Baseline Peripheral Blood Mononuclear Cells Research Articles

HCV protein‐induced cytokine predicts treatment outcomes in chronic hepatitis C virus infection

AbstractBackground & AimsImmune‐mediated processes are thought to influence the efficacy of treatment in chronic hepatitis C virus (HCV) infection. This study evaluated the association of baseline immune responses with the achievement of a sustained viral response (SVR) upon pegylated interferon and ribavirin treatment.MethodsBaseline serum and peripheral blood mononuclear cells (PBMC) cytokine activity was assessed. Metabolic and liver injury parameters were evaluated as underlying factors.ResultsBaseline demographics and disease parameters did not differ between the SVR− (n = 14) and SVR+ (n = 25) cohorts except for body mass index (BMI) values and liver injury scores. Baseline circulating TNF‐α levels were three‐fold higher with subsequent treatment failure vs. success (P = 0.124). Baseline peripheral blood mononuclear cells (PBMC, n = 25) were cultured with HCV core and non‐structural (NS)3 proteins. Core (P = 0.0003) and NS3 (P = 0.018) induced greater TNF‐α production within the SVR−, compared with the SVR+, cohorts. Similar findings were noted for interleukin (IL)‐1β and IL‐10 synthesis. Furthermore, HCV core‐induced TNF‐α synthesis correlated with patient BMI values (r = 0.489, P = 0.015). Core (r = 0.432, P = 0.065) and NS3 (r = 0.530, P = 0.020)‐induced TNF‐α displayed a positive relationship with serum adiponectin concentrations. In addition, lipopolysaccharide stimulated cytokine synthesis associated with BMI and adiponectin levels. Although unable to predict treatment outcomes, NS3‐induced IL‐6 synthesis and serum leptin concentrations corresponded to liver injury scores.ConclusionAn enhanced PBMC susceptibility to core and NS3‐induced TNF‐α synthesis at baseline was associated with treatment failure. This phenomenon appeared to involve the interaction of virally generated TNF‐α activity and metabolic disease. In contrast, IL‐6 activity and leptin levels may indicate liver damage.

Sensitive testing of plasma HIV-1 RNA and Sanger sequencing of cellular HIV-1 DNA for the detection of drug resistance prior to starting first-line antiretroviral therapy with etravirine or efavirenz

This study investigated strategies that may increase the yield of drug resistance testing prior to starting antiretroviral therapy (ART), and whether transmitted and polymorphic resistance-associated mutations (RAMs) correlated with virological outcomes. We carried out retrospective testing of baseline samples from patients entering the SENSE trial of first-line ART in Europe, Russia and Israel. Prior to randomization to etravirine or efavirenz plus two nucleos(t)ide reverse transcriptase inhibitors (NRTIs), plasma samples underwent routine Sanger sequencing of HIV-1 RT and protease ((plasma)SS) in order to exclude patients with transmitted RAMs. Retrospectively, Sanger sequencing was repeated with HIV-1 DNA from baseline peripheral blood mononuclear cells (PBMCSS); baseline plasma samples were retested by allele-specific PCR targeting seven RT RAMs (AS-PCR) and ultra-deep RT sequencing (UDS). By (plasma)SS, 16/193 (8.3%) patients showed ≥ 1 transmitted RAM affecting the NRTIs (10/193, 5.2%), non-nucleoside reverse transcriptase inhibitors (4/193, 2.1%) or protease inhibitors (2/193, 1.0%). No additional RAMs were detected by AS-PCR (n = 152) and UDS (n = 24); PBMCSS (n = 91) yielded two additional samples with one RAM each. Over 48 weeks, 4/79 (5.1%) patients on etravirine and 7/78 (9.0%) on efavirenz experienced virological failure; none had baseline RAMs. Conversely, 11/79 (13.9%) patients randomized to etravirine had one polymorphic RAM from the etravirine score in baseline plasma (V90I, V106I or E138A), without any impact on virological outcomes. The detection of resistance increased marginally with PBMC testing but did not increase with sensitive plasma testing. A careful consideration is required of the cost-effectiveness of different strategies for baseline HIV drug resistance testing.

