The in vitro red blood cell microvesiculation exerts procoagulant activity of blood cell storage in Southeast Asian ovalocytosis.

Southeast Asian ovalocytosis (SAO) is characterized by the misfolding of band 3 protein in red blood cells (RBC). The abnormal structure of the band 3 protein results in dysmorphic RBC and related functions. Previous data showed that in vitro storage under hypothermic conditions alters band 3 protein structure and function. Microvesiculation includes shedding of RBC membranes, called RBC-derived microparticles/extracellular vesicles (RMP/EVs), and storage lesions. Unfortunately, there is no evidence of RBC microvesiculation under in vitro storage conditions in heterozygous SAO individuals. This study determined the generation of REVs and procoagulant activity during the storage of SAO blood samples in southern Thailand. Venous blood was collected from eight SAO and seven healthy individuals, preserved in citrate phosphate dextrose-adenine 1 (CPDA-1) at 4 °C for 35 days. The absolute numbers of REVs and PS-expressing RBCs were analyzed using flow cytometry. The procoagulant activity of the produced extracellular vesicles was determined by a clotting time assay. The results showed a significant increase in the number of REVs and PS-expressing RBCs in the SAO blood samples. Significantly correlated PS externalization and procoagulant activity were observed in the SAO blood samples. These lines of evidence indicate that the abnormality of the Band 3 protein is possibly involved in aberrant microvesiculation, exerting procoagulant activity in vitro. Increased pools of REV production and abnormal storage lesions in SAO blood samples should be a concern. Notably, the mechanisms underlying membrane vesiculation depend on the extent of blood cell storage under hypothermic conditions.

Characterisation and immuno-stimulating activity of polysaccharides from Thai medicinal plants.

Water-soluble polysaccharides were isolated from the tubers of Butea superba Roxb. and Pueraria candollei Wall. Ex Benth. var. mirifica (Shaw et Suvat.) C. Niyomdham, the leaves of Centella asiatica (L.) Urb, Ocimum basilicum L., Psidium guajava and Andrographis paniculata (Burn. f.) Nees, the stems of Cymbopogon citratus (Stapf ExG), and the fruits of Psidium guajava and Scaphium scaphigerum. The immunological impacts of the polysaccharides on T-lymphocyte proliferation in vitro was investigated by flow cytometric (immunofluorescence) analysis using staphylococcal enterotoxin B (SEB) as a positive control. It was found that the polysaccharides enhanced T-lymphocyte proliferation, ranging from 4.5 to 27.0% at a concentration of 100 microg mL(-1), while the activity of SEB was 13.3%. The medicinal plants showing the highest immuno-stimulating activity were the tubers of Butea superba Roxb. The water-extracted tubers contained 60.0% (w/w) carbohydrates with 6.6% (w/w) uronic acid. The major constituent monosaccharides of the tubers were 28.2 mol% galactose, 10.5 mol% arabinose and 36.4 mol% glucose.

Effects of green tea on iron accumulation and oxidative stress in livers of iron-challenged thalassemic mice.

Liver is affected by secondary iron overload in transfusions dependent b-thalassemia patients. The redox iron can generate reactive oxidants that damage biomolecules, leading to liver fibrosis and cirrhosis. Iron chelators are used to treat thalassemias to achieve negative iron balance and relieve oxidant-induced organ dysfunctions. Green tea (GT) (Camellia sinensis) catechins exhibit anti-oxidation, the inhibition of carcinogenesis, the detoxification of CYP2E1-catalyzed HepG2 cells and iron chelation. The purpose of this study was to investigate the effectiveness of GT in iron-challenged thalassemic mice. Heterozygous BKO type-thalassemia (BKO) mice (C57BL/6) experienced induced iron overload by being fed a ferrocene-supplemented diet (Fe diet) for 8 weeks, and by orally being given GT extract (300 mg/kg) and deferiprone (DFP) (50 mg/kg) for a further 8 weeks. Liver iron content (LIC) was analyzed by TPTZ colorimetric and Perl's staining techniques. Concentrations of liver reduced glutathione (GSH), collagen and malondialdehyde (MDA) were also measured. Dosages of the GT extract and DFP lowered LIC in the Fe diet-fed BKO mice effectively. The extract did not change any concentrations of liver glutathione, collagen and MDA in the BKO mice. Histochemical examination showed leukocyte infiltration in the near by hepatic portal vein and high iron accumulation in the livers of the iron-loaded BKO mice, however GT treatment lowered the elevated iron deposition. In conclusion, green tea inhibits or delays the deposition of hepatic iron in regularly iron-loaded thalassemic mice effectively. This will prevent the iron-induced generation of free radicals via Haber-Weiss and Fenton reactions, and consequently liver damage and fibrosis. Combined chelation with green tea would be investigated in beta-thalassemia patients with iron overload.

