Boonrat Tassaneetrithep

DC-SIGN (CD209) Mediates Dengue Virus Infection of Human Dendritic Cells

Dengue virus is a single-stranded, enveloped RNA virus that productively infects human dendritic cells (DCs) primarily at the immature stage of their differentiation. We now find that all four serotypes of dengue use DC-SIGN (CD209), a C-type lectin, to infect dendritic cells. THP-1 cells become susceptible to dengue infection after transfection of DC-specific ICAM-3 grabbing nonintegrin (DC-SIGN), or its homologue L-SIGN, whereas the infection of dendritic cells is blocked by anti–DC-SIGN antibodies and not by antibodies to other molecules on these cells. Viruses produced by dendritic cells are infectious for DC-SIGN– and L-SIGN–bearing THP-1 cells and other permissive cell lines. Therefore, DC-SIGN may be considered as a new target for designing therapies that block dengue infection.

Cell Type Specificity and Host Genetic Polymorphisms Influence Antibody-Dependent Enhancement of Dengue Virus Infection

ABSTRACT Antibody-dependent enhancement (ADE) is implicated in severe, usually secondary, dengue virus (DV) infections. Preexisting heterotypic antibodies, via their Fc-gamma receptor (FcγR) interactions, may increase disease severity through enhanced target cell infection. Greater numbers of infected target cells may contribute to higher viremia and excess cytokine levels often observed in severe disease. Monocytes, macrophages, and immature and mature dendritic cells (DC) are considered major cellular targets of DV. Apheresis of multiple donors allowed isolation of autologous primary myeloid target cell types for head-to-head comparison of infection rates, viral output, and cytokine production under direct infection (without antibody) or ADE conditions (with antibody). All studied cell types except immature DC supported ADE. All cells undergoing ADE secreted proinflammatory cytokines (interleukin-6 [IL-6] and tumor necrosis factor alpha [TNF-α]) at enhancement titers, but distinct cell-type-specific patterns were observed for other relevant proteins (alpha/beta interferon [IFN-α/β] and IL-10). Macrophages produced type I interferons (IFN-α/β) that were modulated by ADE. Mature DC mainly secreted IFN-β. Interestingly, only monocytes secreted IL-10, and only upon antibody-enhanced infection. While ADE infection rates were remarkably consistent in monocytes (10 to 15%) across donors, IL-10 protein levels varied according to previously described regulatory single nucleotide polymorphisms (SNPs) in the IL-10 promoter region. The homozygous GCC haplotype was associated with high-level IL-10 secretion, while the ACC and ATA haplotypes produced intermediate and low levels of IL-10, respectively. Our data suggest that ADE effects are cell type specific, are influenced by host genetics, and, depending on relative infection rates, may further contribute to the complexity of DV pathogenesis.

Performance evaluation of the Alere PIMA CD4 test for monitoring HIV-infected individuals in resource-constrained settings.

Background: Enumeration of CD4+ T-lymphocytes is important in the management of HIV. However, standard laboratory systems based on flow cytometry are expensive, complicated, and thus unavailable to most resource-limited settings where a low-cost and fully automated point-of-care CD4 testing system is required. In attempts to address this issue, a study was conducted to validate the Alere PIMA point-of-care CD4 test. Method: Duplicate values of the absolute number of CD4+ T-lymphocytes in 203 HIV-infected blood samples obtained using the PIMA system were compared with the two predicate single-platform FACSCount and the dual-platform FACSCan (Becton Dickinson Biosciences). Results: The overall absolute CD4+ T-lymphocyte count obtained using the PIMA system correlated highly with the FACSCount (r2 = 0.957; mean bias, -54.2 cells/μL; limit of agreement, -190.9 to +82.5 cells/μL) and the FACSCan (r2 = 0.957; mean bias -44.0 cells/μL; limit of agreement, -179.7 to +91.6 cells/μL). Good correlation and low biases were also observed for samples with CD4+ T-lymphocyte count ranges of 0 to 200 and 0 to 350 cells/μL. Additionally, there was no significant difference in absolute CD4+ T-lymphocyte counts noted between the duplicate samples using the PIMA system. Conclusions: This new point-of-care product is a simple and reliable system and should contribute significantly to the simplification of performing CD4 testing and thus increase access for patients in resource-limited settings. The inability to obtain values for the frequency (%) of CD4+ T-lymphocyte count is one limitation of the PIMA system, the addition of which would be of value for clinical staging or monitoring in HIV-infected pediatric patients.

