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.
Showing the most recent 10 out of 28 publications
