Lorena Salazar

The immunological response and post-treatment survival of DC-vaccinated melanoma patients are associated with increased Th1/Th17 and reduced Th3 cytokine responses

IntroductionImmunization with autologous dendritic cells (DCs) loaded with a heat shock-conditioned allogeneic melanoma cell lysate caused lysate-specific delayed type hypersensitivity (DTH) reactions in a number of patients. These responses correlated with a threefold prolonged long-term survival of DTH+ with respect to DTH− unresponsive patients. Herein, we investigated whether the immunological reactions associated with prolonged survival were related to dissimilar cellular and cytokine responses in blood.Materials and methodsHealthy donors and melanoma patient’s lymphocytes obtained from blood before and after vaccinations and from DTH biopsies were analyzed for T cell population distribution and cytokine release.Results/discussionPeripheral blood lymphocytes from melanoma patients have an increased proportion of Th3 (CD4+ TGF-β+) regulatory T lymphocytes compared with healthy donors. Notably, DTH+ patients showed a threefold reduction of Th3 cells compared with DTH− patients after DCs vaccine treatment. Furthermore, DCs vaccination resulted in a threefold augment of the proportion of IFN-γ releasing Th1 cells and in a twofold increase of the IL-17-producing Th17 population in DTH+ with respect to DTH− patients. Increased Th1 and Th17 cell populations in both blood and DTH-derived tissues suggest that these profiles may be related to a more effective anti-melanoma response.ConclusionsOur results indicate that increased proinflammatory cytokine profiles are related to detectable immunological responses in vivo (DTH) and to prolonged patient survival. Our study contributes to the understanding of immunological responses produced by DCs vaccines and to the identification of follow-up markers for patient outcome that may allow a closer individual monitoring of patients.

Multiple imputation procedures allow the rescue of missing data: An application to determine serum tumor necrosis factor (TNF) concentration values during the treatment of rheumatoid arthritis patients with anti-TNF therapy

Longitudinal studies aimed at evaluating patients clinical response to specific therapeutic treatments are frequently summarized in incomplete datasets due to missing data. Multivariate statistical procedures use only complete cases, deleting any case with missing data. MI and MIANALYZE procedures of the SAS software perform multiple imputations based on the Markov Chain Monte Carlo method to replace each missing value with a plausible value and to evaluate the efficiency of such missing data treatment. The objective of this work was to compare the evaluation of differences in the increase of serum TNF concentrations depending on the -308 TNF promoter genotype of rheumatoid arthritis (RA) patients receiving anti-TNF therapy with and without multiple imputations of missing data based on mixed models for repeated measures. Our results indicate that the relative efficiency of our multiple imputation model is greater than 98% and that the related inference was significant (p-value < 0.001). We established that under both approaches serum TNF levels in RA patients bearing the G/A -308 TNF promoter genotype displayed a significantly (p-value < 0.0001) increased ability to produce TNF over time than the G/G patient group, as they received successively doses of anti-TNF therapy.

G2 checkpoint-dependent DNA repair and its response to catalase in Down syndrome and control lymphocyte cultures.

The amount of DNA lesions repaired in G2 and also G2 timing are controlled by the DNA damage‐dependent checkpoint. Down syndrome (DS) lymphocytes showed twice as much constitutive DNA damage in G2 than control ones, when recording it as chromosomal aberrations in metaphase, after caffeine‐induced checkpoint abrogation. During G2, DS lymphocytes repaired 1.5 times more DNA lesions than control ones. However the DS cells displayed a decreased threshold for checkpoint adaptation, as the spontaneous override of the G2 to mitosis transition block induced by the checkpoint took place in the DS cells when they had three times more DNA lesions than controls. Catalase addition to cultures scavenges hydrogen peroxide diffused from cells, resulting in subsequent intracellular depletion (Antunes and Cadenas, 2000). The intracellular H2O2 level seemed to regulate the G2 checkpoint. Thus, in controls, H2O2 depletion (induced by 3.2–50 μg/mL catalase) prevented its functioning: chromosomal damage increased while G2 shortened. Conversely, in the DS lymphocytes, 12.5 μg/mL catalase lengthened G2 and decreased chromosomal damage, in spite that the amount of DNA repaired in G2 was half of that repaired in the catalase‐free DS lymphocytes.

Abstract B18: Induction of Th1/Th17 and inhibition of Th3 cellular profile by DC-based immunotherapy is associated with immunological and clinical anti-tumor responses in melanoma patients

The ability of DCs to induce a primary immune response makes them ideal candidates for cancer vaccines against tumors. Recently, in a serie of phase I/II clinical studies, we showed the effectiveness of autologous DCs loaded with a conditioned allogeneic melanoma cell lysate named TRIMEL. Our results demonstrated a correlation between positive tumor-specific delayed type hypersensitivity (DTH) reaction induced by DC-vaccination, and improved long-term patient survival in late-stage melanoma patients. In fact, 60% of patients showed a positive DTH reaction showing a three-fold prolonged survival compared to non-responder patients (33 and 11 months respectively). Herein, we investigated whether cellular and cytokine profiles in patients PBL before and after vaccination reflects differential responses associated to the clinical outcome. Analysis of T cell populations in blood showed that melanoma patients (n=28) have an increased proportion of Th3 (CD4+TGF-β+) regulatory T lymphocytes (2.64 % ± 0.16) compared to normal donors (n=14) (1.72 % ± 0.22). However, we didn9t observed differences in TGF-β concentrations at serum level. Moreover, we analyzed vaccine effect on cytokine and cellular responses in PBL. Interestling, although there was not differences in Th3 population proportions between responder (n=14) and non-responder patients (n=14) before vaccination, after treatment responder patients showed a three-fold reduction of CD4+TGf-β+ T cells compared to non-responders (1.65 % ± 0.19 and 5.54 % ± 0.28 respectively). Furthermore, our results show that DC-vaccination resulted in a three-fold augment of IFN-γ releasing by Th1 population and a two-fold increase of IL-17 producing Th17 lymphocyte population in responder patients, but not in non-responders. A direct correlation between IFN-γ and IL-17 production in responder patients blood was observed (p Citation Information: Clin Cancer Res 2010;16(7 Suppl):B18