Soluble Interleukin-7 receptor levels and risk of acute graft-versus-host disease after allogeneic haematopoietic stem cell transplantation.

Interleukin-7 is a cytokine essential for T cell homeostasis. IL-7 binds to cellular IL-7 receptors in competition with a soluble form of the receptor (sIL-7Rα). We hypothesized that altered sIL-7Rα levels may cause adverse outcomes in patients undergoing HSCT. In parallel, we investigated the impact of the IL-7Rα SNP rs6897932, which has been associated with release of IL-7R. The sIL-7Rα levels decreased during HSCT (from 114ng/ml before to 48ng/ml at day +14 (P<0.0001)). This pattern was inversely mirrored by IL-7. The IL-7/sIL-7Rα ratio at day +14 was significantly higher in patients developing grades II-IV aGVHD (OR=4.3, P=0.026). Furthermore, donor carriage of the rs6897932 T allele was associated with reduced sIL-7Rα levels, increased risk of grades II-IV aGVHD (OR=2.4, P=0.055) and increased transplant-related mortality (CC=4.5%, CT=21.4% and TT=27.3%, P=0.0037). In conclusion, this study suggests an impact of sIL-7Rα levels and rs6897932 donor genotype on alloreactivity and outcome after HSCT.

Cytotoxicity assessment, inflammatory properties, and cellular uptake of Neutraplex lipid-based nanoparticles in THP-1 monocyte-derived macrophages.

Current antiretroviral drugs used to prevent or treat human immunodeficiency virus type 1 (HIV-1) infection are not able to eliminate the virus within tissues or cells where HIV establishes reservoirs. Hence, there is an urgent need to develop targeted delivery systems to enhance drug concentrations in these viral sanctuary sites. Macrophages are key players in HIV infection and contribute significantly to the cellular reservoirs of HIV because the virus can survive for prolonged periods in these cells. In the present work, we investigated the potential of the lipid-based Neutraplex nanosystem to deliver anti-HIV therapeutics in human macrophages using the human monocyte/macrophage cell line THP-1. Neutraplex nanoparticles as well as cationic and anionic Neutraplex nanolipoplexes (Neutraplex/small interfering RNA) were prepared and characterized by dynamic light scattering. Neutraplex nanoparticles showed low cytotoxicity in CellTiter-Blue reduction and lactate dehydrogenase release assays and were not found to have pro-inflammatory effects. In addition, confocal studies showed that the Neutraplex nanoparticles and nanolipoplexes are rapidly internalized into THP-1 macrophages and that they can escape the late endosome/lysosome compartment allowing the delivery of small interfering RNAs in the cytoplasm. Furthermore, HIV replication was inhibited in the in vitro TZM-bl infectivity assay when small interfering RNAs targeting CXCR4 co-receptor was delivered by Neutraplex nanoparticles compared to a random small interfering RNA sequence. This study demonstrates that the Neutraplex nanosystem has potential for further development as a delivery strategy to efficiently and safely enhance the transport of therapeutic molecules into human monocyte-derived macrophages in the aim of targeting HIV-1 in this cellular reservoir.

Cardio-Metabolic Disease Risks and Their Associations with Circulating 25-Hydroxyvitamin D and Omega-3 Levels in South Asian and White Canadians.

