重型乙型肝炎前期患者外周血免疫活性细胞的特点

目的 分析重型乙型肝炎前期(以下简称重肝前期)患者外周血免疫活性细胞的特点.方法 选取重肝前期患者48例,慢性乙型肝炎患者35例,健康志愿者20例,采用流式细胞仪检测外周血淋巴细胞CD3+、CD3+/CD4+、CD3 +/CD8+、CD4 +/CD25 +/CD45+等亚群表达百分比,计算各淋巴细胞亚群绝对值,并进行统计学分析.结果 与慢性乙肝组及健康对照组相比,重肝前期组CD3+T细胞、CD8+T细胞、CD4+ CD25+调节性T细胞(Tregs)的绝对值均明显下降(P＜0.01或P＜0.05);重肝前期组CD4+T细胞绝对值与慢性乙肝组差异无统计学意义(P＞0.05),但CD4+T细胞百分率明显高于慢性乙肝组(P＜0.05).此外,CD4 +/CD8+比值各组间存在显著差异,重肝前期组显著高于慢性乙肝组和健康对照组(P＜0.01或P＜0.05).结论 重型乙型肝炎前期存在一定程度的细胞免疫功能紊乱,其特征为CD4+T细胞占优势,CD8+T细胞和CD4+ CD25+ Tregs绝对值下降.     

HBV感染PBMC后对宿主细胞CD25 mRNA表达的影响

HBV除对肝细胞具有高度亲和性外，还可侵犯外周血单个核细胞（PBMC）,这不仅是HBV肝外感染的直接证据，也是HBV潜伏或隐匿的场所。CD25是IL-2Rα链，主要存在于T淋巴细胞表面，是T淋巴细胞活化的标志，在抗病毒免疫反应中起重要作用。为探讨HBV感染PBMC后对宿主细胞CD25mRNA表达的影响，本文选择249例慢性乙肝患者进行观察，现将结果报道如下。     

慢性乙型肝炎患者外周血CD8+/CD28+淋巴细胞及其亚型的研究

目的 探讨慢性乙型肝炎患者外周血CD8+/CD28+淋巴细胞及其亚型与其临床状态和HBV复制的关系.方法 采用流式细胞技术多色荧光分析法,检测研究对象的外周血CD8+淋巴细胞、CD45RO、CD45RA以及CD28的表达及HBV病毒载量.结果 ①慢性HBV携带组CD8+淋巴细胞CD28的表达率(10.64±5.09%)明显低于慢性乙肝组和正常对照组;而CD45RA和CD45RO的表达差异无统计学意义;②慢性乙肝组CD8+/CD45RO+/CD28+的表达(10.99±7.33%)明显高于正常对照组,而慢性乙肝组和慢性HBV携带组CD8+/CD45RA+/CD28+表达率均明显低于正常对照组;③HBeAg阴性慢性乙肝CD8+/CD45RO+/CD28+表达率明显高于阳性组,而CD8+/CD45RA+/CD28+表达率两组无差别.结论 慢性HBV携带者处于病毒携带状态可能与CD8细胞上协同分子CD28的表达低下有关;CD8+/CD45RO+/CD28+亚型淋巴细胞数的升高与慢性乙肝的病情进展及血清HBeAg转换有关.     

