肝癌组织HNF4α mRNA定量检测方法的建立与初步应用

目的 建立一种定量检测肝细胞癌（HCC）组织肝细胞核因子4α（HNF4α）mRNA的方法。方法 取手术获得的HCC组织16例,提取总RNA,逆转录PCR扩增获得全长HNF4α mRNA对应cDNA产物,经TA克隆后,测序确认。根据获得序列,设计检测HNF4α mRNA 引物及MGB荧光探针。以体外转录HNF4α mRNA为标准品,建立一步法 逆转录荧光定量PCR检测方法,同时设定β-actin mRNA为内参对照,最终计算得到组织HNF4α mRNA水平。采用免疫组化染色检测HNF4α蛋白表达,比较HNF4α mRNA水平与HNF4α蛋白表达的对应结果。结果 HCC组织HNF4α mRNA定量标准曲线斜率为-3.237,相关系数r值达0.994,β-actin mRNA定量标准曲线斜率为-3.037,相关系数r值为0.996,表明建立的方法可用于检测HNF4α mRNA定量（V-4α值）,16例HCC组织HNF4α mRNA与HNF4α 蛋白表达结果呈一致性对应关系。结论 我们初步成功建立了定量检测HCC组织HNF4α mRNA水平方法,其意义还需要进一步研究。     

干扰素α-2b与聚乙二醇干扰素α-2a治疗慢性乙型肝炎患者疗效比较研究*

目的 观察干扰素α-2b与聚乙二醇干扰素α-2a（PEG-IFNα-2a）治疗慢性乙型肝炎（CHB）患者的临床疗效。方法 将116例CHB患者分成两组,每组58例。给予对照组普通干扰素α-2b治疗,给予观察组PEG-IFNα-2a,疗程均为48 w。检测血清层黏连蛋白（LN）、透明质酸（HA）、Ⅳ型胶原（Ⅳ-C）、Ⅲ型前胶原肽（PⅢP）及血清白介素4（IL-4）、白介素6（IL-6）、白介素10（IL-10）和干扰素γ（IFN-γ）变化。结果 在治疗结束时,观察组HBV DNA转阴率、HBeAg转阴率、HBeAg血清转换率和ALT复常率分别为75.9%、29.3%、22.4%和70.7%,显著高于对照组的51.7%、19.0%、10.3%和55.2%（P<0.05）;观察组血清LN为（148.5±46.0） μg/L,显著低于对照组【（162.6±26.7） μg/L,P<0.05】,血清HA为（158.7±67.9）μg/L,显著低于对照组【（201.4±55.1） μg/L,P<0.05】,血清Ⅳ-C为（108.3±33.4） μg/L,显著低于对照组【（119.2±62.4） μg/L,P<0.05】,血清PⅢP为（145.8±47.2） μg/L,显著低于对照组【（155.3±36.7）μg/L,P<0.05】;观察组血清IL-4水平为（1.5±0.6） pg/ml,显著低于对照组【（3.9±1.6） pg/ml,P<0.05】,而IL-6水平为（6.9±1.3） pg/ml,显著高于对照组【（3.6±0.9） pg/ml,P<0.05】,IL-10水平为（22.6±13.4） pg/ml,显著低于对照组【（17.3±11.4） pg/ml,P<0.05】,IFN-γ水平为（37.2±10.2） pg/ml,显著高于对照组【（28.3±10.5） pg/ml,P<0.05】;观察组血小板计数减少发生率为51.7%,显著高于对照组的12.1%（P<0.05）。结论 PEG-IFNα-2a治疗CHB患者临床疗效优于普通干扰素α-2b,能持久有效地抑制HBV复制,改善血清肝纤维化指标,但不良反应较大,应及时处理。     

干扰素α2b联合拉米夫定治疗慢性乙型肝炎患者血清HBeAg阴转和肝功能的变化*

目的分析抗病毒治疗对慢性乙型肝炎患者HBeAg阴转及肝功能的影响。方法选择2013年2月至2013年11月在本院进行治疗的慢性乙型肝炎患者130例,随机分为观察组和对照组各65例。给予对照组患者干扰素α2b治疗,观察组在此基础上联合拉米夫定治疗。采用ELISA法检测血清乙型肝炎病毒标记物;采用实时荧光定量PCR法检测HBV DNA,分别在治疗6个月和12个月观察患者肝功能、HBV DNA阴转率和HBeAg转阴率的变化。结果在治疗6个月时,观察组谷丙转氨酶和谷草转氨酶水平分别为（45.7±23.8） U/L和（62.3±23.7） U/L,显著低于对照组的[（55.8±25.3） U/L和（73.2±25.3） U/L,P<0.05];在治疗12个月时,观察组谷丙转氨酶和谷草转氨酶水平分别为（32.7±19.4） U/L和（46.4±6.3） U/L,显著低于对照组的[（44.6±17.3） U/L和（52.8±5.2） U/L,P<0.05];两组患者血清HBV DNA阴转率和HBeAg阴转率的差异无统计学差异（P>0.05）。结论抗病毒治疗能够有效地改善慢性乙型肝炎患者肝功能,促进HBeAg阴转,使用干扰素α2b联合拉米夫定治疗的效果更为显著,且安全性较高。     

