Methods for the determination of the interferon-induced enzyme 2'-5' oligoadenylate synthetase in mononuclear blood cells

The determination of 2'-5'-oligoadenylate synthetase in peripheral blood mononuclear cells is used as a biological response parameter during therapy with interferon and in the diagnosis of diseases related to the interferon system. In this communication, some general aspects concerning the preparation of 2'-5'-oligoadenylate synthetase from peripheral blood mononuclear cells and the incubation conditions of the 2'-5'-oligoadenylate synthetase reaction are reported. Four analytical procedures for the determination of the products formed during the 2'-5'-oligoadenylate synthetase reaction were comparatively investigated and the advantages and limitations of the assays are discussed. As an example of possible clinical application, the levels of 2'-5'-oligoadenylate synthetase were determined in the mononuclear cell fraction of peripheral blood from healthy persons as well as from children with chronic myelogenous leukaemia.     

Production of tumor necrosis factor alpha and interleukin 1 beta by peripheral blood mononuclear cells from chronic hepatitis type C patients during interferon therapy.

To elucidate the role of tumor necrosis factor alpha (TNF-alpha) and interleukin 1 beta (IL-1 beta) in chronic hepatitis type C (CH(C], TNF alpha and IL-1 beta released from peripheral blood mononuclear cells were measured in 6 CH(C) patients before and during interferon therapy. During the therapy, TNF-alpha levels were significantly high in two patients whose serum ALT levels turned normal, while IL-1 beta levels did not show significant change. These results suggest that TNF-alpha takes part in improvement of CH(C).     

α-2b干扰素治疗HBeAg阳性慢性乙型肝炎临床分析

目的 观察普通α-2b干扰素（IFN）和聚乙二醇干扰素（PEG IFN）α-2b治疗HBeAg阳性慢性乙型肝炎抗病毒的临床疗效.方法 HBeAg阳性慢性乙型肝炎患者64例,36例给予普通α-2b干扰素抗病毒治疗,每次500万U,皮下注射,每周3次; 28例给予聚乙二醇干扰素α-2b 1.0 μg/kg皮下注射,每周1次.疗程均为52周.结果 治疗结束时,普通干扰素组与PEG IFN组的完全应答率分别为52.8%、60.7%;部分应答率分别为27.8%、21.4%;无应答率分别为19.4%、17.9%,两组比较差异无统计学意义（P〉0.05）.治疗结束时普通干扰素α-2b组,PEG-IFN α-2b组丙氨酸氨基转移酶（ALT）复常率分别是81.7%、85.9%,二者比较差异无统计学意义（P〉0.05）.普通干扰素α-2b组与PEG-IFN α-2b组在52周治疗结束时HBV DNA阴转率分别为49.3%、57.6%,二者比较差异无统计学意义（P〉0.05）.结论 两种不同剂型的α-2b干扰素治疗HBeAg阳性慢性乙型肝炎的应答率相似,普通干扰素具有更好的成本效益.     

Novel alpha interferon (IFN-alpha) variant with improved inhibitory activity against hepatitis C virus genotype 1 replication compared to IFN-alpha2b therapy in a subgenomic replicon system

Hepatitis C virus (HCV) treatment is based on the association of pegylated alpha interferon (IFN-α) and ribavirin. To improve the level of sustained virological response to treatment, especially in patients infected with HCV genotype 1, new IFNs with improved efficacy and toxicity profiles may be developed. In this report, we show that, in the BM4-5 cell line harboring an HCV subgenomic replicon, a novel and naturally occurring human IFN-α17 variant, GEA007.1, which was discovered by using an original population genetics-based drug discovery approach, inhibits HCV genotype 1 RNA replication more efficiently than does IFN-α2b. Moreover, we show that complete viral clearance is obtained in BM4-5 cells after long-term treatment with GEA007.1, while HCV subgenomic RNA is still detected in cells treated with other IFN-α variants or with standard IFN-α2b. Eventually, we demonstrate that the better inhibitory activity of GEA007.1 compared to that of standard IFN-α is likely to be due to stronger and faster activation of the JAK-STAT signaling pathway and to broader expression of IFN-α-responsive genes in cells. Our results demonstrate a superior inhibitory activity of GEA007.1 over that of IFN-α2b in the HCV replicon system. Clinical trials are required to determine whether GEA007.1 could be a potent “next generation” IFN for the treatment of HCV infection, especially in nonresponders or relapsing patients infected with HCV genotype 1 who currently represent a clinical unmet need.     