乙肝病毒相关肝病患者肝移植后外周血单个核细胞及肝组织中乙型肝炎病毒cccDNA的检测

背景：肝移植后受者体内绝大部分病毒负荷被清除,植入新肝后其复发性肝炎病原体从肝外进入肝内的途径及其复制规律目前尚无定论。 目的：检测肝移植前后外周血单个核细胞和肝组织中乙型肝炎病毒cccDNA及血清中乙型肝炎病毒DNA的表达。 方法：采用淋巴细胞分离液从乙肝病毒相关终末期肝病37例患者外周血中分离出单个核细胞,采用荧光定量PCR检测肝移植前后及移植后乙肝复发3个时期外周血单个核细胞和肝组织中cccDNA及血清乙型肝炎病毒DNA表达。 结果与结论：肝移植前,单个核细胞cccDNA阳性12例,肝组织cccDNA阳性6例,检出率分别为32%和16%,单个核细胞、肝组织中cccDNA拷贝范围分别为(3.028~6.508)×10⁴,(4.158~6.234)×10⁴ 拷贝/mL。肝移植后,单个核细胞cccDNA阳性1例,无血清乙型肝炎病毒DNA检测阳性病例。6例肝移植后乙肝复发病例中外周血单个核细胞cccDNA阳性4例,肝组织活检cccDNA阳性1例,6例血清乙型肝炎病毒 DNA均为阳性。提示乙肝病毒相关终末期肝病患者肝移植后乙肝复发途径可能是残留乙肝病毒在外周单个核细胞中以cccDNA为模板复制,然后再迁移到肝脏。     

HBV转基因小鼠肝脏NKT细胞功能与PD1、CD28表达分析

目的研究HBV转基因小鼠肝脏中NKT细胞的功能与表面PD1、CD28表达的关系。方法分离小鼠肝脏、脾脏、胸腺和腹膜淋巴结单个核细胞,利用流式细胞检测技术,分别检测其淋巴细胞中NKT细胞的频率,同时检测肝脏NKT细胞PD1、CD28的表达及IFN-γ、IL-4的分泌功能,比较肝脏、脾脏、胸腺和腹膜淋巴结这几个主要免疫组织淋巴细胞中NKT细胞所占的比例,并分析肝脏NKT细胞PD1、CD28的表达与细胞功能的关系。结果与正常同品系小鼠比较,HBV转基因小鼠肝脏、脾脏、胸腺和腹膜淋巴结NKT细胞数量明显减少(P<0.05),与脾脏、胸腺和腹膜淋巴结相比,肝脏淋巴细胞中含有大量的NKT细胞;与正常同品系小鼠比较,HBV转基因小鼠肝脏NKT细胞PD1的表达明显增多(P<0.05),CD28的表达明显减少(P<0.05),肝脏NKT细胞IFN-γ、IL-4的分泌功能明显降低(P<0.05)。结论肝脏中含有大量的NKT细胞,HBV转基因小鼠肝脏NKT细胞的功能存在明显的缺陷,并提示PD1的增加和CD28的降低可能与NKT细胞功能的下调密切相关。     

慢性乙型肝炎患者HBV特异性细胞毒性T细胞PD-1的表达研究

目的 检测慢性乙型病毒性肝炎患者外周血中HBV特异性CD8 细胞毒性T淋巴细胞表面细胞程序性死亡受体1(programmed cell death 1,PD-1)的表达情况.方法 采集慢性乙型肝炎患者外周血,直接离体情况下利用MHC-I-肽-五聚体技术标定HBV表位特异性细胞毒性T淋巴细胞以及荧光抗体标记细胞表面PD-1分子,经流式细胞仪检测分析.结果 在慢性乙型肝炎患者,HBV核心抗原18-27特异性CTL表达PD-1上调,达(79.0±12.5)%,显著高于总CD8 T细胞(27.7±14.8)%,以及CMV特异性CTL(20.6±5.9)%.结论 慢性乙肝患者HBV特异性CTL高表达PD-1分子,可能与慢性乙肝患者HBV特异性CTL功能低下密切相关.     