聚乙二醇干扰素α-2b治疗慢性乙型肝炎患者外周血T淋巴细胞亚群和血清细胞因子水平变化*

目的 研究聚乙二醇干扰素α-2b治疗慢性乙型肝炎患者外周血T淋巴细胞亚群和血清细胞因子水平的变化。 方法 2014年1月~2016年1月我院收治的184例慢性乙型肝炎患者,92例接受聚乙二醇干扰素α-2b联合恩替卡韦治疗48 w,另92例只接受恩替卡韦治疗。使用流式细胞仪检测外周血T淋巴细胞亚群,采用放射免疫法检测血清IL-6、INF-ɑ、IL-4,采用酶联免疫吸附法检测血清IL-17、TGF-β1和HBV标记物,采用荧光定量PCR法检测血清HBV DNA、核转录因子RORγt、Foxp3、IL-17mRNA。 结果 在停药随访24 w,联合组与恩替卡韦组血清HBV DNA阴转率分别为86.96%和84.78%（P>0.05）;联合组血清HBeAg阴转率为28.89%（13/45）,与恩替卡韦组的15.22%（7/46）比,无显著性差异（P>0.05）;联合组血清ALT水平为（34.6±11.6） U/L,显著低于恩替卡韦组【（64.6±20.5） U/L,P<0.05】;联合组外周血CD3+、CD4+细胞和CD4+/CD8+比值分别为（75.6±14.5）%、（42.7±10.3）%和（1.4±0.6）,显著高于恩替卡韦组【（66.8±14.4）%、（36.7±8.5）%和（1.0±0.5）,P<0.05】,CD8+细胞百分比为（29.3±7.3） %,显著低于恩替卡韦组【（34.8±8.5） %,P<0.05】,两组NK细胞百分比比较无显著性差异（P>0.05）;治疗前两组血清IL-6、IL-17、IL-4、INF-ɑ、TGF-β1水平比较无显著性差异（P>0.05）,治疗后联合组血清IL-6水平为（6.8±1.2）pg/ml,显著高于恩替卡韦组【（3.5±0.8） pg/ml,P<0.05】,IL-17水平为（0.7±0.3） pg/ml,显著低于恩替卡韦组【（2.8±0.9） pg/ml,P<0.05】,IL-4水平为（1.4±0.5）pg/ml,显著低于恩替卡韦组【（3.8±1.5）pg/ml,P<0.05】,INF-ɑ水平为（4.0±1.3） pg/ml,显著高于恩替卡韦组【（2.6±0.9）pg/ml,P<0.05】,两组血清TGF-β1水平比较无显著性差异（P>0.05）;治疗前两组血清Foxp3、IL-17和RORγt mRNA水平比较无显著性差异（P>0.05）,治疗后联合组血清RORγt水平为（0.86±0.31）,显著低于恩替卡韦组【（1.56±0.43）,P>0.05】,而两组血清Foxp3和IL-17mRNA水平比较无显著性差异（P>0.05）。 结论 聚乙二醇干扰素α-2b能通过调控细胞因子和核转录因子水平,从多个环节调控慢性乙型肝炎患者免疫功能,发挥抗病毒作用。     

α疱疹病毒ICP4的转录调控机制

ICP4基因是α疱疹病毒的一个立即早期基因,其编码蛋白具有重要的调控作用,是病毒复制和基因表达的必须基因。本文在解析ICP4结构特点的基础上阐述了ICP4的转录激活或转录抑制作用、对病毒潜伏感染的影响、与其他蛋白的相互作用及其他一些重要功能,为深入研究α疱疹病毒的复制与基因表达调控和ICP4的功能提供参考。     

