RESUMO
Red blood cells (RBCs) infected with Plasmodium falciparum are protected from complement-mediated lysis by surface membrane glycosyl-phosphatidylinositol (GPI)-anchored proteins, which include decay accelerating factor (DAF or CD55) and CD59. To determine if P. falciparum avoids or replicates less efficiently in GPI protein-deficient cells at a higher risk for complement-mediated lysis, we compared P. falciparum infectivity among control RBCs with those from subjects with paroxysmal nocturnal hemoglobinuria (PNH), a condition in which RBCs express variable levels of DAF (negative and positive) and CD59 (negative [-], intermediate [I], and high [H]). Co-cultures of 19 matched samples of control and PNH RBCs were infected with P. falciparum to directly compare parasitic invasion. Each PNH RBC sample was then assessed for P. falciparum infectivity across the spectrum of GPI protein deficiency. Identification methods included biotin-streptavidin for RBC populations, fluorescein isothiocyanate-labeled antibodies to DAF and CD59, hydroethidine for parasite DNA, and flow cytometry. The mean +/- SD parasitemias in co-cultured PNH and control RBCs were 24.7 +/- 6.9% versus 21.0 +/- 5.9% (P = 0.12). For individual PNH samples, parasitemias were significantly higher in DAF (-) cells versus DAF (+) cells (25.0 +/- 8.9% versus 19.1 +/- 8.7%; P < 0.001) and in CD59 (-) cells versus I/H cells (22.5 +/- 6.4% versus 17.6 +/- 4.2%; P < 0.0003). Across the CD59 spectrum, mean parasitemias were highest in CD59 (-) cells (24.5 +/- 6.4%), followed by CD59-H cells (19.5 +/- 5.4%), and CD59-I cells (16.4 +/- 4.8%). Expression of DAF in 12 (63%) of 19 infected PNH samples was reduced. Thus, P. falciparum does not selectively avoid RBCs with fewer GPI proteins and parasite replication in PNH cells is at least as robust as in normal RBCs.
Assuntos
Proteínas Inativadoras do Complemento/deficiência , Membrana Eritrocítica/metabolismo , Hemoglobinúria Paroxística/sangue , Glicoproteínas de Membrana/deficiência , Proteínas de Membrana/deficiência , Plasmodium falciparum/fisiologia , Animais , Divisão Celular/fisiologia , DNA de Protozoário/análise , Citometria de Fluxo , Glicosilfosfatidilinositóis/metabolismo , HumanosRESUMO
BACKGROUND: Thalassemia is a hereditary hemolytic anemia caused by mutations in the globin gene complex. Circulatory disturbances including arterial and venous thrombosis have also been noted in these patients. Aggregability of abnormal RBC and the high level of membrane-derived microparticles stemming from activated platelets and other blood cells are thought to be responsible for the associated thrombotic risk. Destruction of RBC is also thought to be an important pathophysiological consequence, particularly through the formation of circulating vesicles. To our knowledge, there has been no attempt to quantitatively evaluate the number of RBC vesicles in thalassemia. This prompted us to study the level of RBC vesicles in the peripheral blood of thalassemia patients using quantitative flow cytometry. METHODS: Whole blood from each subject was doubly stained for RBC and platelet or annexin V markers, together with the known density TruCount beads. RBC vesicles were gated according to their forward/side scatter and RBC marker. Percentage of RBC vesicles and their absolute number were analyzed by flow cytometry. RESULTS: Our data indicated that RBC vesicles were annexin V-positive. The number of annexin V-positive events was higher than their intact RBCs. RBC vesicles were present in both normal and thalassemic blood samples, but the numbers of RBC vesicles were significantly higher in thalassemia. Both the percentage and the absolute number of RBC vesicles were especially marked in splenectomized subjects with beta-thalassemia/Hemoglobin E. When clinical and hematological indices were compared with RBC vesicles, there was an inverse relationship between the degree of severity in thalassemia patients and the number of RBC vesicles. CONCLUSION: Flow cytometric quantitation of RBC vesicles is simple, reliable and may offer new insights in to study of the relationship between defective hemoglobin synthesis, RBC perturbation and pathophysiological complications in thalassemia.
