Lymphocyte subpopulations in malaria infected individuals living in an endemic area.

Development of partial immunity in people living in malaria endemic area is complex. For better understanding, the lymphocyte subpopulations from infected patients were evaluated by flow cytometer before any antimalarial treatment. In P. vivax infection, the frequency of T-helper type 1 (Th1) was decreased significantly (p = 0.042). In contrast, the number of T- helper type 2 (Th2) was increased significantly (p = 0.001). These trends have also been observed in P. faciparum infection. The Th2 predominant response to the natural malaria infection is likely due to persistent stimulation by Plasmodium species. In P. falciparum infection, CD8+ cytotoxic lymphocytes were significantly reduced (p = 0.007). However, such changes were not found in P. vivax infection. This might suggest that CD8+ cell responses to different Plasmodium spp in a different way. Both Th2 activation and CD8+ cell suppression may reflect less protective effects and chronic malaria infection could be established.

Effects of hydroxypyridinone iron chelators in combination with antimalarial drugs on the in vitro growth of Plasmodium falciparum.

Using standard in vitro drug susceptibility methods, we assessed the antimalarial activity of 3 orally administered iron chelators (hydroxypyridinones) alone and in combination with conventional antimalarials drugs (quinine, mefloquine, artesunate, tetracycline, atovaquone) against a chloroquine-resistant Plasmodium falciparum isolate. When tested alone, all iron chelators and antimalarial compounds inhibited the growth of the parasites. IC50 values for iron chelators were 60-70 microM, whereas the IC50 values for antimalarial drugs were in nM ranges, with artesunate being the most potent. The derived isobolograms for the interaction of hydroxypyridinones and antimalarial drugs showed addition or mild antagonism, similar to desferroxamine (Sum of Fractional Inhibitory Concentration, sigma FIC < 0.5 or > 4.0). Despite the absence of synergy with conventional drugs, intrinsic antimalarial activity of hydroxypyridinones supports the continued assessment of these iron chelators as treatment adjuncts.

Purification of human hemopoietic stem cells for transplantation in Thailand.

Stem cell transplantation (SCT) has become the therapy of choice for many hematologic and immunologic disorders. At present, only 25% of patients have suitable HLA-identical donors. In an attempt to increase the donor pool for SCT in Thailand and Southeast Asia, we developed a program whereby parents and mismatched siblings can be used as donors. In this preliminary study, after granulocyte-colony-stimulating factor (G-CSF) was given to adult donors, peripheral blood stem cells (PBSC) were collected and CD34+ cells purified using a CliniMACS immunomagnetic device (Miltenyi Biotec, Germany). In seven experiments, purified CD34+ cells could be obtained from G-CSF-stimulated PBSC in large numbers (1.71 +/- 0.19 x 10(8)), with high purity (93 +/- 2.4%) and excellent recovery (64.28% - 85.62%). Immune reactive T and NK cells were adequately depleted to less than 0.2%. The purification procedure can be completed within 3 hours. In conclusion, a clinical stem cell purification program using this novel device is now established in Thailand and for the first time in Southeast Asia. This should allow further development of advanced SCT therapy including haploidentical and mismatched CD34+ SCT for patients' lacking HLA-identical donors in this region.

B cells are the principal circulating mononuclear cells infected by dengue virus.

Although dengue virus infects a variety of cells in vitro, little is known about cell types infected in vivo. Since blood is a readily accessible tissue, we chose to determine which circulating blood cells are infected by dengue viruses. We collected blood mononuclear cells from acutely ill dengue patients and separated the cells by flow cytometry into subsets for virus isolation. Cells were sorted into groups corresponding to the cluster designations CD3, CD14, CD16 and CD20. Virus was isolated from sorted groups by inoculation into Toxorhynchites splendens mosquitos. The majority of the virus was recovered from the CD20 or B cell positive subset. Little virus was isolated from monocytes, NK cells or T cells. Virus was isolated from B cells regardless of the age or sex of the patient, virus serotype isolated, or the patient's history of dengue virus infection. The location of cell associated virus was determined by proteolytic digestion of surface virus. There was an equal distribution of virus between the intracellular compartment and the surface of B cells. The intracellular localization of virus was confirmed by immunocytochemistry. Since this study focused on circulating cells, no inferences were made regarding infection of cells in solid tissues.