人源化小鼠异位肺癌动物模型的构建

目的：探讨一种可用于研究肺癌免疫的人源化小鼠动物模型。方法：采用人外周血单个核细胞输注非肥胖糖尿病/严重联合免疫缺陷小鼠体内建立人源化小鼠,在此小鼠模型基础上再构建异位肺癌荷瘤模型。结果：10只人源化小鼠异位荷瘤模型全部构建成功,免疫重构的小鼠外周血及脾脏中均可检测到大量人CD3＋T、CD4＋T、CD8＋T细胞。人源化荷瘤小鼠肿瘤组织内可发现人CD3＋T、CD4＋T、CD8＋T淋巴细胞浸润。结论：该模型为我们了解肺癌发生发展与免疫系统的关系,也为研究免疫治疗干预措施等提供了有价值的工具。     

慢性HBV感染患者肝组织内CD4+CD25+调节性T淋巴细胞的表达

目的 观察慢性HBV感染患者免疫耐受期与免疫清除期肝组织中CD4+ CD25+调节性T细胞的表达及分布情况.方法 应用免疫组织化学法检测19例免疫耐受期及12例免疫清除期慢性乙型肝炎患者肝组织中FoxP3的表达,6例正常肝组织为对照.结果 FoxP3阳性信号位于淋巴细胞胞核内,阳性细胞主要聚集在汇管区,肝窦内亦可见散在单个淋巴细胞呈阳性.在免疫耐受期及免疫清除期患者肝组织中FoxP3较正常肝组织明显增加(P<0.01),免疫清除期患者肝组织内Fox P3明显高于免疫耐受期患者(P<0.01).免疫清除期患者肝组织中FoxP3阳性标记指数与ALT、HBeAg及HBV-DNA水平无明显相关性.免疫耐受期与免疫清除期两组相比,在年龄、ALT、TBIL、PTA、HBeAg及HBV-DNA水平方面差异均有统计学意义.结论 肝组织中CD4+ CD25+调节性T细胞在慢性HBV感染慢性化和抑制免疫,控制肝脏炎症反应方面可能起了重要作用.     

慢性乙型肝炎患者外周血CD4~+CD25~+Treg与CD4~+和CD8~+ T淋巴细胞亚群的相关研究

目的:探讨慢性乙型肝炎患者外周血中CD4+CD25+调节性T细胞的含量和CD4+CD8+T淋巴细胞亚群分布,两者之间相关性及与HBV的相关性。方法:采用流式细胞术检测50例慢性乙型肝炎患者和20例健康对照者外周血中CD4+CD25high、CD4+CD25+Foxp3+Treg细胞表达及CD3/CD4/CD8 T淋巴细胞亚群,荧光定量PCR法检测HBV DNA含量。结果:慢性乙型肝炎患者外周血中CD4+CD25highTreg明显高于健康对照组(P0.01),且随HBV DNA载量增加,患者外周血中CD4+CD25highTreg细胞的水平逐渐升高。慢性乙型肝炎患者外周血中CD4+CD25+Foxp3+Treg细胞也相应增高,且与CD4+CD25highTreg细胞的变化成正相关(r=0.890,P0.001)。与健康对照组比较,患者组CD4+T细胞百分率及CD4+/CD8+比值均降低,而CD3+T细胞和CD8+T细胞百分率差异无显著性(P0.05)。CD4+CD25highTreg细胞与HBV DNA取对数后成正相关(r=0.782,P0.001),与谷丙转氨酶(ALT)成正相关(r=0.432,P0.005);与CD3+、CD4+、CD8+T细胞水平及CD4+/CD8+比值均无相关性(P0.05)。CD3+、CD4+、CD8+T淋巴细胞及CD4+/CD8+比值与HBV DNA载量之间亦无相关性(P0.05)。结论:慢性乙型肝炎患者外周血中CD4+CD25+Treg细胞增高,且与HBV的复制水平及ALT增高具有一致性,而T细胞亚群是否可作为监测CHB患者免疫状态的指标需进一步探讨。     

