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: 6647595
• 负责人: MILTON W TAYLOR
• 金额: $ 32.5万
• 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 2006-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6647595
• 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，以及非裔美国人与其他人之间的差异是由于感染/治疗方案期间细胞因子反应的遗传差异。人类细胞因子表达阵列将用于分析不同丙型肝炎患者组之间特异性细胞因子表达的差异。将从基线、给药后6、15、24小时以及每周一次直至四周采集的PBMC样本制备RNA。人细胞因子表达阵列将用来自患者组（应答者、无应答者、非裔美国人、高加索人）的cDNA运行。初步数据表明，通过该方法可以检测非裔美国人患者与其他患者之间以及应答者与非应答者之间细胞因子基因表达的差异。将通过RT-PCR确认所有阵列数据。在治疗后早期时间点采集的血清样品的分析显示IL-6和IL-1 Ra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。这种细胞因子似乎不是由PBMC合成的，但表明对干扰素的急性期应答。其他细胞因子，特别是那些被DNA芯片发现差异表达的细胞因子，将在上述时间点从个体的血清样本中进行检测，我们将血清细胞因子、DNA表达芯片、病毒滴度和治疗结果相关联。.使用在上述时间点从PBMC分离的RNA，我们将通过RT-PCR测量干扰素诱导基因的诱导，所述干扰素诱导基因先前显示参与抗病毒应答。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RNA酶L和PKR进行酶测定。丙型肝炎病毒是否存在于一类免疫细胞（淋巴细胞，巨噬细胞）中存在争议。将在进入治疗前从患者中建立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拟体内事件的体外系统。这种实验的组合应该确定在对治疗和病毒耐药性的不同反应中重要的因素。