RESUMO
PURPOSE: The PD-1 (programmed cell death-1) receptor is expressed on the surface of activated T cells. Its ligand, programmed cell death ligand-1 (PD-L1), is expressed on the surface of dendritic cells or macrophages. The PD-1/PD-L1 interaction ensures prevention of autoimmunity by activating the immune system only when needed. In cancers, PD-L1 expressed on the tumour cells binds to PD-1 receptors on the activated T cells, leading to inhibition of the cytotoxic T cells and immunosuppression. PD-1/PD-L1 pathway is upregulated in EBV infection that is known to worsen the CLL prognosis. Therefore, we aimed to study the association between PD-1 and PD-L1 expressions, EBV status and the CLL prognosis. METHODS AND PATIENTS: The study was conducted on 80 newly diagnosed CLL patients and 80 controls. We analyzed PD-1 and PD-L1 expressions and EBV-DNA load by real-time PCR. The cytogenetic abnormalities and expression of ZAP70 and CD38 were detected by FISH and Flow cytometry, respectively. RESULTS: PD-1/PD-L1 expressions were significantly upregulated in CLL patients compared to controls. In addition, their mRNA levels were significantly higher in EBV( +) versus EBV( -) patients. High expression of PD-1/PD-L1 was associated with poor prognostic markers (RAI stages of CLL, del 17p13, ZAP70, and CD38 expression), failure of complete remission, shorter progression-free survival, and overall survival. CONCLUSION: High expression of PD-1 and PD-L1, together with high EBD-DNA load were linked to worse prognosis in CLL. In addition, PD-1 and PD-L1 might represent suitable therapeutic targets for patients suffering from aggressive CLL.
Assuntos
Antígeno B7-H1/genética , Infecções por Vírus Epstein-Barr , Expressão Gênica , Herpesvirus Humano 4/genética , Leucemia Linfocítica Crônica de Células B/metabolismo , Receptor de Morte Celular Programada 1/genética , ADP-Ribosil Ciclase 1/análise , Autoimunidade , Antígeno B7-H1/metabolismo , Estudos de Casos e Controles , DNA Viral/sangue , Infecções por Vírus Epstein-Barr/imunologia , Feminino , Humanos , Terapia de Imunossupressão , Leucemia Linfocítica Crônica de Células B/imunologia , Leucemia Linfocítica Crônica de Células B/mortalidade , Masculino , Glicoproteínas de Membrana/análise , Pessoa de Meia-Idade , Prognóstico , Receptor de Morte Celular Programada 1/metabolismo , Intervalo Livre de Progressão , RNA Mensageiro/metabolismo , Análise de Sobrevida , Linfócitos T/metabolismo , Linfócitos T Citotóxicos , Regulação para Cima , Carga Viral , Proteína-Tirosina Quinase ZAP-70/análiseRESUMO
Sepsis caused by Gram-negative bacteria is the consequence of an unrestrained infection that continuously releases lipopolysaccharide (LPS) into the bloodstream, which triggers an uncontrolled systemic inflammatory response leading to multiorgan failure and death. After scrutinizing the immune modulation exerted by a recombinant Fasciola hepatica fatty acid binding protein termed Fh15, our group demonstrated that addition of Fh15 to murine macrophages 1 h prior to LPS stimulation significantly suppresses the expression of proinflammatory cytokines tumor necrosis factor alpha (TNF-α) and interleukin-1ß (IL1-ß). The present study aimed to demonstrate that Fh15 could exert a similar anti-inflammatory effect in vivo using a mouse model of septic shock. Among the novel findings reported in this article, (i) Fh15 suppressed numerous serum proinflammatory cytokines/chemokines when injected intraperitoneally 1 h after exposure of animals to lethal doses of LPS, (ii) concurrently, Fh15 increased the population of large peritoneal macrophages (LPMs) in the peritoneal cavity (PerC) of LPS-injected animals, and (iii) Fh15 downregulated the expression on spleen macrophages of CD38, a cell surface ectoenzyme with a critical role during inflammation. These findings present the first evidence that the recombinant parasitic antigen Fh15 is an excellent modulator of the PerC cell content and in vivo macrophage activation, endorsing Fh15's potential as a drug candidate against sepsis-related inflammatory response.IMPORTANCE Sepsis is a potentially life-threatening complication of an infection. Sepsis is mostly the consequence of systemic bacterial infections leading to exacerbated activation of immune cells by bacterial products, resulting in enhanced release of inflammatory mediators. Lipopolysaccharide (LPS), the major component of the outer membrane of Gram-negative bacteria, is a critical factor in the pathogenesis of sepsis, which is sensed by Toll-like receptor 4 (TLR4). The scientific community highly pursues the development of antagonists capable of blocking the cytokine storm by blocking TLR4. We report here that a recombinant molecule of 14.5 kDa belonging to the Fasciola hepatica fatty acid binding protein (Fh15) is capable of significantly suppressing the LPS-induced cytokine storm in a mouse model of septic shock when administered by the intraperitoneal route 1 h after a lethal LPS injection. These results suggest that Fh15 is an excellent candidate for drug development against endotoxemia.