小鼠NKT细胞在5型腺病毒感染所致肝损伤早期的免疫调节作用

目的 使用5型腺病毒(AdS)感染小鼠模型来研究肝脏NKT细胞(natural killer Tcell)在肝损伤早期的免疫调节机制.方法 C57BL/6小鼠尾静脉注射1.5×109PFU和3×109PFUAdS病毒以构建两个剂量组病毒感染的小鼠肝损伤模型,通过观察病毒感染后5 d内小鼠肝组织病理学及小鼠血清丙氨酸转氨酶/天门冬氨酸转氨酶(ALT/AST)水平改变来判断肝损伤程度,使用流式细胞术(FACS)分析感染5 d内肝单个核细胞亚群比例、NKT细胞表面FasL表达水平以及NKT细胞合成IL-4和IFN-γ水平的变化,应用RT-PCR检测小鼠肝内趋化因子及趋化因子受体表达水平.结果 高滴度(3×109 PFU)的AdS病毒感染小鼠1 d后,小鼠肝脏内NKT细胞明显增加,其表面FasL表达上调,肝脏NKT细胞合成IL-4和IFN-γ的水平明显增加,肝组织内淋巴细胞浸润明显;低滴度AdS病毒(1.5×109PFU)感染小鼠后,肝脏NKT细胞比例变化不明显,CD8+T细胞在肝脏的浸润明显弱于高滴度AdS病毒感染;RT-PCR检测结果 显示:3×109PFU AdS病毒感染2 d后,小鼠肝内活化后可调节的及正常的T细胞分泌的趋化因子(RANTES)、人干扰素诱导蛋白10(IP-10)以及巨噬细胞炎症蛋白(MIP)-1β表达增加,3d后相关趋化因子受体CCR5、CCR1、CXCR3表达上调.结论 NKT细胞在淋巴细胞向肝脏趋化的过程中起重要的作用,这种作用与病毒感染诱导NKT细胞合成IL-4和IFN-γ及上调其表面的FasL,从而促进肝细胞内IP-10、Mig等趋化因子的产生有关.     

四种细胞移植重建C57BL雌鼠免疫功能的对比

背景：据报道肌肉来源的细胞能重建造血功能,还需要进一步观察其他系细胞是否也能重建造血功能。 目的：对比不同细胞对C57BL雌鼠造血功能恢复的作用。 方法：摘眼球取绿色荧光蛋白转基因雄鼠外周血,溶血后制成白细胞,再取脾脏、肝脏制成脾细胞和肝细胞悬液,同时制备骨髓细胞。将C57BL雌鼠半致死量射线照射后,回输4种细胞,并在移植后18,39,53 d检测外周血绿色荧光蛋白阳性细胞。同时标记CD4-PE和CD8-PE,并在移植后100 d杀鼠取骨髓检测Y染色体阳性率。 结果与结论：移植后18,39,53 d均在C57BL雌鼠外周血检测到绿色荧光蛋白阳性细胞;C57BL雌鼠外周血绿色荧光蛋白阳性细胞百分比：回输脾细胞和骨髓细胞>回输肝细胞>回输外周血白细胞。同时在回输脾细胞和骨髓细胞的C57BL雌鼠外周血中检测到绿色荧光蛋白和CD4双阳性细胞、绿色荧光蛋白和CD8双阳性细胞,而回输肝细胞和外周血白细胞的C57BL雌鼠外周血中几乎未检测到双阳性细胞。移植后100 d检测到的Y染色体阳性率与外周血中绿色荧光蛋白阳性率呈正相关。结果说明绿色荧光蛋白转基因雄鼠的外周血白细胞、脾细胞、肝细胞和骨髓细胞均有重建造血功能的作用,其中脾细胞与骨髓细胞作用相似,作用大于肝细胞,肝细胞作用大于白细胞。     

Mouse hepatitis viral infection induces an extrathymic differentiation of the specific intrahepatic alpha beta-TCRintermediate LFA-1high T-cell population.