2'-,5'-Oligoadenylate synthetase response ratio predicting virological response to PEG-interferon-alpha2b plus ribavirin therapy in patients with chronic hepatitis C

OBJECTIVE: Although all the mechanisms of elimination of hepatitis C virus (HCV) by Interferon (IFN) have not been fully elucidated, the 2'-5'-oligoadenylate (2-5A) system is one of the mechanisms of the antiviral effect of IFN. Consequently, the measurement of 2'-5'-oligoadenylate synthetase (2-5AS) activity could be useful for the evaluation of IFN treatment. This retrospective study was aimed at assessing whether 2-5AS activity functions as a clinical marker of virological response to PEG-interferon-alpha2b (PEG-IFN) plus ribavirin therapy of chronic hepatitis C. METHODS: The 32 patients included in this study had high viral loads of serum HCV-RNA of genotype 1b with chronic hepatitis C. All the patients received a regimen of PEG-IFN plus ribavirin for 48 weeks, and were then divided into two groups: one group (effective group) with undetectable serum HCV-RNA levels at 24 weeks (n = 22) of therapy, the other group (ineffective group) with persistent presence of HCV-RNA in serum at 24 weeks (n = 10). The 2-5AS activity in serum was measured 2, 8 and 12 weeks before initial administration. RESULTS: The 2-5AS response ratio (measured value/measured value of baseline 2-5AS) at 2, 8 and 12 weeks after the administration in the effective group was significantly higher than that in the ineffective group. CONCLUSIONS: These results suggest that the ratio of 2-5AS is closely related to the antiviral effect, and that the measurement of 2-5AS response ratio may be a useful clinical parameter of virological response to PEG-IFN plus ribavirin therapy of chronic hepatitis C.     

Specific binding of human alpha interferon to high-affinity cell-surface binding sites on peripheral blood mononuclear cells

To characterize receptors for alpha interferon (IFN-alpha) on human cells, we studied the binding of radioiodinated recombinant DNA-derived human IFN-alpha to human peripheral blood mononuclear cells (PBMCs) from normal individuals and from patients with chronic type B hepatitis. At 1 degree C, binding reached equilibrium after 2 to 3 hours of incubation, and saturation of specific binding occurred at a concentration of approximately 4000 fmol/ml. Binding of labeled IFN-alpha was specific; it was inhibited by an excess of unlabeled IFN-alpha or IFN-beta but not by cholera toxin or IFN-gamma. Scatchard analysis of binding data yielded for normal PBMCs an apparent dissociation constant (Kd) of 1.54 +/- 0.49 x 10(-9) mol/L (mean +/- SD) and an apparent maximum binding capacity (Bmax) of 7.35 +/- 1.22 x 10(-11) mol/L. Corresponding values for patients with chronic type B hepatitis who had not received treatment were similar, suggesting that such patients should respond normally to endogenous interferon. Analysis of data on the binding of labeled IFN-alpha to normal PBMCs from experiments in which a high specific activity ligand or subpopulations of PBMCs had been used revealed that receptors for IFN-alpha on PBMCs are heterogenous. In patients with chronic type B hepatitis who were receiving IFN-alpha therapy, the apparent Kd was increased (3.02 +/- 0.91 x 10(-9) mol/L) without any appreciable change in the apparent Bmax or any appreciable changes in the proportions of subpopulations of PBMCs. This decreased affinity induced by IFN-alpha treatment does not necessarily reflect an effect on a single binding site.(ABSTRACT TRUNCATED AT 250 WORDS)     

Treatment of patients with chronic hepatitis B who have failed previous antiviral treatment with pegylated interferon alpha2a (40 kda; PEGASYS)