Y型聚乙二醇干扰素α-2b注射液治疗HCV基因2/3型慢性丙型肝炎患者疗效和安全性的多中心随机对照试验研究*

目的 以标准剂量的聚乙二醇干扰素（PegIFN）α-2a联合利巴韦林作为阳性对照,评价新型试验药物Y型PegIFNα-2b注射液联合利巴韦林治疗2型/3型慢性丙型肝炎（CHC）患者的疗效和安全性。方法 采用多中心、随机开放、阳性药对照的Ⅲ期临床试验,筛选符合要求的2型/3型CHC患者,按照2:1的比例随机分配到Y型PegIFNα-2b组和PegIFNα-2a组,同时口服利巴韦林,疗程24 w,停药随访24 w。采用Abbott RealTime HCV Genotype II检测HCV基因型,采用Cobas TaqMan实时定量PCR法检测血清HCV RNA水平。详细记录不良事件。主要疗效指标为持续病毒学应答（SVR）,并进行非劣效检验。结果 本试验实际入组2型/3型CHC患者255例,实际治疗241例。全分析集（FAS）数据显示,158例试验组和83例对照组患者SVR分别为85.4%（95% CI 79.94%~90.94%）和79.5%（95% CI 70.84%~88.20%,P=0.2402）;对符合方案分析集（PPS）人群分析显示,试验组和对照组患者SVR分别为87.9%（95% CI 82.45%~93.27%）和85.9%（95% CI 77.82%~94.01%,P=0.7060）,率差的95%可置信区间均符合非劣效标准;对PPS人群分析显示,85.8%受试者获得了早期病毒学应答（RVR）,RVR的阳性预测值为90.1%;试验组和对照组不良事件发生率相似,分别为95.6%和95.2%,严重不良事件发生率分别为3.8%和3.6%。结论 应用PegIFNα联合利巴韦林治疗2型/3型CHC患者,新型试验药物Y型PegIFNα-2b具有与对照药物PegIFNα-2a相似的疗效和安全性。     

原发性胆汁性肝硬化患者肝组织转化生长因子β-1表达水平变化

目的 探讨原发性胆汁性肝硬化（PBC）患者肝组织转化生长因子-β1（TGF-β1）表达的变化。 方法 52例PBC患者接受超声引导下肝穿刺活检,选择病理科留取的正常肝组织20份作为对照,另选择20例健康人采集静脉血。采用免疫组化法检测肝组织TGFβ-1表达,采用ELISA法检测血清肿瘤坏死因子α（TNF-α）和白介素6（IL-6）水平。 结果 正常肝组织不表达或仅有少量TGF-β1表达,而PBC患者肝实质细胞胞浆内呈TGF-β1高表达;PBC患者血清TNF-α和IL-6水平分别为（28.71±13.54） pg/ml和（21.3±9.4） pg/ml,显著高于健康人【分别为（21.3±15.4） pg/ml和（2.1±1.6） pg/ml,P<0.01】;7例PBC肝组织肝纤维化S0期患者肝组织TGF-β1表达及血清TNF-α和IL-6水平分别为（0.5±0.2）10-2、（7.1±4.1） pg/ml和（5.1±1.0） pg/ml,显著低于12例S1期【（4.2±1.3） 10-2、（18.6±6.2） pg/ml、（11.5±3.6） pg/ml,P<0.01】、18例S2期【（6.9±1.2） 10-2、（27.3±9.9） pg/ml、（19.4±4.1） pg/ml,P<0.01】、9例S3期【（13.3±15.1） 10-2、（39.7±15.18） pg/ml、（27.3±8.1） pg/ml,P<0.01】和6例S4期【（21.2±17.1） 10-2、（53.4±17.3） pg/ml、（47.8±11.0） pg/ml,P<0.01】;15例Child-Pugh A级患者肝组织TGF-β1表达及血清TNF-α和IL-6水平分别为（1.9±1.6） 10-2、（12.2±3.1） pg/ml和（7.3±2.5） pg/ml,显著低于25例Child-Pugh B级【（15.9±13.6） 10-2、（32.9±8.6） pg/ml、（21.8±6.3） pg/ml,P<0.01】或12例C级患者【（22.6±18.5） 10-2、（49.1±19.3） pg/ml、（45.5±12.7） pg/ml,P<0.01】。 结论 PBC患者肝组织TGFβ-1表达及血清TNF-α和IL-6水平显著升高,可能与肝组织纤维化增生和/或肝功能受损有关。     