OBJECTIVES: This study compared cardio-metabolic disease risk factors and their associations with serum vitamin D and omega-3 status in South Asian (SAC) and White Canadians (WC) living in Canada's capital region. METHODS: Fasting blood samples were taken from 235 SAC and 279 WC aged 20 to 79 years in Ottawa, and 22 risk factors were measured. RESULTS: SAC men and women had significantly higher fasting glucose, insulin, homeostasis model assessment for insulin resistance (HOMA-IR), apolipoprotein B (ApoB), ratios of total (TC) to HDL cholesterol (HDLC) and ApoB to ApoA1, leptin, E-selectin, P-selectin, ICAM-1 and omega-3 (p < 0.05), but lower HDLC, ApoA1, vitamin D levels than WC (p < 0.05). SAC women had higher CRP and VEGF than WC women. Adequate (50-74.9 nmol/L) or optimal (≥ 75 nmol/L) levels of 25(OH)D were associated with lower BMI, glucose, insulin, HOMA-IR, TG, TC, low density lipoprotein cholesterol (LDLC), ApoB/ApoA1 ratio, CRP, leptin, and higher HDLC, ApoA1, omega-3 index, L-selectin levels in WC, but not in SAC. Intermediate (>4%-<8%) or high (≥ 8%) levels of omega-3 indices were related to lower E-selectin, P-selectin, ICAM-1 and higher HDLC, 25(OH)D levels in WC, but not in SAC. The BMIs of ≤ 25 kg/m2 were related to lower LDLC, ApoB, VEGF, creatinine and higher 25(OH)D in WC, but not in SAC. CONCLUSIONS: The associations of vitamin D, omega-3 status, BMI and risk factors were more profound in the WC than SAC. Compared to WC, vitamin D status and omega-3 index may not be good predictive risk factors for the prevalence of CVD and diabetes in SAC.

Soluble IL-7R alpha (sCD127) inhibits IL-7 activity and is increased in HIV infection.

Soluble CD127 (sCD127) appears to play an important role in the immunopathogenesis of several chronic infections, multiple sclerosis, and various cancers. The function of sCD127 and whether it influences IL-7 bioavailability or activity is unknown. In this study, we demonstrated that recombinant and native sources of sCD127 significantly inhibited IL-7-mediated STAT5 and Akt phosphorylation in CD8(+) T cells. IL-7-mediated proliferation and Bcl-2 expression were similarly reduced by sCD127. In each case, native sCD127 inhibited IL-7 activity to a greater degree than rsCD127. Anti-IL-7 activity was inherent to human plasma and could be reversed by depletion of CD127, revealing for the first time the biological activity of naturally occurring sCD127. Plasma sCD127 concentrations were increased in HIV(+) individuals compared with HIV(-) controls, correlated with IL-7 levels, and remained unchanged in HIV(+) individuals following 1 y of effective antiretroviral therapy. Determining the regulation and function of sCD127 may be critical for understanding both the pathogenesis of diseases in which IL-7 likely has a role (e.g., HIV infection, cancer) and its potential impact on IL-7 as a therapeutic approach.

QASI, an international quality management system for CD4 T-cell enumeration focused to make a global difference.

BACKGROUND: A significant worldwide mobilization effort to treat people with HIV disease began in 2003. Most guidelines for initiating antiretroviral therapy require reliable and reproducible CD4 T-cell counting. Therefore, any effort that improves global availability of quality managed assessment schemes for CD4 T-cell enumeration is a positive achievement for the clinical management of AIDS on a worldwide scale. METHODS: The Canadian QASI-Quality Management System (QMS) has been in operation for over a decade. More recently, QMS has fine-tuned its strategy to optimize its global impact in the fight against the HIV/AIDS pandemic. Three modifications were implemented: (1) introduction of skills and knowledge transfer workshops pertaining to the initiation of national quality management programs for CD4 counting, (2) introduction of a road map to establish domestic EQAP for countries that are ready, and (3) introduction of a statistical analysis package which permits continuous monitoring of global impact of the QASI-QMS. RESULTS: Based on QASI-QMS distribution of specimens over four consecutive participation cycles, there was decreased interlaboratory variation for both low and medium CD4 T-cell levels. After three cycles of consecutive participation, there is an average of 38 and 26% error reduction reported for the mid and low CD4 levels, respectively. CONCLUSION: The program improvements mentioned earlier appear to have had a profound effect with regard to enhancing the performance of laboratories participating in the QASI-QMS. Specifically, there is a significant reduction in interlaboratory variability of CD4 T-cell counts resulting from continuous participation in the QASI-QMS.