Comparison of 5 Flow Cytometric Immunophenotyping Systems for Absolute CD4+ T-Lymphocyte Counts in HIV-1-Infected Patients Living in Resource-Limited Settings

Enumeration of CD4+ T lymphocytes is important in management of HIV-infected patients. However, CD4 testing by current gold standard bead-based flow cytometer (FCM) system is expensive for developing countries. This study compared 2 affordable volumetric FCMs with the 3 predicate FCM systems. CD4+ T-lymphocyte counts on blood samples from 150 HIV-1-infected Thai patients were determined in parallel by 5 FCM systems: the 2 single-platform volumetric FCM systems, Guava and CyFlowgreen; the 2 standard single-platform bead-based systems (2-color FACSCount and the TriTEST/TruCOUNT tube using a FACSCalibur FCM); and the dual-platform TriTEST system. Correlation and agreement were analyzed using linear regression and Bland-Altman analysis. Results from these 2 volumetric systems gave similar results and excellent correlation: R2 > 0.93; mean biases ranged from +6.3 to +24.1 cells per microliter more for the Guava. In contrast, the CyFlowgreen showed the lowest values with R2 > 0.97; mean biases ranged from −9.8 to −27.6 cells per microliter. This indicates that the absolute CD4+ T-lymphocyte counts determined by CyFlowgreen are <FACSCount<DP TriTEST<TriTEST/TruCOUNT<Guava. Although the use of these 2 volumetric FCMs could make CD4+ T-lymphocyte enumeration more affordable in resource-poor settings, variations among these systems should be considered if these are to be interchanged.

Effects of cytochrome P450 2C19 and paraoxonase 1 polymorphisms on antiplatelet response to clopidogrel therapy in patients with coronary artery disease.

Clopidogrel is an antiplatelet prodrug that is recommended to reduce the risk of recurrent thrombosis in coronary artery disease (CAD) patients. Paraoxonase 1 (PON1) is suggested to be a rate-limiting enzyme in the conversion of 2-oxo-clopidogrel to active thiol metabolite with inconsistent results. Here, we sought to determine the associations of CYP2C19 and PON1 gene polymorphisms with clopidogrel response and their role in ADP-induced platelet aggregation. Clopidogrel response and platelet aggregation were determined using Multiplate aggregometer in 211 patients with established CAD who received 75 mg clopidogrel and 75–325 mg aspirin daily for at least 14 days. Polymorphisms in CYP2C19 and PON1 were genotyped and tested for association with clopidogrel resistance. Linkage disequilibrium (LD) and their epistatic interaction effects on ADP-induced platelet aggregation were analysed. The prevalence of clopidogrel resistance in this population was approximately 33.2% (n = 70). The frequencies of CYP2C19*2 and *3 were significantly higher in non-responder than those in responders. After adjusting for established risk factors, CYP2C19*2 and *3 alleles independently increased the risk of clopidogrel resistance with adjusted ORs 2.94 (95%CI, 1.65–5.26; p<0.001) and 11.26 (95%CI, 2.47–51.41; p = 0.002, respectively). Patients with *2 or *3 allele and combined with smoking, diabetes and increased platelet count had markedly increased risk of clopidogrel resistance. No association was observed between PON1 Q192R and clopidogrel resistance (adjusted OR = 1.13, 95%CI, 0.70–1.82; p = 0.622). Significantly higher platelet aggregation values were found in CYP2C19*2 and *3 patients when compared with *1/*1 allele carriers (p = 1.98×10−6). For PON1 Q192R genotypes, aggregation values were similar across all genotype groups (p = 0.359). There was no evidence of gene-gene interaction or LD between CYP2C19 and PON1 polymorphisms on ADP-induced platelet aggregation. Our findings indicated that only CYP2C19*2 and *3 alleles had an influence on clopidogrel resistance. The risk of clopidogrel resistance increased further with smoking, diabetes, and increased platelet count.

The Use of Glutaraldehyde-Fixed Chicken Red Blood Cells as Counting Beads for Performing Affordable Single-Platform CD4+ T-Lymphocyte Count in HIV-1-Infected Patients

CD4+ T-lymphocyte count is an important marker in management of HIV-1-infected patients. The standard single-platform (SP) flow cytometric (FCM) CD4+ testing that uses the known reference microbeads is expensive; more affordable alternatives are therefore needed. We evaluated the use of glutaraldehyde-fixed chicken red blood cells (CRBCs) as counting beads as an alternative for enumerating CD4+ T-lymphocyte counts in 87 HIV-1-infected patients. Linear regression analyses revealed an excellent correlation of the SP FCM using CRBCs with the standard SP bead-based FCM method (percentages, r2 > 0.99; absolute counts, r2 > 0.98) over the entire range including the clinically relevant range. Mean percent bias for the CRBC method was +0.35% [limits of agreement (LOA): −1.86% to +2.57%]. For absolute CD4+ T-lymphocytes, the mean biases was −47.76 cells per microliter (LOA: −191.34 to +98.81 cells/μL) with much lower bias for CD4+ T-lymphocyte counts <200 cells per microliter (LOA: −31.92 to +22.95 cells/μL). The use of CRBCs is comparable with the use of commercial microbeads. This has resulted in major cost savings to resource-limited countries where the health care system is under increasing pressure to operate cost effectively. This can greatly facilitate and ensure the success of the ongoing antiretroviral therapy program in these countries.