Immune response to Plasmodium vivax has a potential to reduce malaria severity.

Plasmodium falciparum infection causes transient immunosuppression during the parasitaemic stage. However, the immune response during simultaneous infections with both P. vivax and P. falciparum has been investigated rarely. In particular, it is not clear whether the host's immune response to malaria will be different when infected with a single or mixed malaria species. Phenotypes of T cells from mixed P. vivax-P. falciparum (PV-PF) infection were characterized by flow cytometry, and anti-malarial antibodies in the plasma were determined by an enzyme-linked immunosorbent assay. We found the percentage of CD3+delta2+-T cell receptor (TCR) T cells in the acute-mixed PV-PF infection and single P. vivax infection three times higher than in the single P. falciparum infection. This implied that P. vivax might lead to the host immune response to the production of effector T killer cells. During the parasitaemic stage, the mixed PV-PF infection had the highest number of plasma antibodies against both P. vivax and P. falciparum. Interestingly, plasma from the group of single P. vivax or P. falciparum malaria infections had both anti-P. vivax and anti-P. falciparum antibodies. In addition, antigenic cross-reactivity of P. vivax or P. falciparum resulting in antibodies against both malaria species was shown in the supernatant of lymphocyte cultures cross-stimulated with either antigen of P. vivax or P. falciparum. The role of delta2 +/- TCR T cells and the antibodies against both species during acute mixed malaria infection could have an impact on the immunity to malaria infection.

Preliminary in vivo efficacy studies of a recombinant rhesus anti-alpha(4)beta(7) monoclonal antibody.

Recent findings established that primary targets of HIV/SIV are lymphoid cells within the gastrointestinal (GI) tract. Focus has therefore shifted to T-cells expressing alpha(4)beta(7) integrin which facilitates trafficking to the GI tract via binding to MAdCAM-1. Approaches to better understand the role of alpha(4)beta(7)+ T-cells in HIV/SIV pathogenesis include their depletion or blockade of their synthesis, binding and/or homing capabilities in vivo. Such studies can ideally be conducted in rhesus macaques (RM), the non-human primate model of AIDS. Characterization of alpha(4)beta(7) expression on cell lineages in RM blood and GI tissues reveal low densities of expression by NK cells, B-cells, naïve and TEM (effector memory) T-cells. High densities were observed on TCM (central memory) T-cells. Intravenous administration of a single 50mg/kg dose of recombinant rhesus alpha(4)beta(7) antibody resulted in significant initial decline of alpha(4)beta(7)+ lymphocytes and sustained coating of the alpha(4)beta(7) receptor in both the periphery and GI tissues.

Cytoadherence between endothelial cells and P. falciparum infected and noninfected normal and thalassemic red blood cells.