Development of a quantitative bead capture assay for soluble IL-7 receptor alpha in human plasma.

BACKGROUND: IL-7 is an essential cytokine in T-cell development and homeostasis. It binds to the IL-7R receptor, a complex of the IL-7Ralpha (CD127) and common gamma (CD132) chains. There is significant interest in evaluating the expression of CD127 on human T-cells as it often decreased in medical conditions leading to lymphopenia. Previous reports showed the usefulness of CD127 as a prognostic marker in viral infections such as HIV, CMV, EBV and HCV. A soluble CD127 (sCD127) is released in plasma and may contribute to disease pathogenesis through its control on IL-7 activities. Measuring sCD127 is important to define its role and may complement existing markers used in lymphopenic disease management. We describe a new quantitative assay for the measurement of sCD127 in plasma and report sCD127 concentrations in healthy adults. METHODOLOGY/PRINCIPAL FINDINGS: We developed a quantitative bead-based sCD127 capture assay. Polyclonal CD127-specific antibodies were chosen for capture and a biotinylated monoclonal anti-CD127 antibody was selected for detection. The assay can detect native sCD127 and recombinant sCD127 which served as the calibrator. The analytical performance of the assay was characterized and the concentration and stability of plasma sCD127 in healthy adults was determined. The assay's range was 3.2-1000 ng/mL. The concentration of plasma sCD127 was 164+/-104 ng/mL with over a log variation between subjects. Individual sCD127 concentrations remained stable when measured serially during a period of up to one year. CONCLUSIONS/SIGNIFICANCE: This is the first report on the quantification of plasma sCD127 in a population of healthy adults. Soluble CD127 plasma concentrations remained stable over time in a given individual and sCD127 immunoreactivity was resistant to repeated freeze-thaw cycles. This quantitative sCD127 assay is a valuable tool for defining the potential role of sCD127 in lymphopenic diseases.

Involvement of interferon-gamma in microglial-mediated loss of dopaminergic neurons.

Growing evidence implicates microglia in the loss of dopaminergic neurons in Parkinson's disease (PD). However, factors mediating microglial activation in PD are poorly understood. Proinflammatory cytokines, such as interferon-gamma (IFN-gamma), orchestrate the actions of microglia. We report here that PD patients express significantly elevated levels of IFN-gamma in their blood plasma. After this initial finding, we found that IFN-gamma-deficient mice displayed attenuated 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced substantia nigra pars compacta dopaminergic cell loss along with reduced loss of striatal tyrosine hydroxylase and dopamine transporter fiber density. MPTP-induced depletion of striatal dopamine and its metabolite DOPAC (3,4-dihydroxyphenylacetic acid), as well as deltaFosB, a marker of postsynaptic dysfunction, were also attenuated in these knock-out mice. Consistent with the role for IFN-gamma in microglial activation, MPTP-induced morphological activation of microglia was abrogated compared with wild-type mice. To examine more mechanistically the role of IFN-gamma in microglial activation, we evaluated the interactions between microglia and dopaminergic neurons in an in vitro mixed microglia/midbrain neuron rotenone-induced death paradigm. In this in vitro paradigm, dopaminergic neurons are selectively damaged by rotenone. Exogenous IFN-gamma ligand alone and without rotenone resulted in dopaminergic cell loss, but only in the presence of microglia. The addition of an IFN-gamma neutralizing antibody attenuated neuronal loss as a result of rotenone treatment. The presence of only wild-type microglia and not those deficient in IFN-gamma receptor elicited significant dopaminergic cell loss when exposed to rotenone. Neurons deficient in IFN-gamma receptor, however, did not display increased resistance to death. Finally, levels of IFN-gamma message increased in microglia in response to rotenone. Together, these data suggest that IFN-gamma participates in death of dopaminergic neurons by regulating microglial activity.

A combined HIV-1 protein bead array for serology assay and T-cell subset immunophenotyping with a hybrid flow cytometer: a step in the direction of a comprehensive multitasking instrument platform for infectious disease diagnosis and monitoring.