Assuntos
Contagem de Eritrócitos , Citometria de Fluxo/métodos , Talassemia beta/sangue , Adolescente , Adulto , Anexina A5/sangue , Biomarcadores/sangue , Humanos , Contagem de Plaquetas , Valores de Referência , Análise de Regressão , Talassemia alfaRESUMO
This study represents a comprehensive evaluation of normative values for lymphocyte immunophenotype subsets using flow cytometry techniques in a Japanese population. Lymphocyte reference ranges were determined for percentage and absolute count of T, B, and NK cells in healthy adult Japanese using an extensive two-color immunophenotyping panel and consistently applied quality control methodology. Reference values were also determined for activation markers on CD3+ lymphocytes CD3+/CD25+, CD3+/CD38+ and CD3+/HLA-DR+. Differences in age and gender were observed for specific lymphocyte subsets. Comparison of the Japanese study with a Thai multi-center study that used similar methodology also demonstrated ethnic differences in lymphocyte reference ranges. The results in this study strongly suggest that reference values derived from studies in one population may not be applied to another population even when similar protocols for reagents, instruments and procedures are used although such studies do appear useful for epidemiological comparisons.
Assuntos
Citometria de Fluxo , Imunofenotipagem , Valores de Referência , ADP-Ribosil Ciclase/sangue , ADP-Ribosil Ciclase 1 , Adulto , Fatores Etários , Antígenos CD/sangue , Antígenos de Diferenciação de Linfócitos B/sangue , Antígenos de Diferenciação de Linfócitos T/sangue , Linfócitos B/metabolismo , Biomarcadores/sangue , Feminino , Antígenos HLA-DR/sangue , Humanos , Japão , Células Matadoras Naturais/metabolismo , Ativação Linfocitária , Contagem de Linfócitos , Masculino , Glicoproteínas de Membrana , Pessoa de Meia-Idade , Receptores de Interleucina-2/sangue , Fatores Sexuais , Linfócitos T/metabolismoRESUMO
BACKGROUND: Severe injury is associated with changes in monocytes that may contribute to poor outcomes. Longitudinal characterization of monocyte response patterns after trauma may provide added insight into these immunological alterations. METHODS: Venous blood obtained seven times during post-injury days 1 through 13 from 61 patients with an injury severity score >20 was assessed by flow cytometry for monocytes (CD14+) expressing HLA-DR or CD71 (transferrin receptor) and for circulating levels of interleukin (IL) 1alpha, IL-1beta, IL-6, soluble CD14 (sCD14), tumor necrosis factor-alpha (TNF-alpha), prostaglandin E(2) (PGE(2)), thromboxane B(2) (TXB(2)), and endotoxin. Urine neopterin was measured by high-pressure liquid chromatography, expressed as a neopterin-creatinine ratio. RESULTS: Trauma patients had leucocytosis days 1 through 13, monocytosis days 5 through 13, reduced proportions of CD14+HLA-DR+ cells days 2 through 5, and elevated proportions of CD14+CD71+ cells days 1 through 13. Neopterin was elevated all days, peaking on day 10. sCD14 was elevated days 2 through 13, and there were sporadic elevations of IL-1alpha, IL-1beta, IL-6, TNF-alpha, PGE(2), TXB(2), and endotoxin. Sepsis syndrome patients (n = 6) had larger and more prolonged reductions in CD14+HLA-DR+ cells and higher neopterin values, in comparison with uneventful patient outcomes. CONCLUSIONS: Altered proportions of monocytes expressing HLA-DR and CD71 and elevated sCD14 and urine neopterin levels, for up to 2 weeks after severe injury, underscores an extended period of profound immunological effects. Additional studies to more fully assess temporal monocyte response patterns after severe injury, including activation, may be warranted.
Assuntos
Antígenos CD/metabolismo , Antígenos HLA-DR/metabolismo , Monócitos/imunologia , Monócitos/metabolismo , Receptores da Transferrina/metabolismo , Índice de Gravidade de Doença , Ferimentos e Lesões/imunologia , Adulto , Biomarcadores , Dinoprostona/sangue , Endotoxinas/sangue , Feminino , Humanos , Interleucina-1/sangue , Interleucina-6/sangue , Contagem de Leucócitos , Receptores de Lipopolissacarídeos/sangue , Masculino , Neopterina/urina , Solubilidade , Tromboxano B2/sangue , Fatores de Tempo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BD Biosciences is a leader in the use of flow cytometry for determining immune system status and for counting CD4 cells in patients with human immunodeficiency virus (HIV) infection. The company has gained this position through many years of basic research and product development in immunology and cell biology, dye chemistry, immunoassays, instrumentation, and software. Some of the highlights of these developments and their historical perspective are described in this review.
