Differential cytokine responses in Hep C patients.

我们假设丙型肝炎患者对治疗的反应不同的基础，以及非裔美国人和其他人之间的差异是由于感染/治疗方案期间细胞因子反应的遗传差异所致。人类细胞因子表达阵列将被用来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将从基线采集的PBMC样本中提取，在治疗后6、15、24小时，每周一次，最长为四周。人类细胞因子表达阵列将与来自患者组(应答者、无应答者、非裔美国人、高加索人)的cDNA一起运行。初步数据表明，这种方法可以检测到非裔美国人患者和其他患者以及应答者和无应答者之间细胞因子基因表达的差异。所有的阵列数据将通过RT-PCR进行确认。对治疗后早期采集的血清样本的分析显示，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小时内回到基线。这些细胞因子似乎不是由PBMC合成的，但表明对干扰素有急性时相反应。其他细胞因子，特别是那些通过DNA阵列发现差异表达的细胞因子，将在上述时间点在个人血清样本中进行检测。我们将把血清细胞因子、DNA表达阵列、病毒滴度和治疗结果联系起来。。利用在上述时间点从PBMC中提取的RNA，我们将通过RT-PCR来测量干扰素诱导基因的诱导，这些基因以前被证明参与了抗病毒反应。它们包括寡聚A合成酶、Mx蛋白和吲哚胺2-3双加氧酶。核糖核酸酶L和蛋白酪氨酸激酶受体将进行酶活性测定。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)中是有争议的。在进入治疗之前，将从患者身上建立PBMC培养，并评估病毒RNA的存在。这些培养物将用干扰素-α(或干扰素-α和利巴韦林)处理，并检测细胞因子的产生，特别是那些通过DNA表达阵列鉴定的细胞因子以及推测的病毒滴度的变化。我们将尝试建立一个模拟体内事件的体外系统。这一组合实验应该确定对治疗和病毒耐药性的不同反应起重要作用的因素。

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.