Evaluation of monoclonal antibody reagents from three different manufacturers using flow cytometric two-color immunophenotyping.

We evaluated a flow cytometric (FCM) two-color immunophenotyping of CD3+/CD4+ T-helper and CD3+/CD8+ T-suppressor lymphocytes in whole blood samples from HIV-infected individuals using monoclonal antibody reagents from three different manufacturers. Lymphocytes were firstly determined using CD45/CD14 in association with a forward scatter/side scatter gating strategy. CD3+/CD4+ and CD3+/CD8+ were then determined and compared. Reagents from all manufacturers showed good separation of lymphocytes, monocytes and granulocytes with high purity and recovery. There was a good correlation of the percentage of CD3+/CD4+ and CD3+/CD8+ lymphocytes amongst each of the manufacturer's reagents, but the fluorescent intensities of positive cells were not the same. This difference can result in poor discrimination of positive and negative non CD3 cells leading to erroneous results.

An in vitro study on thalassemic erythroid precursors in liquid culture.

Thalassemia is an inherited hematological disorder which can generally be classified according to the affected globin imbalance (alpha- or beta-globin) into two main types, i.e. alpha-thalassemia and beta-thalassemia, respectively. There is a wide range of cellular abnormalities associated with thalassemic erythrocytes such as hypochromia, microcytosis, reduced cellular deformability and membrane oxidative damage. The red cell abnormalities lead to premature destruction with marrow erythroid hyperplasia and ineffective erythropoiesis. The abnormalities in thalassemic red blood cells have been found along the erythroid differentiation pathway other than the mature stage as previously shown in bone marrow erythroid precursors and in reticulocytes, the penultimate stage of erythroid differentiation. However, there is a lag in our understanding of the more primitive erythroid stages due to the difficult and hazardous marrow aspiration and heterogeneity of cells derived. We have utilized a novel method of Two-Phase Liquid Culture (TPLC) of beta-thalassemia/HbE erythroid precursors instead of conventional semisolid culture. This type of liquid culture can given higher cell yield with quite synchronous cell differentiation stages and easily be applied for other cellular analytical techniques. The peripheral blood mononuclear cells (PBMC) obtained from non-splenectomized and splenectomized beta-thalassemia/HbE patients were first cultured in medium supplemented with 5637 conditioned medium for a 6-day period (phase I) and then transferred to medium supplemented with recombinant human erythropoietin to allow the terminal differentiation of erythroid precursors (phase II). During the phase I or II, the cultured cells were periodically sampled to determine the cell number, cytocentrifuged on glass slides and stained with Wright stain for morphological assessment of their differentiation stages and analyzed flow cytometrically by staining with fluoresceinated anti-transferrin receptor (anti-CD71) and R-phycoerythrin-conjugated anti-glycophorin A. After assessment by flow cytometry, the remaining stained cells were cytocentrifuged on glass slides and photographed by a fluorescent microscope and a laser scanning confocal microscope. The results of morphological assessment, flow cytometric analysis and microscopic pictures will be presented.

Genotypic and phenotypic implications in paroxysmal nocturnal hemoglobinuria (PNH): a preliminary investigation.