BACKGROUND: A new generation of bench-top flow cytometers with digital signal processing to perform suspension array technology (SAT) based bead array assays as well as leukocyte immunophenotyping is now available. These hybrid instruments provide an opportunity for the development of a more cost effective multitasking platform to support infectious disease treatment in resource limited countries. METHODS: We report the development and testing of two modules compatible with the hybrid flow cytometers. The first module is an eleven HIV-1 protein bead array (PBA) for the detection of circulating antibodies and the second is a cell based T-cell enumeration assay. RESULTS: The HIV-1 PBA was tested in parallel with two enzyme immunoassays (EIAs) for the detection of plasma antibodies from 4 HIV-1 seroconversion panels and a low antibody titer panel. The PBA as well as the two EIAs performed equally for the detection of antibody positive samples from all seroconversion panels. One antibody positive sample from the low antibody titer panel was missed by the PBA together with one of the two EIAs tested. A parallel analysis of the HIV-1 PBA with Western blot (a confirmatory test for HIV infection) using plasma from nine HIV-1(+) individuals showed that the HIV-1 PBA detected more of the gp41 and gp120 antibody positive samples. Preliminary CD4 T-cell immunophenotyping results from 14 HIV(+) and 10 HIV(-) whole blood specimens with the hybrid flow cytometer platform compared well to conventional flow cytometry data. CONCLUSION: The successful combination of bead and cell based assays on a single hybrid instrument demonstrated the potential utility of a multitasking platform. The results presented are providing groundwork for future development of more cost effective modular architecture for a flexible flow cytometry based platform.

Characterization of the Cyno-EBV LMP1 homologue and comparison with LMP1s of EBV and other EBV-like viruses.

EBV latent membrane protein 1 (LMP1) is essential for EBV-mediated transformation and has been associated with several cases of malignancies. EBV-like viruses in Cynomolgus monkeys (Macaca fascicularis) have been associated with high lymphoma rates in immunosuppressed monkeys. In the study, the entire coding region of the Cyno-EBV LMP1 gene was cloned, sequenced and expressed in human embryonic kidney (HEK) cells 293. The Cyno-EBV LMP1 homologue sequence predicted a 588 amino acid (a.a.) protein with a short 19 a.a. N-terminus, six transmembrane domains and a long carboxy tail of 404 a.a. The protein contained a series of seven 9 a.a.-tandem repeats and two 20 a.a.-repeats, which harbored two potential TRAF binding motifs, PxQxT/S. These repeats shared no homology with the repeats in any other LMP1. However, the proline-rich sequence GPxxPx(6) found within the 11 a.a.-repeats of EBV LMP1 was conserved in Cyno-EBV carboxy tail and contained two consensus JAK/STAT sequences PxxPxP. A cluster of eight histidine residues was found in proximity to the last transmembrane domain of Cyno-EBV LMP1 and was exploited as a natural protein tag in expression studies. Western blot analysis revealed a major polypeptide of 110 kDa. Comparative functional studies showed that Cyno-EBV LMP1 expressed in HEK 293 cells shares the same ability as EBV LMP1 to induce NFkappaB driven CAT activity.

Evaluation of a universal template for single-platform absolute T-lymphocyte subset enumeration.

The single-platform absolute T-lymphocyte subset analysis was evaluated utilizing a universal protocol in a Canadian multicenter study with the collaboration of the members of the Canadian HIV Trials Network (CTN). Participants used flow cytometers and reagents of their choice for labeling and lysing whole blood. Over a 2-year period, CTN laboratories performed single-platform absolute T-lymphocyte subset enumerations on fresh and commercial stabilized blood products using commercially available microfluorospheres TruCount and Flow-Count. This multicenter evaluation demonstrated that the application of a universal template for single-platform analysis provides a generic approach that embraces a wide array of immunophenotyping settings available in clinical laboratory.