Secondary nested RT-PCR for detection of occult HCV infection To detect occult HCV infection, secondary nested RT-PCR of the 5��-untranslated region of HCV RNA was performed as described elsewhere [32]�C[34]. Total RNA was extracted the from PBMCs using the RNeasy mini kit (Qiagen, Valencia, CA) with on-column deoxyribonuclease digestion. For cDNA synthesis, 200 to 400 ng RNA was reverse transcribed with ReverTraAce-�� kit (Toyobo, Osaka, Japan) using an HCV RNA-specific primer. These cDNA samples served as templates in the direct round of PCR amplification (primer pairs used: 5��-CTG TGA GGA ACT ACT GTC TTC and 5��-GCG GTT GGT GTT ACG TTT), and the nested round reaction (primer pairs used: 5��-GCA GAA AGC GTC TAG CCA TGG C and 5��-CTG CAA GCA CCC TAT CAG GCA GT) was performed with the products of the first round PCR.

For positive cases in secondary nested RT-PCR of HCV RNA, genotypes of HCV were determined as follows. Briefly, the final PCR product of 243 bp was purified and cloned into the TOPO TA cloning vector (Invitrogen, Carlsbad, CA), and genotype was ascertained by automatically sequencing five clones from each patient. Results Presence of HCV-specific T cell responses in seronegative, aviremic patients Among 77 hemodialysis patients with chronic renal disease, one patient was seropositive without viremia, and five patients were seropositive with viremia. The other 71 patients were seronegative and aviremic, meaning they exhibited no clinical evidence of current or past HCV infection. There were no seronegative patients with viremia.

HCV-specific T cell responses were evaluated by direct ex vivo IFN-�� ELISpot assay of PBMCs in all patients except the seropositive, aviremic patient. For comprehensive analysis, we stimulated PBMCs with 49 matrixed mixes of 600 overlapping peptides covering the complete HCV polyprotein sequence (Figure S1A). All of the viremic patients and a majority of seronegative, aviremic patients exhibited insignificant levels of HCV-specific T cell responses (Figure 1A). In contrast, eight (11.3%) of the seronegative, aviremic patients displayed obvious T cell responses against HCV (Figure 1A). This was an unexpected result as these HCV-specific T cells were detected in patients without evidence of current or past infection. Figure 1B shows results of the IFN-�� ELISpot assay in response to each peptide mix.

Figure 1 HCV-specific T cell responses in seronegative, aviremic hemodialysis patients. Conducting the IFN-�� ELISpot assay with a matrixed mix of overlapping peptides enabled us to identify T cell epitopes within the HCV polyprotein for each patient (Figure S1B and Table 1). T cell responses to the identified epitopes were confirmed Entinostat by IFN-�� ICS (Figure 1C). We also identified which T cell subset, CD4+ or CD8+, was responsible for the HCV-specific IFN-�� production in each patient (Figure 1C and Table 1).