BACKGROUND: Although nucleot(s)ide analogues can effectively suppress hepatitis B virus (HBV) replication, many patients experience relapse of hepatitis after cessation of treatment. We aimed to investigate the efficacy of pegylated interferon alpha2a (PEG-IFN-alpha2a) in these difficult-to-treat patients. METHODS: Chronic hepatitis B patients who have received antiviral drugs for > or =12 months and stopped for > or =6 months were treated by 48-week PEG-IFN-alpha2a. Virological response was defined as HBV DNA <10,000 copies/ml and hepatitis B e antigen (HBeAg) seroconversion (for HBeAg-positive patients). RESULTS: A total of 40 patients, 29 HBeAg-positive and 11 HBeAg-negative, with median log10 HBV DNA 7.3 copies/ml and alanine aminotransferase 110 IU/ml were studied. The last antiviral treatment was given for 92 +/- 61 weeks and stopped for 176 +/- 88 weeks. At the end of treatment, 22 (12 HBeAg-positive and 10 HBeAg-negative; 55%) patients had virological response and 16 (7 HBeAg-positive and 9 HBeAg-negative; 40%) patients had undetectable HBV DNA (<100 copies/ml). At 24 weeks post-treatment, 14 (8 HBeAg-positive and 6 HBeAg-negative; 35%) patients had virological response and 9 (5 HBeAg-positive and 4 HBeAg-negative; 23%) patients had undetectable HBV DNA. Two (5%) patients had lost hepatitis B surface antigen. HBV DNA levels at week 24 best predicted sustained virological response (area under curve 0.76, 95% confidence interval 0.60-0.92, P=0.007). At HBV DNA cutoffs of 3 logs and 5 logs at week 24, the sensitivity/specificity for sustained virological response were 50%/85% and 86%/62%, respectively. CONCLUSIONS: PEG-IFN-alpha2a was effective in the treatment of chronic hepatitis B patients who have failed previous antiviral treatment.     

Antiproliferative effect of hepatitis C virus on mitogen-stimulated peripheral blood mononuclear cells: potential role in viral persistence in Egyptian patients

OBJECTIVES: We aimed to study the effect of hepatitis C virus (HCV) and sera of chronic HCV patients on phytohemagglutinin (PHA)-stimulated normal donor PBMCs and to study the effect of chronic HCV infection on some cytokine profile. SUBJECTS AND METHODS: (3)H-Thymidine uptake was utilized to study effect of pelleted virus and patients sera on PBMCs proliferation in vitro. The study included 337 Egyptian chronic liver patients from Ain Shams University Hospitals and 90 healthy control subjects. The patients' group included chronic hepatitis C (250 subjects), and other chronic liver diseases (87 subjects). All subjects' sera were subjected to RT-PCR for HCV RNA detection, IL-4, IL-1beta, and TNF-alpha measurement by EIA, and biochemical measurement of ALT and albumin. RESULTS: Treatment of PHA-stimulated normal donor PBMCs with pelleted virus led to decrease (dose response) in their rate of proliferation. This was partially reversed after addition of HCV RNA positive patients' sera. HCV RNA positive patients were significantly higher in IL-4 and ALT, and lower in IL-1beta and albumin than HCV RNA negative patients. CONCLUSION: HCV infection suppresses early immune response. This leads to increased IL-4 Th2 cytokine.     

慢性乙型肝炎患者外周血单个核细胞内乙型肝炎病毒脱氧核糖核酸与肝组织损伤程度相关性分析

目的研究慢性乙型肝炎患者外周血单个核细胞(PBMC)内乙型肝炎病毒脱氧核糖核酸(HBV-DNA)与肝组织损伤程度的相关性。方法采用聚合酶链反应方法测定119例慢性乙型肝炎患者PBMC内HBV-DNA定量,并对肝组织进行病理学诊断。结果119例乙型肝炎患者PBMC内HBV-DNA检出率为71.4%,轻度、中度、重度慢性乙型肝炎患者PBMC内HBV-DNA阳性率分别为58.1%、69.4%、84.6%;慢性乙型肝炎患者PBMC内HBV-DNA检出率与肝组织损伤程度呈正相关(rs=0.912,P<0.05)。结论PBMC中HBV-DNA阳性率随肝损害加重而升高。     

Dynamics of apoptotic activity during antiviral treatment of patients with chronic hepatitis C