Phase I Study of Topotecan, Ifosfamide, and Etoposide (TIME) with Autologous Stem Cell Transplant in Refractory Cancer: Pharmacokinetic and Pharmacodynamic Correlates

To determine the maximum tolerated dose (MTD) of topotecan in combination with ifosfamide, mesna, and etoposide (TIME), followed by autologous hematopoietic cell transplant (HCT), in patients with chemotherapy-refractory malignancies. Patients were treated with (in mg/m(2)/d) ifosfamide 3,333, mesna 3,333, and topotecan 3.3 to 28.3 during days -8 through -6 and etoposide 500 (days -5 through -3) followed by HCT on day 0. Once MTD was defined, we expanded this dosing cohort to include patients with high-risk lymphoma due to activity seen during dose escalation. Topotecan pharmacokinetic analyses were carried out, and topoisomerase I levels and activity were measured. The topotecan MTD in this regimen was 64 mg/m(2) (21.3 mg/m(2)/d). Mucositis was dose limiting and correlated with topotecan dose level and area under the curve (AUC). Dose level was also correlated with length of hospitalization, number of days of parenteral nutrition, and neutrophil and platelet engraftment. Topotecan AUC was significantly correlated with time to platelet recovery. The baseline peripheral blood mononuclear cell topoisomerase I level was found to be a significant positive predictor for overall and progression-free survival. Topotecan AUC was positively correlated with dose level, with a trend toward decreasing clearance with increasing dose. Topotecan can be a useful drug in the high-dose setting given its activity in some malignancies when given in standard dose. Pharmacokinetic monitoring may be a valuable tool for optimizing the use of topotecan and to avoid toxicity seen with high-systemic exposures. Baseline topoisomerase I levels may have an important role in predicting topotecan efficacy.

Cellular HIV-1 DNA quantification and short-term and long-term response to antiretroviral therapy

The aim of our study was to determine whether HIV-1 DNA level before antiretroviral therapy (ART) was associated with short- and long-term virological and immunological responses. Patients starting first-line protease inhibitor-containing regimens were enrolled in a prospective multicentre cohort in 1998-99. HIV-1 DNA was quantified using real-time PCR at baseline and after 1 year of ART. The association between HIV-1 DNA and virological and immunological responses after 1 and 7 years on ART was studied in multivariate regression models along with other biological and clinical variables. Virological failure (VF) at month 12 (M12) was defined as a plasma HIV-1 RNA >500 copies/mL. Time to death or two plasma HIV-1 RNA >500 copies/mL between M12 and M84 was studied for long-term VF. HIV-1 DNA levels were measured in 148 patients. The median baseline peripheral blood mononuclear cell (PBMC) HIV-1 DNA was 3.7 log(10) copies/10(6) PBMCs. At M12, the median PBMC HIV-1 DNA was 2.99 log(10) copies/10(6) PBMCs. The median decrease in PBMC HIV-1 DNA between M0 and M12 was -0.7 log(10) copies/10(6) PBMCs. Higher baseline PBMC HIV-1 DNA and plasma HIV-1 RNA were independently associated with a higher risk of VF at M12. Only the baseline plasma HIV-1 RNA was independently associated with long-term virological response. The baseline CD4 cell count was the only parameter associated with short- and long-term immunological responses. HIV-1 DNA impacted the virological response in our cohort. Further research is warranted to study the impact of HIV-1 DNA with currently recommended first-line cART.

Abstract 82: A comprehensive characterization of cholecalciferol supplementation induced changes in the vitamin D3 metabolic pathway in metastatic colorectal cancer patients

