Affordable flow cytometry for enumeration of absolute CD4(+ )T-lymphocytes to identify subtype C HIV-1 infected adults requiring antiretroviral therapy (ART) and monitoring response to ART in a resource-limited setting

BACKGROUND: The World Health Organization (WHO)'s "3 × 5 program" has spurred efforts to place 3 million people on combination antiretroviral therapy (ART) for treatment of AIDS in resource-limited countries. Paradoxically, the cost of CD4(+ )T-lymphocyte count essential for decision-making to commence HIV positive adults on ART as well as for monitoring responses to ART remains unaffordable in most resource-limited countries. Thus, low-cost methods for enumerating CD4(+ )T-lymphocyte are urgently needed. OBJECTIVE: To evaluate Cyflow cytometry (Cyflow SL, Partec, Munster, Germany) for enumeration of absolute CD4(+ )T-lymphocyte in subtype C HIV-1 seropositive subjects using FACSCount (Becton and Dickinson, Immunocytometry Systems, San Jose, CA, USA) as the "predicate method". METHODS: A total of 150 HIV-1 seropositive subjects were included in the evaluation exercise. Fifty-eight specimens were collected from pregnant HIV-1 seropositive women (subtype C drug resistance study). Twenty-seven specimens were collected from women and their spouses with AIDS followed in a Duke ART study to assess the immunologic and virologic responses to generic ART, comprising Stavudine, Lamivudine and Nevirapine (Stalanev, Varichem Labs, Harare, Zimbabwe). Sixty-five specimens were collected from AIDS patients enrolled in an ongoing Kaposi Sarcoma (KS) study to investigate impact of ART on KS progression. Enumeration of CD4(+ )T-lymphocytes using FACSCount is routinely conducted for all the three studies. The Medical Research Council of Zimbabwe and Medicines Control Authority of Zimbabwe approved the studies. Whole blood was collected in EDTA vacutainer tubes and aliquoted into two tubes (200 μL in each). CD4(+ )T-lymphocyte counts were enumerated using a Cyflow counter, in the Department of Immunology and a FACSCount in the Department of Obstetrics and Gynaecology within 6 hours of phlebotomy following manufacturers' instructions. RESULTS: Using linear regression analysis, there was a very strong correlation (R = 0.991) between the overall CD4(+ )T-lymphocyte counts obtained by FACSCount and those obtained by Cyflow. When data analysis was stratified by study groups, there was a strong correlation between the FACSCount and Cyflow CD4(+ )T-lymphocyte counts from subjects in the three independent studies; Subtype C resistance (R(2 )= 0.987), Duke ART (R(2 )= 0.980) and KS (R(2 )= 0.994), Table 1. Using Bland-Altman plots, the overall, absolute CD4(+ )T lymphocytes obtained by the two methods were in excellent agreement (mean difference 1.21, 95% Confidence Interval {CI): -2.1 to 3.3). For the 0–250 CD4(+ )T-lymphocytes range, the CD4 counts obtained using FACSCount were also in good agreement with those obtained using Cyflow counter (mean difference = 2.6 cells/μL, 95% CI: -1.1 to 6.3). Similarly, in the 251–500 (mean difference 1.0, cells/μL, 95% CI: -3.7 to 5.6) and the 501–1200 (mean difference = 0.29 cells/μL, 95% CI: -8.1 to 8.7) CD4 T-lymphocytes range, good agreement was observed. CONCLUSION: The Cyflow counter is as accurate as the FACSCount in enumerating absolute CD4(+ )T-lymphocytes in the range 1–1200 cells/μL. Cyflow cytometry is relatively affordable, easy to use technology that is useful not only in identifying HIV seropositive individuals who require ART but also for monitoring immunologic responses to ART

Circulating Endothelial Progenitor Cells in Kidney Transplant Patients

Background: Kidney transplantation (RTx) leads to amelioration of endothelial function in patients with advanced renal failure. Endothelial progenitor cells (EPCs) may play a key role in this repair process. The aim of this study was to determine the impact of RTx and immunosuppressive therapy on the number of circulating EPCs. Methods: We analyzed 52 RTx patients (58613 years; 33 males, mean 6 SD) and 16 age- and gender-matched subjects with normal kidney function (57617; 10 males). RTx patients received a calcineurin inhibitor (CNI)-based (65%) or a CNI-free therapy (35%) and steroids. EPC number was determined by double positive staining for CD133/VEGFR2 and CD34/VEGFR2 by flow cytometry. Stromal cell-derived factor 1 alpha (SDF-1) levels were assessed by ELISA. Experimentally, to dissociate the impact of RTx from the impact of immunosuppressants, we used the 5/6 nephrectomy model. The animals were treated with a CNI-based or a CNI-free therapy, and EPCs (Sca+cKit+) and CD26+ cells were determined by flow cytometry. Results: Compared to controls, circulating number of CD34+/VEGFR2+ and CD133+/VEGFR2+ EPCs increased in RTx patients. There were no correlations between EPC levels and statin, erythropoietin or use of renin angiotensin system blockers in our study. Indeed, multivariate analysis showed that SDF-1 – a cytokine responsible for EPC mobilization – is independently associated with the EPC number. 5/6 rats presented decreased EPC counts in comparison to control animals. Immunosuppressive therapy was able to restore normal EPC values in 5/6 rats. These effects on EPC number were associated with reduced number of CD26+ cells, which might be related to consequent accumulation of SDF-1. Conclusions: We conclude that kidney transplantation and its associated use of immunosuppressive drugs increases the number of circulating EPCs via the manipulation of the CD26/SDF-1 axis. Increased EPC count may be associated to endothelial repair and function in these patients.

