DCs Pulsed with Novel HLA-A2-Restricted CTL Epitopes against Hepatitis C Virus Induced a Broadly Reactive Anti-HCV-Specific T Lymphocyte Response

OBJECTIVE: To determine the capacity of dendritic cells (DCs) loaded with single or multiple-peptide mixtures of novel hepatitis C virus (HCV) epitopes to stimulate HCV-specific cytotoxic T lymphocyte (CTL) effector functions. METHODS: A bioinformatics approach was used to predict HLA-A2-restricted HCV-specific CTL epitopes, and the predicted peptides identified from this screen were synthesized. Subsequent IFN-γ ELISPOT analysis detected the stimulating function of these peptides in peripheral blood mononuclear cells (PBMCs) from both chronic and self-limited HCV infected subjects (subjects exhibiting spontaneous HCV clearance). Mature DCs, derived in vitro from CD14(+) monocytes harvested from the study subjects by incubation with appropriate cytokine cocktails, were loaded with novel peptide or epitope peptide mixtures and co-cultured with autologous T lymphocytes. Granzyme B (GrB) and IFN-γ ELISPOT analysis was used to test for epitope-specific CTL responses. T-cell-derived cytokines contained in the co-cultured supernatant were detected by flow cytometry. RESULTS: We identified 7 novel HLA-A2-restricted HCV-specific CTL epitopes that increased the frequency of IFN-γ-producing T cells compared to other epitopes, as assayed by measuring spot forming cells (SFCs). Two epitopes had the strongest stimulating capability in the self-limited subjects, one found in the E2 and one in the NS2 region of HCV; five epitopes had a strong stimulating capacity in both chronic and self-limited HCV infection, but were stronger in the self-limited subjects. They were distributed in E2, NS2, NS3, NS4, and NS5 regions of HCV, respectively. We also found that mDCs loaded with novel peptide mixtures could significantly increase GrB and IFN-γ SFCs as compared to single peptides, especially in chronic HCV infection subjects. Additionally, we found that DCs pulsed with multiple epitope peptide mixtures induced a Th1-biased immune response. CONCLUSIONS: Seven novel and strongly stimulating HLA-A2-restricted HCV-specific CTL epitopes were identified. Furthermore, DCs loaded with multiple-epitope peptide mixtures induced epitope-specific CTLs responses

Analysis of FOXP3+ Regulatory T Cells That Display Apparent Viral Antigen Specificity during Chronic Hepatitis C Virus Infection

We reported previously that a proportion of natural CD25+ cells isolated from the PBMC of HCV patients can further upregulate CD25 expression in response to HCV peptide stimulation in vitro, and proposed that virus-specific regulatory T cells (Treg) were primed and expanded during the disease. Here we describe epigenetic analysis of the FOXP3 locus in HCV-responsive natural CD25+ cells and show that these cells are not activated conventional T cells expressing FOXP3, but hard-wired Treg with a stable FOXP3 phenotype and function. Of ∼46,000 genes analyzed in genome wide transcription profiling, about 1% were differentially expressed between HCV-responsive Treg, HCV-non-responsive natural CD25+ cells and conventional T cells. Expression profiles, including cell death, activation, proliferation and transcriptional regulation, suggest a survival advantage of HCV-responsive Treg over the other cell populations. Since no Treg-specific activation marker is known, we tested 97 NS3-derived peptides for their ability to elicit CD25 response (assuming it is a surrogate marker), accompanied by high resolution HLA typing of the patients. Some reactive peptides overlapped with previously described effector T cell epitopes. Our data offers new insights into HCV immune evasion and tolerance, and highlights the non-self specific nature of Treg during infection

Fibromyalgia syndrome improved using a mostly raw vegetarian diet: An observational study

<p>Abstract</p> <p>Background</p> <p>Fibromyalgia engulfs patients in a downward, reinforcing cycle of unrestorative sleep, chronic pain, fatigue, inactivity, and depression. In this study we tested whether a mostly raw vegetarian diet would significantly improve fibromyalgia symptoms.</p> <p>Methods</p> <p>Thirty people participated in a dietary intervention using a mostly raw, pure vegetarian diet. The diet consisted of raw fruits, salads, carrot juice, tubers, grain products, nuts, seeds, and a dehydrated barley grass juice product. Outcomes measured were dietary intake, the fibromyalgia impact questionnaire (FIQ), SF-36 health survey, a quality of life survey (QOLS), and physical performance measurements.</p> <p>Results</p> <p>Twenty-six subjects returned dietary surveys at 2 months; 20 subjects returned surveys at the beginning, end, and at either 2 or 4 months of intervention; 3 subjects were lost to follow-up. The mean FIQ score (n = 20) was reduced 46% from 51 to 28. Seven of the 8 SF-36 subscales, bodily pain being the exception, showed significant improvement (n = 20, all P for trend < 0.01). The QOLS, scaled from 0 to 7, rose from 3.9 initially to 4.9 at 7 months (n = 20, P for trend 0.000001). Significant improvements (n = 18, P < 0.03, paired t-test) were seen in shoulder pain at rest and after motion, abduction range of motion of shoulder, flexibility, chair test, and 6-minute walk. 19 of 30 subjects were classified as responders, with significant improvement on all measured outcomes, compared to no improvement among non-responders. At 7 months responders' SF-36 scores for all scales except bodily pain were no longer statistically different from norms for women ages 45–54.</p> <p>Conclusion</p> <p>This dietary intervention shows that many fibromyalgia subjects can be helped by a mostly raw vegetarian diet.</p