0-4 weeks) increases the risk of chronicity.     

IL-22和SOCS3在3型鼠肝炎病毒诱导的小鼠慢性病毒性肝炎发病中的作用探讨

探讨鼠3型肝炎病毒（MHV-3）诱导的慢性病毒性肝炎C3H/Hej小鼠肝组织白细胞介素-22（IL-22）和细胞因子信号抑制因子3（SOCS3）mRNA水平的变化。方法给C3H/Hej小鼠腹腔注射MHV-3（100 pfu）,诱导慢性病毒性肝炎模型,采用实时荧光定量PCR 法检测MHV-3感染后0、5、10、15和20 d时小鼠肝组织IL-22和SOCS3 mRNA水平的变化。结果模型鼠肝组织IL-22和SOC3 mRNA水平在0 d时分别为（0.26±0.15）%和（6.35±2.21）%,在感染后10、15和20 d后IL-22水平分别为（6.28±2.79）%、（2.50±1.24）%和（2.73±0.85）%,SOC3水平分别为（16.92±4.39）%、（14.06±4.09）%和（13.36±1.89）%,均较0 d时显著增高（P〈0.05）,且在第10 d达到最高峰。结论 IL-22和SOCS3可能参与了MHV-3诱导的小鼠慢性病毒性肝炎的发生发展过程。     

Role of hepatitis C virus infection in spontaneous hepatitis B surface antigen clearance during chronic hepatitis B virus infection.

To examine the role of hepatitis C virus (HCV) infection in spontaneous hepatitis B surface antigen (HBsAg) clearance during the course of chronic hepatitis B virus (HBV) infection, serum specimens from 32 asymptomatic HBsAg carriers and 22 patients with chronic hepatitis type B who underwent spontaneous HBsAg clearance were studied for antibody to HCV (anti-HCV) using commercial EIAs. The results were compared with those of control groups matched for age, sex, hepatitis B e antigen, antibody to hepatitis delta virus, and cirrhosis. Eight (25%) of the asymptomatic carriers and 9 (41%) of the patients with chronic hepatitis were seropositive for anti-HCV in contrast to 1.6% and 9.1% of their respective control groups (P less than .01). Serum alanine aminotransferase level was persistently abnormal after HBsAg clearance in one asymptomatic carrier and in four patients with chronic hepatitis. These patients were seropositive for anti-HCV and at least one of them was negative for HBV-DNA by polymerase chain reaction. The data suggest that HCV superinfection may not only suppress HBV or terminate the HBsAg carrier state but may also assume the role of HBV as the cause of persistent hepatitis or transaminase elevation.     

Polyclonal immunoglobulins from a chronic hepatitis C virus patient protect human liver-chimeric mice from infection with a homologous hepatitis C virus strain

The role of the humoral immune response in the natural course of hepatitis C virus (HCV) infection is widely debated. Most chronically infected patients have immunoglobulin G (IgG) antibodies capable of neutralizing HCV pseudoparticles (HCVpp) in vitro. It is, however, not clear whether these IgG can prevent a de novo HCV infection in vivo and contribute to the control of viremia in infected individuals. We addressed this question with homologous in vivo protection studies in human liver-urokinase-type plasminogen activator (uPA)(+/+) severe combined immune deficient (SCID) mice. Chimeric mice were loaded with chronic phase polyclonal IgG and challenged 3 days later with a 100% infectious dose of the acute phase H77C virus, both originating from patient H. Passive immunization induced sterilizing immunity in five of eight challenged animals. In the three nonprotected animals, the HCV infection was attenuated, as evidenced by altered viral kinetics in comparison with five control IgG-treated animals. Plasma samples obtained from the mice at viral challenge neutralized H77C-HCVpp at dilutions as high as 1/400. Infection was completely prevented when, before administration to na?ve chimeric mice, the inoculum was pre-incubated in vitro at an IgG concentration normally observed in humans. CONCLUSION: Polyclonal IgG from a patient with a long-standing HCV infection not only displays neutralizing activity in vitro using the HCVpp system, but also conveys sterilizing immunity toward the ancestral HCV strain in vivo, using the human liver-chimeric mouse model. Both experimental systems will be useful tools to identify neutralizing antibodies for future clinical use.     

HBV的小鼠模型研究进展

乙型肝炎病毒感染是全球范围内影响人类健康的重要问题,目前人们对HBV及其所致疾?膊 病有了相当深入的认识.但由于缺乏合适的动物模型,乙型肝炎病毒的生物学研究和治疗进展缓慢.小鼠作为一种实验室常用的动物,遗传免疫背景清楚明确,已经成为人们研究乙肝的重要工具.本文简要综述了小鼠模型在乙型肝炎研究中的进展.     

MHV-3诱导的暴发性肝炎小鼠肝脏和慢加急性肝衰竭患者外周血单个核细胞 TLR2表达的变化

目的：研究慢性乙型肝炎患者和慢加急性乙型肝炎肝衰竭（HBV-ACLF）患者外周血单个核细胞（PBMC）Toll 样受体2（TLR2）表达,以及鼠三型肝炎病毒（MHV-3）诱导的暴发性肝炎小鼠肝脏 TLR2表达的变化。方法收集慢性乙型肝炎和 HBV-ACLF 患者外周血,分离 PBMC,采用实时定量 PCR 法检测 PBMC 中 TLR2 mRNA;给 Balb/cJ 小鼠腹腔注射MHV-3（100 pfu）,建立小鼠暴发性肝炎模型,观察感染0、24、48和72 h 后肝脏TLR2水平变化。结果 BALB/cJ 小鼠在感染 MHV-3后,与0 h[（0.39±0.06）%]比,肝细胞 TLR2 mRNA 水平在感染48和72 h 均显著升高[分别为（9.06±1.60）%和（6.42±2.42）%,P＜0.05）],并于48 h 达最高水平,且两时间点细胞 TLR2 mRNA 水平均与血清 ALT 和 AST 水平呈正相关（r=0.804,P＜0.01;r=0.797,P＜0.01）;HBV-ACLF患者 PBMC 中 TLR2 mRNA 水平显著高于慢性乙型肝炎患者[（5.92±5.26）%对（1.15±1.59）%,P＜0.05）]。结论 TLR2参与了 MHV-3诱导的暴发性肝炎小鼠以及 HBV-ACLF 患者肝脏损伤的发病过程。