Abstract Epidemiological data suggest that vitamin D3 deficiency plays a role in the pathogenesis and progression of human colorectal adenomas and adenocarcinomas. We have characterized the changes in plasma vitamin D3 metabolites (25-D3, 24,25-D3 and 1,25-D3), PTH and vitamin D3 binding protein (VDBP) levels, and peripheral blood mononuclear cells (PBMC) CYP24A1 activity in metastatic colorectal cancer (CRC) patients receiving 2000 IU of oral cholecalciferol daily over a 12 month period. Plasma samples were collected on day 0, 14, 30, 60, 90, 180, 270 and 360. Serum 25-D3 and 24,25-D3 were measured by LC/MS/MS method; plasma PTH and 1,25-D3 levels were determined by RIA. Plasma VDBP levels were measured by ELISA. PBMC CYP24A1activity was determined by HPLC method. A total of 50 CRC patients completed the study. Baseline 25-D3 were significantly influenced by season (p= 0.0145) . Baseline plasma 25-D3 levels were <30ng/mL in 66% of patients. Baseline plasma 24,25-D3, 1,25-D3 and VDBP levels were within the normal range. (The normal ranges are 0- 5ng/mL for 24,25-D3, 16 −80pg/mL for 1,25-D3 and 200 - 550μg/mL for VDBP). Mean baseline plasma PTH levels in patients with plasma 25-D3 <20ng/mL and >30ng/mL were 74.82 ± 27.02 and 56.46 ± 18.10pg/mL, respectively (p>0.05). Baseline PBMC CYP24A1activity was not affected by baseline 25-D3 levels. The increase in serum 25-D3 and 24,25-D3 levels during cholecalciferol treatment was characterized by an initial phase of rapid increase (between day 1 and day 90) followed by a phase of stable plasma 25-D3 and 24,25-D3 levels. A linear relation between plasma 25-D3 and 24,25-D3 was observed at baseline and during cholecalciferol supplementation (r2 = 0.55). No consistent changes in PBMC CYP24A1 activity was observed after 90 days of cholecalciferol treatment. The plasma 24,25-D3/25-D3 ratio at day 60 and 90 of cholecalciferol treatment was weakly correlated (r2=0.03 and 0.06, respectively) to changes in plasma 25-D3 levels. No CRC patient subsets with disproportional increase in plasma 24,25-D3 levels relative to 25-D3 levels during cholecalciferol treatment were identified suggesting that that variable 25-D3 responses cannot be explained by increased 24-hydroxylation. The increase in 1,25-D3 was linear, remained below 100pg/mL and did not correlate with plasma 25-D3 levels. A decrease in plasma PTH levels from 62.83 ± 24.84 to 41.61 ± 18.42 pg/mL was observed after 2 months of cholecalciferol supplementation (p<0.0001). Plasma VDBP protein levels were inconsistent across the study duration; no specific patterns or correlations with 25-D3 or 1,25-D3 could be identified. In summary, vitamin D3 deficiency, defined as 25-D3 <30 ng/mL, is prevalent in metastatic CRC patients. Furthermore, CYP24A1-mediated catabolism of 25-D3 to 24,25-D3 does not appear to be a major determinant of systemic 25-D3 levels. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 82.

Association between HIV replication and cholesterol in peripheral blood mononuclear cells in HIV-infected patients interrupting HAART

Cellular cholesterol is essential for HIV replication and may control HIV spread. HIV, in turn, appears to control cholesterol metabolism. To describe the relationships between serum lipids, cellular cholesterol and viral replication during highly active antiretroviral therapy (HAART) interruption. We have correlated virological parameters with the level of circulating lipids in serum and the content of cellular cholesterol in peripheral blood mononuclear cells (PBMCs). The study included 33 patients interrupting HAART with (n = 23) or without (n = 10) atorvastatin treatment. Atorvastatin treatment did not modify PBMC cholesterol levels at week 4 after HAART interruption, although it significantly reduced serum cholesterol (total and LDL, where LDL stands for low density lipoprotein) (P < 0.05). Serum cholesterol or LDL marginally influenced PBMC cholesterol since no significant correlations were found between these parameters either at 0 or 4 weeks after HAART interruption. Analysis of virological data in all patients revealed a negative trend (P = 0.07) between baseline PBMC cholesterol and absolute CD4 T cell counts at baseline but a poor correlation (P = 0.18) with the viral load (VL) at week 4. Separate analysis of control patients showed a correlation between baseline PBMC cholesterol and VL at week 4 (P = 0.01). However, atorvastatin treatment abrogated this correlation by increasing viral replication in individuals with low cellular cholesterol. Our data underscore the potential relevance of PBMC cholesterol in in vivo HIV replication and the complex effects of atorvastatin that seem to be unrelated to PBMC cholesterol.

Differential expression of toll‐like receptor mRNA in treatment non‐responders and sustained virologic responders at baseline in patients with chronic hepatitis C

The contribution of the host immune response to sustained virologic response is not clear in patients with chronic hepatitis C (CHC). The aim of this study was to explore the relationship of the toll-like receptor (TLR) expression with the outcome of antiviral therapy in hepatitis C viral infection. Peripheral blood mononuclear cells (PBMC) were obtained from 15 CHC patients before a 48-week treatment with pegylated interferon (PEG IFN) alpha-2a and ribavirin. A multiplex semi-quantitative reverse-trancriptase polymerase chain reaction (RT-PCR) was used to compare the relative abundance of TLR2-9 transcripts. mRNA levels of TLR2, 3 and 6 were significantly higher in CHC subjects compared with normal controls (n=8). When patients were classified into non-responders (n=8) and sustained virological responders (n=7) according to the virological outcome of the treatment, there was a clear difference in baseline mRNA expression of TLRs and T-helper (Th) 1/2 cytokines. In addition, the mRNA expression of IFN-gamma and nuclear factor of activated T cells (NFAT), which is exclusively expressed in activated T cells, was inversely correlated with that of TLR4, 6 and 9 in non-responders. TLRs mRNA levels are differentially expressed in baseline PBMC of chronic HCV-infected subjects with or without responsiveness to antiviral therapy.

