Differential cytokine responses in Hep C patients.

丙型肝炎患者的细胞因子反应差异。

• 批准号: 6406727
• 负责人: MILTON W TAYLOR
• 金额: $ 16.19万
• 依托单位: TRUSTEES OF INDIANA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-08-01 至 2006-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6406727
• 关键词: African American; cooperative study; cytokine; drug resistance; genetic susceptibility; hepatitis C; human tissue; immunogenetics; immunotherapy; interferon alpha; interferon inducers; leukocytes; microarray technology; patient oriented research; pharmacogenetics; racial /ethnic difference; ribavirin; tissue /cell culture; virus genetics

We hypothesize that the basis for differences in response to treatment in hepatitis C patients, and difference between African Americans and others is due to genetic differences in cytokine responses manifest during the infectious/treatment regimen. Human cytokine expression arrays will be used to analyze differences in expression of specific cytokines between different groups of hepatitis C patients. RNA will be prepared from PBMC samples taken at base line, 6, 15, 24 hours after treatment and at weekly intervals up to four weeks.. Human cytokine expression arrays will be run with cDNA from groups of patients (responders, non- responders, African Americans, Caucasians). Preliminary data indicate that differences in expression of cytokine genes between African American patients and others, as well as between responders and non- responders can be detected by this method. All array data will be confirmed by RT-PCR. Analysis of serum samples taken at early time points after treatment has shown an acute induction of IL-6 and IL-1Ra. Both of these inductions are transient ,and return to base line within 24- 28 hours post treatment. Such cytokines do not appear to be synthesized by PBMC but are indicative of an acute phase response to interferon. Other cytokines, particularly those discovered to be differentially expressed by DNA array will be assayed in serum samples from individuals at the above time points.We shall correlate serum cytokines, DNA expression arrays, viral titer and outcome of treatment. . Using RNA isolated from PBMC at the above time points we shall measure by RT-PCR the induction of interferon induced genes, previously shown to be involved in the anti-viral response. These include oligo A synthetase, Mx protein, and indoleamine 2 3 dioxygenase. Enzyme assay will be performed for RNAse L and PKR. Whether hepatitis C virus resides in a class of immune cells (lymphocytes, macrophages) is controversial. Cultures of PBMC will be established from patients before entering treatment and the presence of viral RNA assessed. These cultures will be treated with IFN-alpha (or IFN-alpha and ribavirin) and assayed for cytokine production, particularly those identified by the DNA expression arrays as well as for changes in presumptive viral titers. We will attempt to establish an in vitro system that mimics in vivo events. This combination of experiments should identify factors that are important in the differential responses to treatment and viral resistance.

我们假设丙型肝炎患者对治疗反应差异的基础以及非裔美国人和其他人之间的差异是由于感染/治疗方案期间表现出的细胞因子反应的遗传差异所致。人类细胞因子表达芯片将用于分析不同组丙型肝炎患者之间特定细胞因子表达的差异。 RNA 将从基线、治疗后 6、15、24 小时以及每周最长 4 周的时间间隔采集的 PBMC 样本中制备。人类细胞因子表达阵列将使用来自患者组（有反应者、无反应者、非裔美国人、白种人）的 cDNA 进行运行。初步数据表明，该方法可以检测非裔美国患者和其他患者之间以及反应者和非反应者之间细胞因子基因表达的差异。所有阵列数据将通过 RT-PCR 进行确认。治疗后早期时间点采集的血清样本分析显示 IL-6 和 IL-1Ra 的急性诱导。这两种诱导都是短暂的，并在治疗后 24-28 小时内恢复到基线。这些细胞因子似乎不是由 PBMC 合成的，但表明对干扰素的急性期反应。其他细胞因子，特别是那些被发现通过 DNA 阵列差异表达的细胞因子，将在上述时间点对来自个体的血清样本进行测定。我们将把血清细胞因子、DNA 表达阵列、病毒滴度和治疗结果关联起来。 。使用在上述时间点从 PBMC 中分离的 RNA，我们将通过 RT-PCR 测量干扰素诱导基因的诱导，先前显示这些基因参与抗病毒反应。这些包括寡聚 A 合成酶、Mx 蛋白和吲哚胺 2 3 双加氧酶。将对 RNAse L 和 PKR 进行酶测定。丙型肝炎病毒是否驻留在一类免疫细胞（淋巴细胞、巨噬细胞）中存在争议。在进入治疗之前，将建立患者的 PBMC 培养物，并评估病毒 RNA 的存在。这些培养物将用 IFN-α（或 IFN-α 和利巴韦林）处理，并测定细胞因子的产生，特别是通过 DNA 表达阵列鉴定的细胞因子以及推定病毒滴度的变化。我们将尝试建立一个模仿体内事件的体外系统。这种实验组合应该能够确定对治疗和病毒耐药性的差异反应很重要的因素。