Comparison of phi29-based whole genome amplification and whole transcriptome amplification in dengue virus

Dengue virus is responsible for 50-100 million new infections annually worldwide. The virus uses error-prone RNA polymerase during genome replication in a host, resulting in the formation of closely related viruses known as quasispecies. The availability of next-generation sequencing technology provides opportunities to analyze viral quasispecies. Before analysis, it is crucial to increase the amount of DNA because of the limited amounts of viral genomic material that can be isolated from a patient. However, using specific primers may overlook the occurrence of possible variations at primer binding sites. To address this problem, the performance of two sequence-independent amplification methods was compared for whole genome amplification (WGA): phi29 DNA polymerase-based WGA and whole transcriptome amplification (WTA). Both methods have the ability to provide complete coverage of the dengue genome from template amounts as low as 1 ng. However, WTA showed greater efficiency in terms of yield (WTA: ~10 μg; phi29-based WGA: ~500 ng) and lower amplification bias. In conclusion, the WTA amplification kit was shown to perform substantially better than phi29 DNA polymerase-based WGA in terms of both final concentration and amplification bias in amplifying small genomes, such as that of the dengue virus.

Expansion of Inefficient HIV-Specific CD8 T Cells during Acute Infection

ABSTRACT Attrition within the CD4+ T cell compartment, high viremia, and a cytokine storm characterize the early days after HIV infection. When the first emerging HIV-specific CD8+ T cell responses gain control over viral replication it is incomplete, and clearance of HIV infection is not achieved even in the rare cases of individuals who spontaneously control viral replication to nearly immeasurably low levels. Thus, despite their partial ability to control viremia, HIV-specific CD8+ T cell responses are insufficient to clear HIV infection. Studying individuals in the first few days of acute HIV infection, we detected the emergence of a unique population of CD38+ CD27− CD8+ T cells characterized by the low expression of the CD8 receptor (CD8dim). Interestingly, while high frequencies of HIV-specific CD8+ T cell responses occur within the CD38+ CD27− CD8dim T cell population, the minority populations of CD8bright T cells are significantly more effective in inhibiting HIV replication. Furthermore, the frequency of CD8dim T cells directly correlates with viral load and clinical predictors of more rapid disease progression. We found that a canonical burst of proliferative cytokines coincides with the emergence of CD8dim T cells, and the size of this population inversely correlates with the acute loss of CD4+ T cells. These data indicate, for the first time, that early CD4+ T cell loss coincides with the expansion of a functionally impaired HIV-specific CD8dim T cell population less efficient in controlling HIV viremia. IMPORTANCE A distinct population of activated CD8+ T cells appears during acute HIV infection with diminished capacity to inhibit HIV replication and is predictive of viral set point, offering the first immunologic evidence of CD8+ T cell dysfunction during acute infection.

Early immune events during acute HIV infection

Background We characterized plasma cytokine and chemokine profiles at baseline and during acute infection in a prospective high-risk cohort (RV217). PBMC collected in parallel allow for the longitudinal analysis of changes in immune cell populations during acute infection. These data can provide insight into the earliest events in host-HIV interaction and inform HIV vaccine development. Methods Semiweekly viral load (VL) testing in individuals at highrisk for HIV acquisition was performed to prospectively identify very early acute HIV infections (Fiebig stage I/II, NAT+/Ab-). Cytokines were assayed in plasma from preinfection through early plasma viral load set point using the Q-Plex Multiplex Array or by traditional ELISA. Peripheral blood cells were longitudinally analyzed with multiparameter flow cytometry. Results African participants were all female and acquired subtype A, C and D recombinant HIV while participants in Thailand were male and acquired HIV subtype E predominantly. Longitudinal plasma samples from acutely infected individuals (n=24) showed similar trends in cytokine profiles with statistically significant increases in expression over time. VL setpoint, IL-10 and MCP-1 expression differed by region. Preliminary PBMC analysis revealed frequency and phenotypic changes in all antigen presenting cell populations. Significant decreases in plasmacytoid dendritic cells were observed at peak VL, which preceded a sharp decline in plasma IFN-a to near baseline values. MCP-1 and IP-10 also rose sharply in conjunction with fluctuations in T cell subsets and APCs. These early changes could impact adaptive cellular and humoral immune responses. Conclusion Understanding cytokine kinetics and VL dynamics during acute HIV infection may help identify key controls of early viral set point. Regional differences may reflect HIV subtype, gender, background cytokine expression levels and co-morbidities as these covaried. Analyses of correlations with immune cell phenotype/function and viral set point are underway. Efforts focus on examination of the evolution of the innate response after infection, prior to peak viremia.