肝泡状棘球蚴组织中HIF-1α、VEGFA的表达及对血管生成的作用

目的 探讨HIF-1α、VEGFA在沙鼠肝泡状棘球蚴组织的表达及血管生成过程中的作用。方法 126只沙鼠随机分成空白组(6只)、假手术组(60只)、模型组(60只),采用开腹直视肝脏穿刺法建立泡状棘球蚴动物模型,假手术组接种同体积的PBS,术后第3 d、7 d、14 d、28 d、42 d、56 d、70 d、84 d、98 d、112 d随机取6只沙鼠,取临近病变的边缘区组织及正常肝组织。采用HE观察病理改变;qRT-PCR、原位杂交、免疫组化检测HIF-1α、VEGFA表达,CD34标记微血管进行MVD计数。结果 HE染色根据病理特点将病程分为早期(14 d内)、中期(14 d~56 d)、晚期(56 d后)。模型组泡状棘球蚴组织随感染时间不同,其HIF-1αmRNA随感染时间呈动态改变,术后第14 d其表达量高于空白组、低于假手术组(F=82.732,P< 0.001);术后第42 d、112 d其相对表达量均高于空白组与假手术组(χ²=11.536,χ²=15.189,P< 0.01);模型组术后第14 d VEGFA mRNA相对表达量低于空白组及假手术组(χ²=15.174,P< 0.01);术后第42 d、112 d,VEGFA mRNA表达量均高于空白组与假手术组(χ²=15.158,χ²=15.158,P< 0.01)。模型组术后第14 d、42 d、112 d HIF-1α表达均高于空白组、假手术组(χ²=8.627,χ²=9.000,F=15.690,P< 0.01);模型组术后第14 d、42 d VEGFA表达高于空白组、假手术组(F=11.250, F=70.059,P< 0.001);模型组术后第14 d、42 d、112 d,其MVD-CD34均高于空白组、假手术组(χ²=12.517,P< 0.01,χ²=13.157,P< 0.01;χ²=13.220,P< 0.01)。结论 沙鼠感染肝泡状棘球蚴中期,病变边缘区HIF-1α、VEGFA表达均升高,同时伴有大量微血管生成,可能存在HIF-1α转录因子激活并上调VEGFA的表达,促进肝泡状棘球蚴组织边缘区的血管新生。;     

聚乙二醇化干扰素α-2a联合利巴韦林治疗代偿期丙型肝炎肝硬化患者临床疗效研究*

目的 探讨应用聚乙二醇化干扰素α-2a联合利巴韦林治疗代偿期丙型肝炎肝硬化患者的临床疗效。方法 2003年1月~2016年12月我院就诊的代偿期丙型肝炎肝硬化患者122例,采用随机数字表法分成两组,每组61例。给予对照组常规护肝治疗,给予观察组聚乙二醇化干扰素α-2a联合利巴韦林治疗24~48 w。随访两组24 w。采用实时荧光定量RT-PCR法检测血清HCV RNA,采用全自动生化分析仪检测血生化指标,采用化学发光法检测血清层粘连蛋白（LN）、Ⅲ型前胶原（PC Ⅲ）、透明质酸（HA）,常规使用Fibroscan行肝脏硬度检测（LSM）。结果 在治疗结束时,观察组血清HCV RNA水平为（2.0±0.4） lg IU/ml,显著低于对照组【（3.8±1.3）lg IU/ml,P<0.05】;血清AST和ALT水平分别（46.03±24.05） U/L和（36.32±20.1） U/L,显著低于对照组【（78.7±21.1） U/L和（51.2±20.9） U/L,P<0.05）;观察组血清LN、PCⅢ和HA水平分别为（126.3±29.0）μg/L、（212.3±43.8）μg/L和（211.4±42.0）μg/L,均显著低于对照组【（140.3±32.1）μg/L、（267.5±39.8）μg/L和（329.6±68.4）μg/L,P<0.05】;观察组LSM为（13.6±2.4） kPa,显著低于对照组【（17.6±5.2）kPa,P<0.05】;在随访时发现,观察组血清ALT复常率和持续病毒学应答率（SVR）均显著高于对照组（分别为93.4%对45.9%和72.1%对9.8%,P<0.05）,而疾病进展发生率为3.3%,显著低于对照组的13.1%（P<0.05）。结论 应用聚乙二醇化干扰素α-2a联合利巴韦林抗病毒治疗代偿期丙型肝炎肝硬化患者可显著提高SVR,延缓肝纤维化进展,稳定肝功能指标。     

慢性乙型肝炎肝衰竭患者血清甲胎蛋白和胸腺素β4水平变化

目的 探讨血清甲胎蛋白（AFP）和胸腺素β4水平在预测慢性乙型肝炎肝衰竭患者转归中的应用价值。方法 2015年1月~2016年7月我院收治的慢性乙型肝炎肝衰竭患者75例,采用ELISA法检测血清AFP和胸腺素β4水平。结果 治疗3个月,在75例慢性乙型肝炎肝衰竭患者中,43例患者（57.3%）死亡,32例患者（42.7%）生存;入院时,生存组血清AFP和胸腺素β4水平分别为（194.2±13.5） μg/L和（914.1±29.5） μg/L,均显著高于死亡组的【（98.4±9.5） μg/L和（504.2±15.4） μg/L,P﹤0.05】;以血清AFP大于或等于71.3 μg/L为截断点,其预测患者在3个月内生存的敏感度为81.5%,特异度为74.9%;以血清胸腺素β4大于472.7μg/L为截断点,其预测患者在3个月内生存的敏感度为76.2%,特异度为69.5%。结论 慢性乙型肝炎肝衰竭患者血清AFP和胸腺素β4水平对患者转归有一定的预测作用,两者水平越高,预后越好。     

Hyaluronan production in human rheumatoid fibroblastic synovial lining cells is increased by interleukin 1β but inhibited by transforming growth factor β1