Mouse hepatitis virus type 3 (MHV3), a coronavirus, is an excellent model for the study of thymic and extrathymic T-cell subpopulation disorders induced during viral hepatitis. It was recently reported that, in addition to the intrathymic T-cell differentiation pathway, an extrathymic differentiation pathway of alpha beta-T-cell receptor (TCR) T lymphocytes exists in the liver, and becomes important under pathological situations such as autoimmune diseases, malignancies or hepatic bacterial infections. In the present study, we compared the phenotypes of resident hepatic, splenic or thymic T-cell subpopulations during the acute viral hepatitis induced by HMV3 in susceptible C57BL/6 mice. The number of liver-resident mononuclear cells (MNC) increased during the viral infection, while cellularity decreased. Single positive (SP) CD4+ cells strongly increased in both the liver and thymus, while double positive (DP) (CD4+ CD8+) cells, present in the liver and thymus of mock-infected mice, decreased in C57BL/6 mice during the viral infection. A shift of alpha beta-TCRintermediate T cells toward alpha beta-TCRhigh was evidenced in the liver and thymus of infected mice, but not in the spleen. The few alpha beta-TCRint double negative (DN) (CD4-CD8-) cells also decreased following viral infection. alpha beta-TCRint or high lymphocytes expressing high levels of leucocyte function antigen-1 (LFA-1) increased in the liver of MHV3-infected mice. In addition, liver-resident T cells expressed strongly the CD44 (Pgp-1) activation marker, suggesting that they were either activated or antigen experienced during the viral infection. No significant change in T-cell subpopulations was detected in the spleen, suggesting that MHV3 infection could induce an early in situ differentiation of resident hepatic T cells rather than a recruitment of lymphocytes from peripheral lymphoid organs.     

Strain-Dependent Migration of CD4 and CD8 Lymphocyte Subsets to Lymph Nodes in NOD (Nonobese Diabetic) and Control Mice

Subpopulations of lymphoid cells were compared with respect to their ability to migrate into peripheral lymphoid organs of nonobese diabetic (NOD) mice and various strains of control mice. In short-term, in vivo homing studies, no major differences in the pattern of homing of B and T cells were observed among all mouse strains studied. On the other hand, CD4 cells localized consistently more efficiently than CD8 cells in both PP and LN of adult NOD and BALB/c mice, whereas both populations migrated roughly equivalently in LN of adult DBA/2, CBA, and C57BL/6 mice. No age-dependent differences in the homing of CD4 and CD8 cells were observed in BALB/c mice. On the contrary, in 2-week-old NOD mice, CD4 and CD8 cells migrated equally well. The preferential entry of CD4 cells in adult NOD and BALB/c did not result from increased blood transit time of CD8 cells. On the other hand, the preferential migration of CD8 cells was observed in the liver, whereas the two T-cell subsets migrated equally well in the lungs. The differences in the homing characteristics of CD4 and CD8 cells among NOD, BALB/c, and C57BL/6 mice were not related to modifications in the level of expression of adhesion molecules such as MEL-14, LFA-1, and Pgp-1.     

Transgenic mice expressing hepatitis B virus X protein are more susceptible to carcinogen induced hepatocarcinogenesis

The hepatitis B virus X (HBx) protein is thought to be implicated in the development of human hepatocellular carcinoma (HCC), but its exact function remains controversial. To investigate whether the expression of the HBx gene alone can induce HCC on an inbred C57BL/6 strain that displays a lower spontaneous rate of liver cancer, and to determine if HBx transgenic mice are more susceptible to the effects of hepatocarcinogens, C57-TgN (HBx) X transgenic mice were bred with normal C57BL/6 mice strain. The F1 mice (about 50% HBx positive and 50% HBx negative) were treated with a single dose of diethylnitrosamine (DEN) at 7 days of age, or were untreated. Mice were killed at appropriate time points and were analyzed for histological change in the liver. The expression of HBx protein were examined by using immunohistochemical staining. Glycogen storage foci were examined by using periodic acid-Schiff (PAS) staining. In HBx transgenic mice untreated with DEN, HBx expression and glycogen storage foci were always observed in the liver after 8 weeks, but not obvious histological pathologic changes. Histological examination of liver tissue confirmed that DEN-treated HBx mice developed approximately twice as many focal lesions of basophilic hepatocytes as treated wild-type littermates. Hepatocellular adenomas and carcinomas were also more frequent in DEN-treated HBx-positive than HBx-negative mice. Taken together, our results suggest that HBx gene expression alone is not sufficient for carcinogenesis, but may act as a promoter for malignant transformation.     