INTRODUCTION: Cell death during antiviral therapy of patients with chronic hepatitis C is not well understood. METHODS: In the present study, apoptotic activity was monitored by quantification of apoptotic cytokeratin-18 neoepitopes in serum from patients with chronic hepatitis C before and 4, 12, 24 and 48 weeks after initiation of antiviral therapy with pegylated interferon-alpha2a and ribavirin and was compared with viral kinetic parameters. RESULTS: After 4 weeks of treatment apoptotic activity decreased significantly compared with baseline. Later during treatment, however, apoptotic activity increased again to levels similar to baseline. Alanine aminotransferase (ALT) activity also showed a significant decrease at week 4 compared with baseline but, in contrast to apoptotic activity, ALT remained at a reduced level during the treatment period. Baseline apoptotic activity was inversely correlated with the infected cell loss while an increase of apoptotic activity within the first 4 treatment weeks compared with baseline was positively correlated with the infected cell loss. CONCLUSIONS: Apoptosis appears to be an important form of cell death during interferon-alpha-based treatment and is associated with infected cell loss and underestimated by ALT activity.     

慢性HCV感染者外周血单个核细胞IL-18和IFNAR1检测的临床意义

目的 分析慢性丙肝（CHC）患者外周血单个核细胞（PBMC）白细胞介素18（IL-18）mRNA的诱导表达及α干扰素受体1（IFN-α receptor1，IFNAAR1）表达，评价慢性丙肝患者PBMC细胞免疫功能。方法 脂多糖（LPS）体外刺激正常对照组和慢性丙型肝炎病毒（Hepatitis C virus，HCV）感染组病人PBMC，RT-PCR检测干扰素治疗前后PBMC IL-18mRNA水平的变化。同时RT-PCR检测不同组PBMC IFNAR1mRNA水平的差异。结果 治疗前干扰素应答组病人（n=12）和无应答组病人（n=26）PBMC IL-18mRNA诱导表达水平显著低于正常对照组（P〈0．01；P〈0．01）；治疗后，应答组病人IL-18mRNA水平随治疗时间延长而升高，1个月时显著高于无应答组病人（P〈0．01），无应答组病人IL18mR-NA水平始终较低。IFN-α2b治疗期间，干扰素应答组病人PBMCIFNAR1mRNA水平从治疗前的高表达而逐渐减少，正常对照和干扰素无应答组病人PBMCIFNAR1mRNA的表达始终较低（治疗前与应答组相比P〈0．01）。结论 慢性HCV感染病人的细胞免疫功能受损，不能正常表达IL-18和IFNAR1，但应答组病人经干扰素治疗后表达能力逐渐恢复。慢性丙肝患者PBMCIL18mRNA和IFNAR1mRNA水平检测也许可以作为干扰素治疗预后的判断指标。     

慢性乙型肝炎患者外周血单个核细胞内与血清HBV DNA载量的相关性及超微结构研究

目的研究慢性乙型肝炎患者外周血单个核细胞(PBMCs)与血清中HBV DNA载量之间的相关性及PBMCs超微结构的变化。方法应用荧光定量聚合酶链反应(FQ-PCR)检测60例慢性乙型肝炎患者PBMCs与血清中HBV DNA的载量;并采用透射电镜分析观察其中20例PBMCs的超微结构所见。结果血清中HBV DNA载量为(5.25±2.11)log10copies/ml,PBMCs中HBV DNA载量为(3.74±1.08)log10copies/ml,两者的HBV DNA表达呈正相关(r=0.84,P<0.0001)。电镜观察到血清HBVDNA载量高(≥105copies/ml)的患者PBMCs胞浆中易见到HBV颗粒,线粒体膨胀、嵴消失,内质网也可见膨胀。病程5年以上或血清HBV DNA载量<103copies/ml的患者,虽然PBMCs中病毒颗粒不多见,但细胞器多有改变。结论慢性乙型肝炎患者PBMCs与血清中HBV DNA载量呈正相关,可作为疗效观察指标。随着病程延长,尽管血清HBV DNA载量明显减少,但PBMCs细胞器损伤明显,提示机体免疫功能受损。     

Apoptosis of peripheral blood mononuclears and evaluation of the effectiveness of antiviral therapy of chronic C hepatitis