OBJECTIVES—To investigate the regulatory roles of interleukin 1β (IL1β), tumour necrosis factor α (TNFα), interferon γ (IFNγ) or transforming growth factor β1 (TGFβ1) on hyaluronan (HA) synthesis by human fibroblastic synovial lining cells.
METHODS—Concentrations of HA in culture supernatants of fibroblastic synovial lining cell line (RAMAK-1 cell line) with or without stimulation by IL1β, TNFα, IFNγ or TGFβ1 were measured by sandwich binding protein assay. Levels of HA synthase mRNA of the cells with or without stimulation were detected by reverse transcribed polymerase chain reaction. Molecular weights of HA in the culture supernatants of the cells with or without stimulation were measured using high performance gel permeation liquid chromatography.
RESULTS—HA synthesis by the cells was not significantly augmented by TNFα or by IFNγ. It was significantly stimulated by IL1β but inhibited by TGFβ1. Molecular weights of HA in the culture supernatants of the cells were unchanged by stimulation with TNFα. They were remarkably increased by stimulation with IL1β and IFNγ, but reduced with TGFβ1.
CONCLUSION—IL1β is an up regulator of HA synthesis, while TGFβ1 is a down regulator. HA production in the synovial lining cells of inflamed joints (for example, rheumatoid arthritis) might be regulated by the balance of these cytokines.

Keywords: synovial lining cells; hyaluronan, interleukin 1β; transforming growth factor β1     

Hepatocyte nuclear factor 4α induces a tendency of differentiation and activation of rat hepatic stellate cells

AIM: To investigate the effect of hepatocyte nuclear factor 4α(HNF4α) on the differentiation and transformation of hepatic stellate cells(HSCs).METHODS: By constructing the recombinant adenovirus vector expressing HNF4α and HNF4αshRNA vector, and manipulating HNF4α expression in HSC-T6 cells, we explored the influence of HNF4α and its induction capacity in the differentiation of rat HSCs into hepatocytes.RESULTS: With increased expression of HNF4αmediated by AdHNF4α, the relative expression of Nanog was downregulated in HSC-T6 cells(98.33 ±12.33 vs 41.33 ± 5.67, P 0.001). Consequently, the expression of G-P-6 and PEPCK was upregulated(G-P-6:14.34 ± 3.33 vs 42.53 ± 5.87, P 0.01; PEPCK: 10.10± 4.67 vs 56.56 ± 5.25, P 0.001), the expression of AFP and ALB was positive, and the expression of Nanog, Type Ⅰ collagen, α-SMA, and TIMP-1 was significantly decreased. HNF4α also downregulated vimentin expression and enhanced E-cadherin expression. The ultrastructure of HNF4α-induced cells had more mitochondria and ribosomes compared with the parental cells. After silencing HNF4α expression,EPCK, E-cadherin, AFP, and ALB were downregulated and α-SMA and vimentin were upregulated.CONCLUSION: HNF4α can induce a tendency of differentiation of HSCs into hepatocyte-like cells. These findings may provide an effective way for the treatmentof liver diseases.     

Recognition of the antigen-presenting molecule MR1 by a Vδ3+ γδ T cell receptor

Unlike conventional αβ T cells, γδ T cells typically recognize nonpeptide ligands independently of major histocompatibility complex (MHC) restriction. Accordingly, the γδ T cell receptor (TCR) can potentially recognize a wide array of ligands; however, few ligands have been described to date. While there is a growing appreciation of the molecular bases underpinning variable (V)δ1⁺ and Vδ2⁺ γδ TCR-mediated ligand recognition, the mode of Vδ3⁺ TCR ligand engagement is unknown. MHC class I–related protein, MR1, presents vitamin B metabolites to αβ T cells known as mucosal-associated invariant T cells, diverse MR1-restricted T cells, and a subset of human γδ T cells. Here, we identify Vδ1/2⁻ γδ T cells in the blood and duodenal biopsy specimens of children that showed metabolite-independent binding of MR1 tetramers. Characterization of one Vδ3Vγ8 TCR clone showed MR1 reactivity was independent of the presented antigen. Determination of two Vδ3Vγ8 TCR-MR1-antigen complex structures revealed a recognition mechanism by the Vδ3 TCR chain that mediated specific contacts to the side of the MR1 antigen-binding groove, representing a previously uncharacterized MR1 docking topology. The binding of the Vδ3⁺ TCR to MR1 did not involve contacts with the presented antigen, providing a basis for understanding its inherent MR1 autoreactivity. We provide molecular insight into antigen-independent recognition of MR1 by a Vδ3⁺ γδ TCR that strengthens an emerging paradigm of antibody-like ligand engagement by γδ TCRs.