BACKGROUND: Cytoadhesion of P. falciparum infected red blood cells (RBCs) to endothelial cells (ECs) is an important phenomenon that causes cerebral malaria in man. Reduced adhesion especially in thalassemia and hemoglobinopathies may be related to a protective mechanism against malaria in such people. METHODS: The cytoadherence assay was performed using both conventional and floating conditions between ECs (ECV 304) and P. falciparum infected and noninfected RBCs from both normal and thalassemia subjects. In floating condition, RBC was fluorescently labeled with anti-glycophorin A antibody, whereas EC was identified by surface expression of PECAM-1, CD-36, ICAM-1, and E-selectin. The condition of floating EC was similar to the condition for subcultivation as they can adhere or bind to any surface. The phosphatidylserine (PS) exposure was also determined by using flow cytometer. RESULTS: The adhesion of noninfected heterozygous thalassemic RBCs (all genotypes) to ECs was significantly increased as compared with normal RBCs (P < 0.02). Interestingly, after P. falciparum infection, the number of normal RBCs bound to ECs was significantly increased as compared with noninfected RBCs (P < 0.01), whereas heterozygous thalassemic RBCs infected by P. falciparum showed no significant difference compared with noninfected RBCs. In addition, we found a similar level of PS exposure in normal and thalassemic infected RBCs, which was related to the cytoadherence phenomenon. CONCLUSION: The reduced adhesion between heterozygous thalassemic RBCs infected by P. falciparum to ECs provides an explanation for their protective mechanism against malaria, as increased adhesion is a high risk for cerebral malaria and nonbinding infected RBCs can be removed by the reticuloendothelial system and other mechanism(s) in vivo.

Flow cytometric analysis of DNA ploidy and S-phase fraction of Stage IIIB cervical carcinoma.

OBJECTIVES: To evaluate the role of flow cytometry-measured DNA ploidy and S-phase fraction as survival prognostic indicators in women with FIGO Stage IIIB squamous cell carcinoma of cervix. METHODS: We retrospectively reviewed the medical and pathological records of women with Stage IIIB squamous cell cervical carcinoma treated between 1993 and 1996. Flow cytometric analysis of DNA ploidy and S-phase fraction was performed by the modified Hedley technique using paraffin-embedded tissue. Survival was calculated using the Kaplan-Meier life-table analysis. RESULTS: Of the 75 cases, 66 were analyzable. Diploid tumors were found in 73%. The mean S-phase fraction was 14% (SD = 5.4). The overall 5-year survival rate was 60%. The survival of patients with aneuploidy tumors was significantly worse than that of the diploid tumors (p = 0.001). The survival of the patients who had S-phase fraction > 12% was significantly worse than those who had S-phase fraction < or =12% (p = 0.04). CONCLUSIONS: In this homogeneous study population, we found that aneuploidy and S-phase fraction >12% correlated with poor survival. Identifying this poor prognostic group would be of benefit in considering additional treatment for a better outcome.

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.

Immunophenotypic discrepancies between granulocytic and erythroid lineages in peripheral blood of patients with paroxysmal nocturnal haemoglobinuria.

In paroxysmal nocturnal haemoglobinuria (PNH), somatic mutation of the PIG-A gene is thought to result in altered expression of glycosylphosphatidylinositol (GPI)-anchored proteins. This study was performed to determine if there were any heterogeneities of cellular phenotypes between two major peripheral blood cells, erythrocytes and granulocytes. Using CD59-based immunocytometry, the patterns of CD59 expression were shown to be conserved in the circulating erythroid cells (reticulocytes and mature erythrocytes) in all 29 patients with PNH. Twenty-one patients had distinct combinations of PNH type I, II, and III cells in different lineages. Only eight patients exhibited similar patterns of CD59 expression between the two lineages. Approximately one third of the patients had PNH type II cells in either or both of the two lineages indicating variable lineage involvement. The proportion of abnormal granulocytes was higher than those of abnormal reticulocytes and erythrocytes. In patients with appropriate erythropoietic responses to haemolysis (RPI > 2.0), shift reticulocytes display predominantly PNH phenotypes. These immature erythroid cells with altered expression of GPI-anchored proteins may dominate the peripheral blood during periods of increased marrow activity resulting in greater phenotypic mosaicism in such patients. Discrepancies in expression of GPI-anchored proteins in PNH which are highly variable between the two lineages may be the result of their different life spans and the influence of complement-mediated cytolysis. The phenomena also indicated the possible occurrence of more than one PNH clones with variable clonal dominance.