Assuntos
Movimento Celular/efeitos dos fármacos , Citocinas/metabolismo , Proteínas de Ligação a Ácido Graxo/administração & dosagem , Proteínas de Helminto/administração & dosagem , Lipopolissacarídeos/imunologia , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/fisiologia , Choque Séptico/patologia , ADP-Ribosil Ciclase 1/análise , Animais , Células Cultivadas , Citosol/química , Modelos Animais de Doenças , Ácidos Graxos/análise , Injeções Intravenosas , Glicoproteínas de Membrana/análise , Camundongos , Proteínas Recombinantes/administração & dosagem , Baço/imunologiaRESUMO
BACKGROUND: Oral preexposure prophylaxis (PrEP) has been established as a pivotal strategy in HIV prevention. However, bacterial sexually transmitted infections (STIs), such as Chlamydia trachomatis and Neisseria gonorrhoeae, are also highly prevalent. Although the presence of STI-related mucosal lesions is a known risk factor for HIV acquisition, the potential increase in risk associated with asymptomatic STIs is not completely understood. Recent data demonstrated higher T-cell activation is a risk factor for sexually acquired HIV-1 infection. We examined the effect of asymptomatic C. trachomatis and N. gonorrhoeae anorectal infection on systemic immune activation, potentially increasing the risk of HIV acquisition. METHODS: We analyzed samples from participants of PrEP Brasil, a demonstration study of daily oral emtricitabine/tenofovir disoproxil fumarate HIV PrEP among healthy MSM, for T-cell activation by flow cytometry. We included 34 asymptomatic participants with anorectal swab for C. trachomatis and/or N. gonorrhoeae infection, whereas negative for other STIs, and 35 controls. RESULTS: We found a higher frequency of human leukocyte antigen DRCD38 CD8 T cells (1.5 vs. 0.9%, Pâ<â0.005) and with memory phenotype in the group with asymptomatic C. trachomatis and/or N. gonorrhoeae infection. Exhaustion and senescence markers were also significant higher in this group. No difference was observed in the soluble CD14 levels. CONCLUSION: Our findings suggest asymptomatic anorectal C. trachomatis and/or N. gonorrhoeae increase systemic immune activation, potentially increasing the risk of HIV acquisition. Regular screening and treatment of asymptomatic STIs should be explored as adjuvant tools for HIV prevention.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Infecções por Chlamydia/patologia , Gonorreia/patologia , Ativação Linfocitária , Doenças Retais/patologia , ADP-Ribosil Ciclase 1/análise , Adulto , Doenças Assintomáticas , Antígenos CD8/análise , Linfócitos T CD8-Positivos/química , Chlamydia trachomatis/isolamento & purificação , Estudos Transversais , Citometria de Fluxo , Antígenos HLA-DR/análise , Humanos , Masculino , Glicoproteínas de Membrana/análise , Neisseria gonorrhoeae/isolamento & purificação , Adulto JovemRESUMO