Characterized by both innate and adaptive immune cell functions, γδ T cells are an unconventional T cell subset. While the functional role of γδ T cells is yet to be fully established, they can play a central role in antimicrobial immunity (1), antitumor immunity (2), tissue homeostasis, and mucosal immunity (3). Owing to a lack of clarity on activating ligands and phenotypic markers, γδ T cells are often delineated into subsets based on the expression of T cell receptor (TCR) variable (V) δ gene usage, grouped as Vδ2⁺ or Vδ2⁻.The most abundant peripheral blood γδ T cell subset is an innate-like Vδ2⁺subset that comprises ∼1 to 10% of circulating T cells (4). These cells generally express a Vγ9 chain with a focused repertoire in fetal peripheral blood (5) that diversifies through neonatal and adult life following microbial challenge (6, 7). Indeed, these Vγ9/Vδ2⁺ T cells play a central role in antimicrobial immune response to Mycobacterium tuberculosis (8) and Plasmodium falciparum (9). Vγ9/Vδ2⁺ T cells are reactive to prenyl pyrophosphates that include isopentenyl pyrophosphate and (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate (8) in a butyrophilin 3A1- and BTN2A1-dependent manner (10–13). Alongside the innate-like protection of Vγ9/Vδ2⁺ cells, a Vγ9⁻ population provides adaptive-like immunobiology with clonal expansions that exhibit effector function (14).The Vδ2⁻ population encompasses the remaining γδ T cells but most notably the Vδ1⁺ and Vδ3⁺ populations. Vδ1⁺ γδ T cells are an abundant neonatal lineage that persists as the predominating subset in adult peripheral tissue including the gut and skin (15–18). Vδ1⁺ γδ T cells display potent cytokine production and respond to virally infected and cancerous cells (19). Vδ1⁺ T cells were recently shown to compose a private repertoire that diversifies, from being unfocused to a selected clonal TCR pool upon antigen exposure (20–23). Here, the identification of both Vδ1⁺ T_naive and Vδ1⁺ T_effector subsets and the Vδ1⁺ T_naive to T_effector differentiation following in vivo infection point toward an adaptive phenotype (22).The role of Vδ3⁺ γδ T cells has remained unclear, with a poor understanding of their lineage and functional role. Early insights into Vδ3⁺ γδ T cell immunobiology found infiltration of Vδ3⁺ intraepithelial lymphocytes (IEL) within the gut mucosa of celiac patients (24). More recently it was shown that although Vδ3⁺ γδ T cells represent a prominent γδ T cell component of the gut epithelia and lamina propria in control donors, notwithstanding pediatric epithelium, the expanding population of T cells in celiac disease were Vδ1⁺ (25). Although Vδ3⁺ IELs compose a notable population of gut epithelia and lamina propria T cells (∼3 to 7%), they also formed a discrete population (∼0.2%) of CD4⁻CD8⁻ T cells in peripheral blood (26). These Vδ3⁺ DN γδ T cells are postulated to be innate-like due to the expression of NKG2D, CD56, and CD161 (26). When expanded in vitro, these cells degranulated and killed cells expressing CD1d and displayed a T helper (Th) 1, Th2, and Th17 response in addition to promoting dendritic cell maturation (26). Peripheral Vδ3⁺ γδ T cells frequencies are known to increase in systemic lupus erythematosus patients (27, 28), and upon cytomegalovirus (29) and HIV infection (30), although, our knowledge of their exact role and ligands they recognize remains incomplete.The governing paradigms of antigen reactivity, activation principles, and functional roles of γδ T cells remain unresolved. This is owing partly due to a lack of knowledge of bona fide γδ T cell ligands. Presently, Vδ1⁺ γδ T cells remain the best characterized subset with antigens including Major Histocompatibility Complex (MHC)-I (31), monomorphic MHC-I–like molecules such as CD1b (32), CD1c (33), CD1d (34), and MR1 (35), as well as more diverse antigens such as endothelial protein coupled receptor (EPCR) and phycoerythrin (PE) (36, 37). The molecular determinants of this reactivity were first established for Vδ1⁺ TCRs in complex with CD1d presenting sulfatide (38) and α-galactosylceramide (α-GalCer) (34), which showed an antigen-dependent central focus on the presented lipids and docked over the antigen-binding cleft.In humans, mucosal-associated invariant T (MAIT) cells are an abundant innate-like αβ T cell subset typically characterized by a restricted TCR repertoire (39–43) and reactivity to the monomorphic molecule MR1 presenting vitamin B precursors and drug-like molecules of bacterial origin (41, 44–46). Recently, populations of atypical MR1-restricted T cells have been identified in mice and humans that utilize a more diverse TCR repertoire for MR1-recognition (42, 47, 48). Furthermore, MR1-restricted γδ T cells were identified in blood and tissues including Vδ1⁺, Vδ3⁺, and Vδ5⁺ clones (35). As seen with TRAV 1-2⁻, unconventional MAITs cells the isolated γδ T cells exhibited MR1-autoreactivity with some capacity for antigen discrimination within the responding compartment (35, 48). Structural insight into one such MR1-reactive Vδ1⁺ γδ TCR showed a down-under TCR engagement of MR1 in a manner that is thought to represent a subpopulation of MR1-reactive Vδ1⁺ T cells (35). However, biochemical evidence suggested other MR1-reactive γδ T cell clones would likely employ further unusual docking topologies for MR1 recognition (35).Here, we expanded our understanding of a discrete population of human Vδ3⁺ γδ T cells that display reactivity to MR1. We provide a molecular basis for this Vδ3⁺ γδ T cell reactivity and reveal a side-on docking for MR1 that is distinct from the previously determined Vδ1⁺ γδ TCR-MR1-Ag complex. A Vδ3⁺ γδ TCR does not form contacts with the bound MR1 antigen, and we highlight the importance of non–germ-line Vδ3 residues in driving this MR1 restriction. Accordingly, we have provided key insights into the ability of human γδ TCRs to recognize MR1 in an antigen-independent manner by contrasting mechanisms.     