A flow cytometric method for measuring neutralization of HIV-1 subtype B and E primary isolates.

BACKGROUND: Clinical trials testing candidate human immunodeficiency virus type 1 (HIV-1) vaccines have required the use of HIV neutralization assays to detect responses to specific geographic subtypes of HIV-1. The variability in results seen with current p24 neutralization assay endpoints prompted us to assess the utility of flow cytometry for monitoring the neutralization of HIV-1 primary isolates. METHODS: A modified neutralization assay was performed using CD8-depleted peripheral blood mononuclear cells (PBMC). The cells were fixed, permeabilized, stained with a directly conjugated HIV-1 p24 monoclonal antibody, and analyzed by flow cytometry. HIV-1 subtype B' and E primary isolates were tested using pooled sera or plasma from subtype B' or E infected patients. RESULTS: Primary isolate cultures (without neutralizing antibody) showed from 18% to 42% p24(+) cells, depending on the virus. Less than 0.2% p24(+) cells were detected in uninfected cultures. Subtype-specific neutralization of viruses was observed using plasma or serum pools; neutralization ranged from 0% to 99% reduction of infected cells. CONCLUSIONS: Flow cytometric detection of intracellular HIV-1 p24 can be used as an endpoint assay to assess neutralization of HIV-1 subtypes B' and E primary isolates. This enumerative method has the advantage of identifying intracellular p24 in specific subsets at an early culture timepoint. It also provides an alternative quantitative endpoint for HIV neutralization assays.

Lymphocyte activation after non-thermal trauma.

BACKGROUND: Severe injury causes immunological changes that may contribute to a poor outcome. Longitudinal characterization of lymphocyte response patterns may provide further insight into the basis of these immunological alterations. METHODS: Venous blood obtained seven times over 2 weeks from 61 patients with injury severity scores above 20 was assessed for lymphocyte phenotypic and activation markers together with serum levels of interleukin (IL) 2, IL-4, soluble IL-2 receptor (sIL-2R), soluble CD4 (sCD4), soluble CD8 (sCD8) and interferon gamma. RESULTS: Severe injury was associated with profound changes in the phenotypic and activation profile of circulating lymphocytes. Activation was indicated by increased numbers of T cells expressing CD25, CD69 and CD71, and raised serum levels of IL-2, sIL-2R, sCD4 and sCD8. Relatively higher levels of sIL-2R and sCD4 were found in patients with sepsis syndrome. CONCLUSION: Polytrauma is associated with dramatic alterations in the phenotypic and activation profile of circulating lymphocytes which are generally independent of clinical course. In contrast, several lymphocyte soluble factors, including sCD4 and sIL-2R, paralleled the clinical course. These data provide new insight into lymphocyte responses after injury and suggest that further assessment of soluble factors as clinical correlates, including those related to lymphocyte activation or generalized inflammation, may be warranted.

Lymphocyte subsets and specific T-cell immune response in thalassemia.

Infection is very common in thalassemia and is one of the major causes of death. To date, it is not quite clear why these patients are susceptible to infection. In this study, lymphocyte immunophenotyping for CD3(+) (T-cells), CD3(+)CD4(+) (T-helper/inducer cells), CD3(+)CD8(+) (T-suppressor/cytotoxic cells), CD3(-)CD19(+) (B-cells), and CD3(-)CD16/56(+) (natural killer cells) subsets and expression of the activation antigen CD69 on CD3(+)CD4(+) and CD3(+)CD8(+) T-cells were determined in the whole blood of thalassemia patients, using a three-color flow cytometric technique. Results showed that only splenectomized beta-thalassemia/hemoglobin (Hb) E patients displayed a marked increase in absolute number of all lymphocytes. In addition, splenectomized beta-thalassemia/Hb E showed a significantly lower percentage of CD3(+) cells, with a corresponding increase in CD19(+) cells. These differences, when compared with normal subjects and other thalassemia patients, may be attributed to splenectomy. alpha-thalassemia patients, on the other hand, showed no significant difference from the normal group. While lymphocyte subsets in splenectomized beta-thalassemia/Hb E patients showed an abnormal distribution, T-cell activation in these patients was not different from the activation seen in normal subjects. This implies that thalassemia patients, during the steady state of disease, appear to have normal T-lymphocyte function with only moderate abnormalities of T- and B-lymphocyte subsets.