A supermatrix analysis of genomic, morphological, and paleontological data from crown Cetacea

<p>Abstract</p> <p>Background</p> <p>Cetacea (dolphins, porpoises, and whales) is a clade of aquatic species that includes the most massive, deepest diving, and largest brained mammals. Understanding the temporal pattern of diversification in the group as well as the evolution of cetacean anatomy and behavior requires a robust and well-resolved phylogenetic hypothesis. Although a large body of molecular data has accumulated over the past 20 years, DNA sequences of cetaceans have not been directly integrated with the rich, cetacean fossil record to reconcile discrepancies among molecular and morphological characters.</p> <p>Results</p> <p>We combined new nuclear DNA sequences, including segments of six genes (~2800 basepairs) from the functionally extinct Yangtze River dolphin, with an expanded morphological matrix and published genomic data. Diverse analyses of these data resolved the relationships of 74 taxa that represent all extant families and 11 extinct families of Cetacea. The resulting supermatrix (61,155 characters) and its sub-partitions were analyzed using parsimony methods. Bayesian and maximum likelihood (ML) searches were conducted on the molecular partition, and a molecular scaffold obtained from these searches was used to constrain a parsimony search of the morphological partition. Based on analysis of the supermatrix and model-based analyses of the molecular partition, we found overwhelming support for 15 extant clades. When extinct taxa are included, we recovered trees that are significantly correlated with the fossil record. These trees were used to reconstruct the timing of cetacean diversification and the evolution of characters shared by "river dolphins," a non-monophyletic set of species according to all of our phylogenetic analyses.</p> <p>Conclusions</p> <p>The parsimony analysis of the supermatrix and the analysis of morphology constrained to fit the ML/Bayesian molecular tree yielded broadly congruent phylogenetic hypotheses. In trees from both analyses, all Oligocene taxa included in our study fell outside crown Mysticeti and crown Odontoceti, suggesting that these two clades radiated in the late Oligocene or later, contra some recent molecular clock studies. Our trees also imply that many character states shared by river dolphins evolved in their oceanic ancestors, contradicting the hypothesis that these characters are convergent adaptations to fluvial habitats.</p

The Isotype of Autoantibodies Influences the Phagocytosis of Antibody-Coated Platelets in Autoimmune Thrombocytopenic Purpura

Autoimmune thrombocytopenic purpura (AITP) is an acquired autoimmune bleeding disorder, characterized by isolated thrombocytopenia because of destruction of auto-antibody-coated platelets by Fc-receptor-mediated phagocytosis. The destruction of autoantibody-sensitized platelets by Fc gamma R-bearing phagocytic cells and the following antigen presentation are considered to play a key role for the pathophysiology of AITP. Although different isotypes of AITP-mediating autoantibodies, e. g. IgG, IgM and IgA, are frequently found in AITP patients, their role in the pathophysiology of AITP remains unclear. Using a flow cytometric monocyte-based phagocytosis assay, we investigated the impact of disease-associated autoantibody isotype in antibody-mediated phagocytosis of platelets. Platelets, labelled with 5-chloromethyl fluorescein diacetate (CMFDA), were incubated with AITP patients' serum characterized by pure IgG or IgM antiplatelet autoantibodies. Labelled platelets were incubated with monocytes. Phagocytosis was defined as the product of percentage of CMFDA-positive monocytes and mean fluorescence intensity of CMFDA. Adherence of platelets to monocytes was quantified by anti-CD61-PerCp in a CMFDA(+) CD14(+) gate. IgG-coated platelets showed a significantly higher phagocytic index than IgM-coated platelets (mean 796 +/- 157 versus 539 +/- 78, P < 0.01). There were no significant differences regarding platelet adherence to monocytes. The isotype of autoantibodies influences the quantity of in vitro phagocytosis of autologous platelets by monocytes. Therefore, the AITP-mediating autoantibody isotype should be considered more carefully in pathophysiologic models and furthermore in diagnostic, therapeutic and prognostic approaches in AITP