Identification of new mutations in the hepatocyte nuclear factor 4alpha gene among families with early onset Type 2 diabetes mellitus.

AIMS: Mutations in hepatocyte nuclear factor (HNF)-4alpha gene located on chromosome 20q have been found to be responsible for the development of early onset Type 2 diabetes mellitus (DM). Through a national campaign, 53 families with autosomal dominant, early onset Type 2 DM (n=654) were assembled to determine the frequency of mutations in the HNF-4alpha gene and their contribution to the development of diabetes. METHODS: Twelve exons and the promoter region of the HNF-4alpha gene were screened in probands of the families by a double gradient, denaturing gradient gel electrophoresis (DG-DGGE) protocol combined with automated bi-directional sequencing of the PCR products of all heterozygous individuals. RESULTS: We detected two new mutations in the HNF-4alpha gene that changed the amino-acid sequence. The first mutation was a Gly-->Ser substitution in codon 115 within a highly conserved DNA binding domain, and all six carriers of this mutation had diabetes and low insulin secretion. The second mutation was an Ile-->Val substitution in codon 454 within the transactivation domain. It was carried by four family members, two of whom also carried a mutation in the HNF-1alpha gene. Of those having only the mutation in HNF-4alpha one had diabetes and the other had normal glucose tolerance and both were obese and hyperinsulinaemic. Thus, it is uncertain that this mutation is responsible for any of the diabetes in this family. CONCLUSION: We have found that mutations in the HNF-4alpha gene account for a small proportion, about 2-4%, of families with early onset, autosomal dominant, Type 2 DM in US Caucasians.     

Cross-α/β polymorphism of PSMα3 fibrils

The formation of ordered cross-β amyloid protein aggregates is associated with a variety of human disorders. While conventional infrared methods serve as sensitive reporters of the presence of these amyloids, the recently discovered amyloid secondary structure of cross-α fibrils presents new questions and challenges. Herein, we report results using Fourier transform infrared spectroscopy and two-dimensional infrared spectroscopy to monitor the aggregation of one such cross-α–forming peptide, phenol soluble modulin alpha 3 (PSMα3). Phenol soluble modulins (PSMs) are involved in the formation and stabilization of Staphylococcus aureus biofilms, making sensitive methods of detecting and characterizing these fibrils a pressing need. Our experimental data coupled with spectroscopic simulations reveals the simultaneous presence of cross-α and cross-β polymorphs within samples of PSMα3 fibrils. We also report a new spectroscopic feature indicative of cross-α fibrils.