Assuntos
Síndrome da Imunodeficiência Adquirida/sangue , Antígenos CD4/sangue , Linfócitos T CD4-Positivos/citologia , Soropositividade para HIV/sangue , Setor de Assistência à Saúde , Ciência de Laboratório Médico , Citometria de Fluxo/métodos , HumanosRESUMO
Artemisinin and its derivatives, artesunate and artemether, are rapidly acting antimalarials that are used for the treatment of severe and uncomplicated multidrug-resistant falciparum malaria. To optimize treatment regimens that use this new class of antimalarials, there is a need for readily available and reproducible assays to monitor drug levels closely in patients. A sensitive and reproducible bioassay for the measurement of the concentrations of artemisinin derivatives in plasma and serum is described. By modifying the in vitro drug susceptibility test, it was found that antimalarial activity in plasma or serum containing an unknown concentration of drug could be equated to the known concentrations of dihydroartemisinin (DHA) required to inhibit parasite growth. Dose-response curves for a Plasmodium falciparum clone (clone W2) and DHA were used as a standard for each assay. Assays with plasma or serum spiked with DHA proved to be reproducible (coefficient of variation, Assuntos
Antimaláricos/sangue
, Artemisininas/sangue
, Plasmodium falciparum/efeitos dos fármacos
, Sesquiterpenos/sangue
, Animais
, Antimaláricos/farmacologia
, Artemisininas/farmacologia
, Artesunato
, Bioensaio
, Biotransformação
, Cromatografia Líquida de Alta Pressão
, Relação Dose-Resposta a Droga
, Eletroquímica
, Temperatura Alta
, Humanos
, Técnicas In Vitro
, Macaca mulatta
, Malária Falciparum/sangue
, Reprodutibilidade dos Testes
, Sesquiterpenos/farmacologia
RESUMO
This study represents a comprehensive evaluation of normative values for lymphocyte immunophenotype subsets using flow cytometry techniques in a Japanese population. Lymphocyte reference ranges were determined for percentage and absolute count of T, B, and NK cells in healthy adult Japanese using an extensive two-color immunophenotyping panel and consistently applied quality control methodology. Reference values were also determined for activation markers on CD3+ lymphocytes CD3+/CD25+, CD3+/CD38+ and CD3+/HLA-DR+. Differences in age and gender were observed for specific lymphocyte subsets. Comparison of the Japanese study with a Thai multi-center study that used similar methodology also demonstrated ethnic differences in lymphocyte reference ranges. The results in this study strongly suggest that reference values derived from studies in one population may not be applied to another population even when similar protocols for reagents, instruments and procedures are used although such studies do appear useful for epidemiological comparisons.
Assuntos
ADP-Ribosil Ciclase/sangue , Adulto , Fatores Etários , Antígenos CD/sangue , ADP-Ribosil Ciclase 1 , Antígenos de Diferenciação de Linfócitos B/sangue , Antígenos de Diferenciação de Linfócitos T/sangue , Linfócitos B/metabolismo , Biomarcadores/sangue , Feminino , Citometria de Fluxo , Antígenos HLA-DR/sangue , Humanos , Imunofenotipagem , Japão , Células Matadoras Naturais/metabolismo , Ativação Linfocitária , Contagem de Linfócitos , Masculino , Glicoproteínas de Membrana , Pessoa de Meia-Idade , Receptores de Interleucina-2/sangue , Valores de Referência , Fatores Sexuais , Linfócitos T/metabolismoRESUMO
Although several animal models for human cerebral malaria have been proposed in the past, name have shown pathological findings that are similar to those seen in humans. In order to develop an animal model for human cerebral malaria, we studied the pathology of brains of Plasmodium coatneyi (primate malaria parasite)-infected rhesus monkeys. Our study demonstrated parazitized erythrocyte (PRBC) sequestration and cytoadherence of knobs on PRBC to endothelial cells in cerebral microvessels of these monkeys. This similar to the findings een in human cerebral malaria. Crebral microvessels with sequestred PRBC were shown by immunohistochemistry to possess CD36, TSP and ICAM-1. These proteins were not evident in cerebral microvessels of uninfected control monkeys. Our study indicates, for the first time, that rhesus monkeys infected with P. coatneyi can be used as a primate model to study human cerebral malaria