The effects of combined antiviral therapy on the apoptosis of peripheral blood mononuclears (PBM) were investigated in 54 patients with chronic hepatitis C (CHC), including patients with chronic viral-and-alcohol hepatitis (CVAH). Simultaneously, serum concentrations of cytokines (tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), and interleukins 10 and 12) using flow cytofluorimetry were taken. The results demonstrate an increase in the intensity of PBM apoptosis in patients with CHC and CVAH vs. controls; there was a tendency of lesser apoptosis intensity in the CVAH subgroup. In both groups IFN-gamma and TNF-alpha concentrations were increased, and there was a direct correlation between the level of apoptosis and TNF-alpha concentration. The study found an increase in the intensity of apoptosis in CHC patients with primary virological response to antiviral therapy with IFN-alpha and riboflavin, which can be considered an additional prognostic factor of the effectiveness of treatment.     

Subpopulation blood lymphocyte composition in blood of patients with chronic hepatitis C during therapy with interferon

The number of lymphocytes expressing CD3, CD4, CD8, CD16, CD25, and CD19 antigens was studied in patients with chronic hepatitis C before and after 12-week therapy with interferon-alpha (IFN-alpha). The percentage of cells expressing CD4+, CD16+, and CD25+ antigens decreased significantly in untreated patients, while the percentage of CD8+ and CD19+ lymphocytes was increased (p < 0.05) vs. the control. After 3-month therapy with IFN-alpha the counts of CD4+ and CD25+ increased significantly in patients with chronic hepatitis C (p < 0.05 and p < 0.01, respectively) in comparison with the initial values. The treatment led to a significant (p < 0.05) decrease in the number of CD8+ cells in the blood. The number of cells expressing CD19+ decreased, but remained high in comparison with the control. These results indicate that cellular immune response is inadequate in patients with chronic hepatitis C. Time course of subpopulation composition of T lymphocytes during effective treatment with IFN-alpha indicates an important role of T-cellular component of immunity in the antiviral defense mechanisms in chronic HCV infection.     

α-干扰素诱导外周血单核细胞向树突状细胞分化

背景：研究表明，树突状细胞接受刺激的性质和微环境及其起源是决定免疫反应中初始CD4^＋T细胞发生Th1或Th2应答的关键因素，树突状细胞是否成熟对其功能的发挥影响很大。 目的：观察广泛应用的细胞因子α-干扰素对人外周血单核细胞向树突状细胞分化成熟的影响。 设计、时间及地点：细胞水平的观察对照实验，于2007-05／12在深圳市人民医院临床医学研究中心实验室完成。 材料：取10名健康成人志愿者的外周血。 方法：采集外周血10mL，肝素抗凝，常规淋巴细胞分离液分离单核细胞，然后将单核细胞与粒-巨噬细胞集落刺激因子、白细胞介素4体外培养7d。分3组进行树突状细胞培养，对照组不加α-干扰素；100U／L α-干扰素组于培养第5天加入100U／L的α-干扰素；300U／Lα-干扰素组于培养第5天加入300U／L的α-干扰素。主要观察指标：培养第7天用流式细胞术检测树突状细胞膜表面CD83和MHC—DR表达水平；应用四氮唑盐法测定树突状细胞刺激同种异体T细胞增殖的能力：采用酶联免疫吸附法检测树突状细胞培养上清中白细胞介素12p40＋p70含量。 结果：100，300U／Lα-干扰素组中CD83^＋和MHC—DR^＋树突状细胞的数量、刺激同种异体T细胞增殖的能力及培养上清液中白细胞介素12p40＋p70水平均显著高于对照组（P〈0．01），并以300U／Lα-干扰素组作用更强。 结论：α-干扰素可以有效促进单核细胞源树突状细胞的功能成熟，并呈剂量依赖效应。     