Amyloids are elongated fibers of proteins or peptides typically composed of stacked cross β-sheets (1, 2). Self-assembling amyloids are notorious for their involvement in human neurodegenerative diseases such as Alzheimer’s and Parkinson’s diseases (1, 2). Phenol soluble modulins (PSMs) are amyloid peptides secreted by the bacteria Staphylococcus aureus (S. aureus) (3–5). Of the PSM family, PSMα3 is of recent interest due to its unique secondary structure upon fibrillation. Whereas other PSM variants undergo conformational changes with aggregation, the α-helical PSMα3 peptide retains its secondary structure while stacking in a manner reminiscent of β-sheets, forming what has been termed cross-α fibrils (3, 4, 6). Although “α-sheet” amyloid fibrils have been previously observed in two-dimensional infrared (2DIR) (7) and associated with PSMs (8), the novel cross-α fibril is distinct from that class of structures. To avoid confusion between these two similarly named but distinct secondary structures, a comparison between the α-sheet domain in cytosolic phosphatase A2 (9) (Protein Data Bank [PDB] identification:1rlw) (10) and cross-α fibrils adopted by PSMα3 (PDB ID:5i55) (3) has been highlighted in SI Appendix, Fig. S1. Interestingly, shorter terminations of PSMα3 have been shown to exhibit β-sheet polymorphs (11). The proposed cross-α fibril structure of the full-length PSMα3 peptide has been confirmed with X-ray diffraction and circular dichroism (4). The present study aims to further characterize these fibrils with linear and nonlinear infrared spectroscopies.S. aureus is an infectious human pathogen with the ability to form communities of microorganisms called biofilms that hinder traditional treatment methods (12–14). PSMs contribute to inflammatory response and play a crucial role in structuring and detaching biofilms (11, 12, 14). While biofilm growth requires the presence of multiple PSMs (14, 15), Andreasen and Zaman have demonstrated that PSMα3 acts as a scaffold, seeding the amyloid formation of other PSMs (5). To effectively inhibit S. aureus biofilm growth, a better understanding of PSMα3 aggregation is needed.The α-helical structure of PSMα3 (12) presents a challenge for probing the vibrational modes and secondary structure of both the monomer and the fibrils. While IR spectroscopy has been used extensively to characterize β-sheets (16–19), the spectral features associated with α-helices are difficult to distinguish from those of the random coil secondary structure (20, 21). This limitation has left researchers to date with an incomplete picture of the spectroscopic features unique to cross-α fibers. The present work combines a variety of 2DIR methods to remove these barriers and probe the active infrared vibrational modes of cross-α fibers.The full-length, 22-residue PSMα3 peptide was synthesized and prepared for aggregation studies following reported methods (3, 4, 11). A total of 10 mM PSMα3 was incubated in D₂O at room temperature over 7 d. These data were compared to the monomer treated under similar conditions. Monomeric samples were prepared at a significantly lower concentration of 0.5 mM to prevent aggregation. Fiber formation was confirmed by transmission electron microscopy (see SI Appendix, Fig. S2 for details). Fourier transform infrared (FTIR) spectra were taken for both the fibrils in solution as well as the low concentration monomers. Spectroscopic simulations of the PSMα3 monomer and fibers were performed on previously reported PDB structures (PDB identification: 5i55) (3) (Fig. 1).Open in a separate windowFig. 1.PDB structures of PSMα3 (A) monomers and (B) cross-α fibers extended along the screw axis. (C) FTIR spectra of 0.5 mM monomeric PSMα3 (blue) compared to the 10 mM PSMα3 fibril (red) in D₂O upon aggregation.     

HCV感染相关干扰素L4真核表达载体的构建与表达

目的 构建HCV感染相关干扰素IFNL4 蛋白融合 His 标签的真核表达载体,并将其在293-T细胞中表达后进行初步纯化鉴定。方法 采用PCR法从含人IFNL4基因序列的质粒pFC14A-p179中扩增出目的片段,将其定向连接到pcDNA3.1-His 真核表达载体中,经酶切和测序鉴定正确后,以脂质体法转染293-T细胞,培养72 h后裂解细胞,采用抗His-Tag抗体行蛋白质印迹法检测目的蛋白的表达。结果 成功构建了真核表达载体pcDNA3.1-IFNL4-His,转染后经Western blot法检测显示20 kDa 位置有目的蛋白条带,大小与目的蛋白相符。结论 我们成功构建了HCV感染相关IFNL4与 His 标签融合的真核表达载体,体外转染293-T细胞后鉴定其工作良好,为后续对干扰素在HCV感染及抗肝纤维化治疗中的研究奠定了基础。     

Characterization of Adaptive-like γδ T Cells in Ugandan Infants during Primary Cytomegalovirus Infection

Gamma-delta (γδ) T cells are unconventional T cells that help control cytomegalovirus (CMV) infection in adults. γδ T cells develop early in gestation, and a fetal public γδ T cell receptor (TCR) clonotype is detected in congenital CMV infections. However, age-dependent γδ T cell responses to primary CMV infection are not well-understood. Flow cytometry and TCR sequencing was used to comprehensively characterize γδ T cell responses to CMV infection in a cohort of 32 infants followed prospectively from birth. Peripheral blood γδ T cell frequencies increased during infancy, and were higher among CMV-infected infants relative to uninfected. Clustering analyses revealed associations between CMV infection and activation marker expression on adaptive-like Vδ1 and Vδ3, but not innate-like Vγ9Vδ2 γδ T cell subsets. Frequencies of NKG2C⁺CD57⁺ γδ T cells were temporally associated with the quantity of CMV shed in saliva by infants with primary infection. The public γδ TCR clonotype was only detected in CMV-infected infants <120 days old and at lower frequencies than previously described in fetal infections. Our findings support the notion that CMV infection drives age-dependent expansions of specific γδ T cell populations, and provide insight for novel strategies to prevent CMV transmission and disease.