乙肝疫苗增强细胞因子诱导的杀伤细胞对于转乙型肝炎病毒基因小鼠的治疗作用

目的 观察乙肝疫苗是否增强细胞因子诱导的杀伤细胞(cytokine-induced killer cell,CIK)对于乙型肝炎病毒转基因小鼠(hepatitis B virus transgenic mice,HBV-Tg)的抗病毒作用.方法 给予HBV-Tg腹腔注射CIK细胞,皮下注射乙肝疫苗,用荧光定量PCR检测外周血中HBV DNA水平变化,流式细胞仪检测外周血中T淋巴细胞亚群,并用HE染色观察肝脏的组织病理改变.结果 CIK细胞降低了HBV-Tg鼠外周血中病毒载量,血中CD3~+、CD4~+及CD8~+细胞增多,乙肝疫苗和CIK细胞联合应用后,这种作用增强.结论 乙肝疫苗增强了CIK对于HBV-Tg小鼠的治疗作用,这种增强作用是否通过增加体内CD3~+、CD4~+及CD8~+细胞,尤其是CD8~+细胞而实现,有待进一步研究.     

黄芪多糖对小鼠骨髓及外周血造血干细胞的增殖及动员作用

探讨APS-P对正常或化疗后小鼠的骨髓、脾及外周血造血干细胞的促增殖及外周动员作用。给正常或经环磷酰胺化疗后的C57BL／6小鼠注射APS-P，然后取骨髓细胞、外周血细胞及脾细胞，并用荧光抗体PerCP-Sca-1，APE-c-kit及PE-Lineage(CD3，CD4，CD8，Trll9，B220 CDllb，Gr-1)进行染色标记，用流式细胞仪检测小鼠造血干细胞(Lin c-kit^ Sca-1^ )数量变化。注射APS-P能使正常及化疗小鼠骨髓、脾及外周血中的造血干细胞有不同程度的增加。这一作用可能是由于APS-P促进骨髓造血干细胞的增殖，而在外周血及脾中的增加则可能是由于APS-P对骨髓造血干细胞的外周动员作用。     

Normal mouse kidneys contain activated and CD3+CD4- CD8- double-negative T lymphocytes with a distinct TCR repertoire

Healthy liver, intestine, lung, and skin harbor resident lymphocytes with conventional and unconventional phenotypes. Lymphocytes also have been detected in healthy mice kidneys; however, these cells have not been well studied and have been largely overlooked. To better characterize the intra-renal lymphocytes, we extensively perfused C57BL/6J mice with PBS and then isolated mononuclear cells for flow cytometry analysis. We observed T cells, B cells, and NK cells in normal mice kidneys after extensive perfusion. Approximately 50% of kidney T lymphocytes expressed intermediate levels of CD3 (CD3int T cells). Similar to liver and lung, a high percentage of unconventional CD3+CD4(-)CD8(-) double-negative T cells was observed in normal mice kidneys, from which 11% expressed B220 antigen. Unlike the spleen and blood, the classic CD4+ and CD8+ T lymphocytes in the kidney had a high proportion of activated CD69+ and effector/memory CD44- CD62L ligand phenotypes. Also, a small percentage of CD4+CD25+forkhead box p3+ and NKT cells was observed in perfused and exanguinated kidneys. In addition, a distinct TCR repertoire was found on intra-renal conventional and unconventional T cells compared with those from the spleen. Finally, after 24 h of renal ischemia reperfusion injury (IRI), increased production of cytokines IFN-gamma and TNF-alpha by CD4+ and CD8+ T cells, isolated from perfused kidneys, was observed. These data suggest that some of these cells harbored in the kidney could be implicated in the immune response of the IRI pathogenic process.     