The genetic and biochemical defects underlying paroxysmal nocturnal hemoglobinuria (PNH) have recently been elucidated. The deficiency of the surface expression of glycosylphosphatidylinositol (GPI)-anchored proteins caused by a somatic mutation of the PIG-A gene, an X-chromosomal gene that participates in the first step of the GPI anchor synthesis, has been shown to be responsible for PNH in all patients. The mutations of PIG-A studied to date are highly heterogeneous. They are however mainly of the frameshift type (61.5%). The characteristic abnormalities of PNH phenotypes has also been shown especially by DAF- and/or CD59-based fluorescent immunocytometry. A great degree of heterogeneity in the patterns and levels of expression of GPI-anchored proteins in various cell types was demonstrated indicating a discrepancy of lineage involvement. In this investigation, major blood cell populations, i.e erythrocytes and granulocytes were analyzed immunophenotypically, the mutations of PIG-A were identified by heteroduplex analysis and nucleotide sequencing and the consequences of PIG-A mutations were observed. All the mutations identified in 9 patients with PNH resulted in complete loss of function as clones of affected granulocytes completely negative for CD59 expression were shown in all patients. Interestingly, granulocytes in these patients contained variable proportions of affected cells varied from 50% to 100% and four of the patients had erythrocytes with diminished expression of GPI-anchored DAF and CD59 coexisting with normal and completely negative cells. Immunophenotypic analysis of reticulocytes in peripheral blood of patients with PNH demonstrated the conserved patterns of DAF and CD59 expression in circulating erythroid cells and the discrepancies between granulocytic and erythroid lineages. These findings suggested that the characteristics of abnormal phenotypes which appear to be highly variable between different hematopoietic lineages are not solely caused by mutation of PIG-A but are influenced by other factor(s).

Lymphocyte immunophenotype reference ranges in healthy Thai adults: implications for management of HIV/AIDS in Thailand.

Lymphocyte immunophenotype reference ranges for T, B, and NK subsets were determined for healthy adult Thais in a multi-center study in Bangkok. Immunophenotyping was by flow cytometry using lysed whole blood. A standard protocol for flow cytometry instrumentation, reagents and quality control was used to minimize site differences and to facilitate comparison of the Thai reference values to those found for Caucasians in previous studies. Major differences were determined for CD3(T), CD4 (T helper/inducer) and CD16+56 (NK) lymphocyte percentages and CD4 lymphocyte absolute counts. Age trends and sex differences were also observed. Compared to Caucasians, Thais, particularly Thai males, had lower CD3 and CD4 T lymphocyte percentages and absolute numbers whereas the percentage of NK lymphocytes was higher. Heterogeneity attributed to biological variation of CD4 T lymphocyte but not other immunophenotype subset distributions was also observed in a well defined geographic population. This study demonstrates the importance of ethnicity, age, sex and possibly environment as factors that influence distribution characteristics of normal lymphocyte immunophenotype reference values. These observations have important implications for the use of lymphocyte subsets-particularly CD3+ CD4+ T lymphocyte measurements as applied to HIV disease staging, AIDS definition and the overall clinical management of HIV/AIDS in Thailand.

Opsonophagocytosis and intracellular killing activity of neutrophils in patients with human immunodeficiency virus infection.

In this study, neutrophils isolated from asymptomatic HIV positive individuals, patients with AIDS-related complex (ARC), ARC patients receiving zidovudine (AZT) and full-blown AIDS patients were assayed for their opsonophagocytic and intracellular killing activities. Progressively decreasing opsonophagocytosis of C. albicans by neutrophils correlated with increasing severity of the disease in all groups of HIV infected individuals, as compared to neutrophils isolated from healthy controls. The intracellular killing of C. albicans by neutrophils of asymptomatic and ARC patients did not differ significantly from controls. Neutrophils of ARC patients receiving AZT and AIDS patients showed a slightly decreased killing activity in comparison to that of neutrophils from healthy controls.

PIG-A gene abnormalities in Thai patients with paroxysmal nocturnal hemoglobinuria.