外周血中CD4＋调节性T细胞检测与慢性丙肝患者抗病毒疗效分析

目的：研究 T 细胞调节性外周血中 CD4＋慢性丙肝患者与抗病毒疗效的关系。方法以收集的经过抗病毒治疗和随访的128例慢性丙肝患者作为试验组,分别抽取治疗前后以及随访24周患者的外周血,采用流式细胞术检测外周血中的CD4＋细胞的百分比,并进行 HCV-RNA 定量和肝功能指标的检测。对照组为同期体检健康者80例。结果试验组和对照组HCV-RNA 定量、AST、ALT、CD4＋（％）在两组之间比较,差异具有统计学意义（P ＜0.05）;治疗前后以及随访24周后,患者的HCV-RNA 定量、AST、ALT、CD4＋（％）的水平均显著的降低（P ＜0.05）。结论抗病毒治疗可以有效地降低慢性丙型肝炎的伤害,提高治愈率,减少对肝脏的损害。外周血中 CD4＋调节性 T 细胞可以作为抗病毒治疗疗效以及丙型肝炎病毒感染的指标。     

干扰素治疗慢性乙肝患者外周血调节性T细胞动态变化和临床意义

目的探讨干扰素抗病毒治疗过程中慢性乙肝患者外周血CD4＋CD25＋调节性T细胞（CD4＋CD25＋Treg）的变化及其临床意义。方法采集干扰素抗病毒治疗的CHB患者46例外周血,在治疗前及治疗后12、24、36、48周,停药后24周时,分别以流式细胞仪检测外周血CD4＋CD25＋Treg比例,实时荧光定量RT-PCR检测血清HBV-DNA载量,酶联免疫吸附法检测HBV标志物,全自动生化分析仪检测ALT水平。结果在46例CHB患者中,完全应答的13例患者CD4＋CD25＋Treg频率在12、24、36、48周及停药后24周时分别为（4.16±0.64）%、（3.98±0.75）%、（3.70±0.55）%、（3.44±0.48）%、（3.07±0.48）%,呈逐步下降趋势,明显低于治疗前（5.73±0.62）%（P〈0.01）。部分应答的18例患者CD4＋CD25＋Treg频率在12、24、36、48周及停药后24周时分别为（4.98±0.62）%（、4.50±0.54）%、（4.10±0.64）%、（4.02±0.64）%（、3.82±0.47）%,呈逐步下降趋势,明显低于治疗前（5.84±0.68）%（P〈0.01）。无应答的15例患者CD4＋CD25＋Treg频率较治疗前无明显变化。完全应答、部分应答组患者Treg频率在12、24、36、48周及停药后24周时与无应答组比较,差异有统计学意义（P〈0.01）。治疗24、48周,停药后24周时,HBeAg阴转率分别为10.8%、26.1%、28.3%,发生HBeAg血清学转换者在治疗12周时CD4＋CD25＋Treg比例明显下降。结论本实验初步认为,干扰素抗病毒治疗过程中及治疗后,有应答的CHB患者的外周血CD4＋CD25＋Treg频率下降。治疗早期CD4＋CD25＋Treg比例下降的患者HBeAg血清学转换率可能更高。     

Selenium supplementation decreases nuclear factor-kappa B activity in peripheral blood mononuclear cells from type 2 diabetic patients

OBJECTIVE: The role of selenium in preventing cardiovascular diseases has been largely described. Oxidative stress and the subsequent activation of nuclear factor-kappa B (NF-kappaB) have been linked to the development of vascular complications. We investigated the effects of selenium supplementation in type 2 diabetic patients on several oxidative stress parameters and NF-kappaB activity. METHODS: We enrolled 56 type 2 diabetic patients with similar glycaemic control: 21 were supplemented by selenium (960 micro g d(-1), 3 months) and 27 received a placebo, and 10 nondiabetic subjects formed the control group. To determine NF-kappaB activation, we used an electrophoretic mobility shift assay followed by a semi-quantitative determination of NF-kappaB in peripheral blood mononuclear cells. RESULTS: Selenium treatment resulted in a significant increase in plasma selenium and red-cell Se GSH px activity. It had no effect on lipid peroxidation measured by malone-dialdehyde (MDA) or on red-cell Cu/Zn SOD. NF-kappaB activity was increased by 80% in diabetic patients. In patients receiving selenium supplementation, selenium NF-kappaB activity was significantly reduced, reaching the same level as the nondiabetic control group. CONCLUSION: In type 2 diabetic patients, activation of NF-kappaB measured in peripheral blood monocytes can be reduced by selenium supplementation, confirming its importance in the prevention of cardiovascular diseases.