T-Helper 2 and 3 type immunity to trophoblast in successful in vitro fertilization-embryo transfer

To determine whether peripheral blood mononuclear cell (PBMC) secretion of T-helper (Th)-1 type cytokines and Th-2 and Th-3 type cytokines in women undergoing in vitro fertilization-embryo transfer (IVF-ET) is associated with therapeutic failure and success, respectively. Cohort study. Academic medical center. One hundred one women undergoing IVF-ET and 19 fertile controls. Peripheral blood was obtained from women undergoing IVF-ET before oocyte retrieval and from 19 nonpregnant fertile controls. The PBMCs were cultured in the presence or absence of a protein extract from either a trophoblast cell line or sperm membrane. Supernatants from PBMC cultures were tested by enzyme-linked immunoabsorbent assay (ELISA) for the Th-1 type cytokines tumor necrosis factor-alpha (TNF)-alpha) and interferon-gamma (IFN)-gamma), the Th-2 type cytokines interleukin (IL)-6 and IL-10, and the Th-3 type cytokine transforming growth factor (TGF)-beta1. Levels of IL-6 and IL-10 were significantly higher in controls than in infertile women with endometriosis, and levels of IL-10 were higher in controls than in women with unexplained infertility. No differences were found in unstimulated levels of TNF-alpha, IFN-gamma, or TGF-beta1 between infertile patients and controls. In trophoblast-stimulated PBMC cultures, levels of TGF-beta1 were significantly lower in subjects who experienced failed compared with ongoing pregnancies. Baseline PBMC secretion of IL-6 and IL-10 is higher in fertile controls than in women with endometriosis, and IL-10 secretion is also higher than in women with unexplained infertility. Trophoblast-stimulated PBMC secretion of TGF-beta1 is positively associated with the establishment of successful pregnancy in women undergoing IVF-ET. Our study provides novel evidence to support a faciliatory role of Th-2 and Th-3 type responses to trophoblast in early pregnancy.

Proviral HIV-DNA predicts viral rebound and viral setpoint after structured treatment interruptions.

In HIV-1-infected patients with long-term undetectable viraemia on highly active antiretroviral treatment (HAART), we found that pre-HAART plasma viraemia and the baseline proviral DNA level were significantly associated with the viraemia setpoint during scheduled treatment interruptions. In long-term treated patients, pre-HAART viraemia may not be available, and in these circumstances proviral DNA, measured at the time of scheduled treatment interruption, can help to identify patients likely to reach a low viraemia setpoint after treatment interruption.

Sequential therapy with dacarbazine and carmustine: a phase I study

Depletion of the DNA-repair protein O6-alkylguanine-DNA alkyltransferase (AGT) increases the sensitivity of cells in culture and of human tumor xenografts to chloroethylnitrosoureas such as carmustine (BCNU). We have previously demonstrated that dacarbazine (DTIC) can deplete AGT activity in cells in culture and in human tumor xenografts. A phase I trial of DTIC followed immediately by BCNU was conducted to determine the DTIC dose resulting in maximal depletion of AGT in the peripheral blood mononuclear cells (PBMC) of cancer patients and to determine the maximally tolerated dose of DTIC given as a 4-h infusion immediately prior to a fixed dose of BCNU. A 4-h infusion of DTIC followed by a 2-h infusion of BCNU was given to 42 patients with refractory solid tumors. Complete depletion of AGT activity was not achieved at DTIC doses of up to 750 mg/m2. The dose-limiting toxicity was hematologic, although at higher doses of BCNU (> or = 100 mg/m2) we observed significant nonhematologic toxicity. Our recommended phase II doses are 1,000 mg/m2 DTIC followed by 75 mg/m2 BCNU. AGT activity in PBMC of the 28 patients studied decreased to a mean of 62% +/- 11% (SE) of the baseline value at 4 h after initiation of the DTIC infusion. At 24 h after initiation of the DTIC infusion, AGT activity in PBMC was depleted to a mean of 65% +/- 14% of the baseline value. There was no direct correlation between the DTIC dose and the extent of AGT depletion. Baseline PBMC AGT levels varied widely among patients.