Deficient biosynthesis of the glycosyl phosphatidyl inositol (GPI)-anchor in blood cells is implicated in the pathogenesis of paroxysmal nocturnal hemoglobinuria (PNH). Abnormal clonal cells appear in various hematopoietic cell lineages, suggesting that PNH arises as a result of somatic mutation occurred at the multipotential hematopoietic stem cell stage. We previously cloned a gene which is responsible for PNH. The gene termed PIG-A (for Phosphatidyl Inositol Glycan-class A) participates in the early step of GPI-anchor biosynthesis. Studies with cell lines and granulocytes from patients with PNH revealed that in all cases so far characterized, PIG-A is the target for the somatic mutation. In the present study, we analyzed PIG-A abnormality in granulocytes from 14 Thai-patients with PNH. PIG-A RNA was reversed transcribed and the coding region was amplified by polymerase chain reaction and cloned into plasmids. The cDNA thus obtained and genomic DNA were analyzed by mutation detection enhancement gel electrophoresis and sequencing. The assessment of function of PIG-A cDNA was based on the ability to correct the phenotype of a PIG-A deficient cell line after transfection. The result showed that all patients had PIG-A abnormality. Three patients had size abnormality of PIG-A transcripts caused by mutations at the splicing sites in the genomic DNA level. Eleven patients had PIG-A transcripts of normal sizes but had mutations in the coding region which included small deletions and insertions. Taken together with the result from Japanese and British patients, the PIG-A somatic mutations in patients with PNH are small mutations widely distributed throughout coding region and the splicing sites.

Flow cytometric three-color determination of CD4 T-lymphocytes on blood specimens from AIDS patients who have a large number of contaminating non-lymphocytes.

A three-color flow cytometric determination of CD4 T-lymphocytes on whole blood specimens from AIDS patients which contain a high proportion of non-lymphocyte elements is described. Peripheral blood cells were stained by a three-color method using monoclonal antibodies conjugated respectively with fluorescein isothiocyanate (FITC)-CD3, phycoerythrin (PE)-CD4 and peridinin chlorophyll protein (PerCP)-CD45. CD45 stains all leukocytes with the highest fluorescence expression of CD45 antigen in lymphocytes. By combining light scatter with CD45 in the fluorescence 3 (FL3) channel, a light scattering window can be drawn to include almost all bright CD45 lymphocytes. This live gate of lymphocytes was then acquired and analysed simultaneously using other irrelevant two-color (FITC/PE) antibodies of CD3 and CD4 in the FITC and PE channels, respectively. This method is easy and straightforward, and gives successful analysis of CD4 T-lymphocytes in AIDS blood specimens contaminated with an unusually large number of non-lymphocytic cells.

Lymphocyte-bearing ferritin in beta-thalassemia/Hb E.

Flow cytometric analysis of lymphocyte subsets and lymphocyte surface ferritin shows no significant difference in the number of total T-cells, B cells, NK cells, helper T-cells (CD 4), suppressor T-cells (CD 8) and CD 4/CD 8 ratio among normal subjects (n = 11) and Hb E trait (n = 6), beta-thalassemia (beta-thal) trait (n = 5), neither in normal and nonsplenectomized patients with beta-thal/Hb E (n = 10) except B cells and CD 4. There is a significant reduction in lymphocytes surface spleen-type and heart-type ferritin in patients with beta-thal/Hb E when compared to normal subjects. No difference can be seen among patients with beta-thal/Hb E, beta-thal trait and Hb E trait. This low percentage of lymphocyte-bearing ferritin suggests a negative relationship between ferritin on the cells' surface and high circulating ferritin normally associated with thalassemic patients.

Bronchoalveolar T-lymphocytes and subsets in pulmonary tuberculosis and bronchogenic carcinoma.

Analysis of T-lymphocytes and their subsets in the blood and bronchoalveolar lavage fluids from 12 patients with active pulmonary tuberculosis, 12 patients with bronchogenic carcinoma and 11 healthy volunteers was aimed at identifying their immunologic functions and interrelationships. In patients with active pulmonary tuberculosis, there was a significant increased in the percentage of T-cells bearing IL-2 receptor both in the blood and bronchoalveolar lavage fluids, whereas patients with bronchogenic carcinoma exhibited an increase in the suppressor T-cells and T-cells bearing IL-2 receptor in the blood only. The presence of T-cells bearing IL-2 receptor is generally accepted to be the hallmark of recently active specific antigen activation of the helper T-lymphocytes together with monokine IL-1 stimulation. Suppressor T-cells, on the other hand, play a role in the immunopathogenesis of lung cancers and lung metastasis.