Role of FcgammaRI (CD64) in erythrocyte elimination and its up-regulation in thalassaemia.

To examine any role of the high affinity Fcgamma class I receptor (FcgammaRI) (CD64) in erythrocyte elimination by mononuclear phagocytes (MP) in thalassaemia (thal), we investigated the in vitro interaction of beta-thalassaemic erythrocytes with monocytes (Mo) whose FcgammaR expression had been modulated by cytokines. Treatment of Mo with interferon (IFN)-gamma or interleukin (IL)-10 which up-regulate FcgammaRI, caused a dose-dependent increase in binding of beta-thalassaemic erythrocytes, whereas stimulation with IL-4 which down-regulates the receptor, reduced this interaction, in a dose-dependent manner, to that of normal erythrocytes. Binding of thalassaemic erythrocytes by IFN-gamma or IL-10-treated Mo was inhibited by FcgammaRI-specific reagents. In addition, Mo expression of FcgammaRI and HLA class II DR was determined by flow cytometry in Thai patients with HbH disease (alpha1/alpha2 or alpha1/Hb Constant Spring) (n = 15) or beta degrees -thal/HbE (n = 16). In both groups of patients FcgammaRI expression was increased as compared to normal controls (n = 14): mean fluorescence intensity (+/-SD) 124.79 +/- 38. 77 in HbH disease and 121.86 +/- 18.23 in beta degrees -thal/HbE versus 91.94 +/- 17.36 in normal controls (P < 0.01 and P < 0.001, respectively). In contrast, HLA class II DR expression was similar in patients and controls. The results suggest that, in thalassaemia, up-regulated FcgammaRI on mononuclear phagocytes plays a role in their interaction with erythrocytes and that this process can be modified by cytokines.

Impairment of Plasmodium falciparum growth in thalassemic red blood cells: further evidence by using biotin labeling and flow cytometry.

Certain red blood cell (RBC) disorders, including thalassemia, have been associated with an innate protection against malaria infection. However, many in vitro correlative studies have been inconclusive. To better understand the relationship between human RBCs with thalassemia hemoglobinopathies and susceptibility to in vitro infection, we used an in vitro coculture system that involved biotin labeling and flow cytometry to study the ability of normal and variant RBC populations in supporting the growth of Plasmodium falciparum malaria parasites. Results showed that both normal and thalassemic RBCs were susceptible to P falciparum invasion, but the parasite multiplication rates were significantly reduced in the thalassemic RBC populations. The growth inhibition was especially marked in RBCs from alpha-thalassemia patients (both alpha-thalassemia1/alpha-thalassemia2 and alpha-thalassemia1 heterozygote). Our observations support the contention that thalassemia confers protection against malaria and may explain why it is more prevalent in malaria endemic areas.

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.

Hematopoietic progenitor cells in the blood and bone marrow in various hematologic disorders.

Hematopoietic progenitor cells are present in the blood and the bone marrow. Changes in the numbers of hematopoietic progenitor cells reflect alteration of pluripotent stem cells. We discuss such changes in common hematologic diseases including aplastic anemia, paroxysmal nocturnal hemoglobinuria (PNH) and thalassemia. In aplastic anemia, the numbers of burst forming units-erythroid (BFU-E) and colony-forming units-granulocyte-macrophage (CFU-GM) are much decreased; the decrease still exists after recovery from therapy. In PNH, the numbers of progenitor cells are low, even in the presence of marrow hypercellularity. In thalassemia, the numbers of progenitor cells are much increased; more pronounced in splenectomized patients.