In vivo staphylococcal superantigen-driven polyclonal Ig responses in mice: dependence upon CD4(+) cells and human MHC class II

Staphylococcal enterotoxin (SE) B and seven other staphylococcal superantigens (SAg), despite promoting vigorous Ig production in human peripheral blood mononuclear cell cultures, are exceedingly poor at eliciting Ig responses in cultures of spleen cells from C57BL/10J (B10) or C3H/HeJ mice. In contrast, SEB elicits Ig responses in cultures of spleen cells from human MHC class II-transgenic mice. Whereas i.p. administration of SEB (0.2-20 microg) to non-transgenic B10 mice elicits very weak in vivo Ig responses, identical treatment of CD4(+) cell-intact (but not CD4(+) cell-depleted) human MHC class II-transgenic mice elicits dramatic increases in both splenic Ig-secreting cells and serum Ig levels. Over a 2-week period, the SEB-induced in vivo Ig responses peak and then plateau or fall in association with a preferential increase in splenic CD8(+) cells. Nevertheless, in vivo depletion of CD8(+) cells has no sustained effect on SEB-driven Ig responses. Taken together, these observations demonstrate that the effects of SAg on in vivo humoral immune responses are highly CD4(+) cell dependent, are substantially CD8(+) cell independent and can be successfully investigated using human MHC class II-transgenic mice. This model system may be useful in investigating the polyclonally activating effects of microbial products (prototypic environmental insults) on the development of systemic autoimmunity.     

Increased susceptibility to apoptosis and attenuated Bcl-2 expression in T lymphocytes and monocytes from patients with advanced chronic hepatitis C

Some viral infections are reported to influence the susceptibility of peripheral blood mononuclear cells (PBMC) to apoptosis, which is related to disease progression. The current study was designed to monitor apoptosis in separated PBMC subsets, CD4+ and CD8+ T lymphocytes, and CD14+ monocytes under apoptotic stimuli in patients with chronic hepatitis C. Apoptosis was induced by serum starvation and by incubation with anti-CD3 antibody and with phorbol 12-myristate 13-acetate. With the escalating severity of liver disease, susceptibility of all PBMC subsets to apoptosis increased under the apoptotic stimulus of serum starvation (P<0.05). Consequently, increased susceptibility to apoptosis was associated with diminished intracellular expression of the antiapoptotic protein Bcl-2 (P<0.05). The current observations demonstrate that the abnormality of PBMC subsets in undergoing apoptosis as a result of the down-regulation of Bcl-2 expression may contribute to viral persistence and progression of liver disease in chronic hepatitis C.     

Green fluorescent protein-transgenic mice: immune functions and their application to studies of lymphocyte development

Green fluorescent protein (GFP) transgenic (GFP+) mice express GFP in most tissues except erythrocytes and hair. Immune responses of GFP+ mouse and their application to studies of lymphocyte development were investigated. Flow cytometric analyses revealed that differentiation patterns of lymphocytes from GFP+ mice are equivalent to those from parental C57BL/6 mice. There was no difference in mature T-cell proliferative ability in response to allogeneic stimulator cells or anti-CD3epsilon stimulation between GFP+ and C57BL/6 mice. Furthermore, the anti-OVA antibody response of GFP+ mice was also the same as that of C57BL/6 mice. Taken together, these results show no immunological differences between GFP+ and C57BL/6 mice. Bone marrow transplantation and in vitro thymus reconstitution experiments were performed in an attempt to apply the GFP+ mice to the analysis of lymphocyte development. When bone marrow cells from GFP+ mice were transplanted. T and B lymphocytes containing GFP developed normally in scid recipients. Next we examined intrathymic T-cell development by hanging drop culture methods. GFP+ and CD4+8+ immature T-cells developed normally from bone marrow cells in the reconstituted thymus. The experimental system using hematopoietic cells from GFP+ mice is a powerful tool for visualizing lymphocyte development.