BACKGROUND: We evaluated the changes in the levels of soluble biomarkers of inflammation and coagulation and T-cell activation among participants of AIDS Clinical Trials Group Study A5217 who were started on antiretroviral therapy (ART) within the first 6 months of HIV infection. METHODS: Cryopreserved specimens were obtained pre-ART (week 0), at the time of virologic suppression (week 36), and at 36 weeks after treatment interruption (week 72). Levels of D-dimer, C-reactive protein (CRP), and soluble CD14 (sCD14) were measured in plasma, whereas T-cell activation levels, defined as the frequencies of CD4 and CD8 T cells coexpressing HLA-DR and CD38, were measured in peripheral blood mononuclear cells. RESULTS: D-dimer levels were significantly lower at viral suppression (P = 0.031), whereas CRP and sCD14 levels remained similar to pre-ART levels. At viral suppression, levels of the soluble markers did not correlate with each other. CD4 T-cell counts pre-ART tended to modestly correlate with levels of D-dimer (r = 0.35; P = 0.058) and CRP (r = 0.33; P = 0.078). At 36 weeks after treatment interruption (week 72), D-dimer levels returned back to pre-ART levels. However, CD8 T-cell activation was significantly lower than pre-ART levels (35.8% at week 0 vs 28.9% at week 72; P = 0.004). CONCLUSIONS: Among the A5217 participants who started ART within the first 6 months of HIV infection, high levels of sCD14 and CRP remain similar to pre-ART levels, suggesting that immune damage occurring in the initial stages of infection persists despite short-term virologic suppression.
Assuntos
Antirretrovirais/uso terapêutico , Biomarcadores/sangue , Infecções por HIV/tratamento farmacológico , Infecções por HIV/patologia , Inflamação/patologia , ADP-Ribosil Ciclase 1/análise , Adulto , Coagulação Sanguínea , Proteína C-Reativa/análise , Linfócitos T CD4-Positivos/química , Linfócitos T CD8-Positivos/química , Feminino , Produtos de Degradação da Fibrina e do Fibrinogênio/análise , Antígenos HLA-DR/análise , Humanos , Receptores de Lipopolissacarídeos/sangue , Ativação Linfocitária , Masculino , Glicoproteínas de Membrana/análise , Plasma/química , Resultado do TratamentoRESUMO
BACKGROUND: Although the anti-HIV-1 effects of vitamin D (VitD) have been reported, mechanisms behind such protection remain largely unexplored. METHODS: The effects of two precursor forms (cholecalciferol/calciol at 0.01, 1 and 100 nM and calcidiol at 100 and 250 nM) on HIV-1 infection, immune activation, and gene expression were analyzed in vitro in cells of Colombian and Italian healthy donors. We quantified levels of released p24 by enzyme-linked immunosorbent assay, of intracellular p24 and cell-surface expression of CD38 and HLA-DR by flow cytometry, and mRNA expression of antiviral and immunoregulatory genes by real-time reverse transcription-polymerase chain reaction. RESULTS: Cholecalciferol decreased the frequency of HIV-1-infected p24CD4 T cells and levels of p24 in supernatants in a dose-dependent manner. Moreover, the CD4CD38HLA-DR and CD4CD38HLA-DR subpopulations were more susceptible to infection but displayed the greatest cholecalciferol-induced decreases in infection rate by an X4-tropic strain. Likewise, cholecalciferol at its highest concentration decreased the frequency of CD38HLA-DR but not of CD38HLA-DR T-cell subsets. Analyzing the effects of calcidiol, the main VitD source for immune cells and an R5-tropic strain as the most frequently transmitted virus, a reduction in HIV-1 productive infection was also observed. In addition, an increase in mRNA expression of APOBEC3G and PI3 and a reduction of TRIM22 and CCR5 expression, this latter positively correlated with p24 levels, was noted. CONCLUSIONS: VitD reduces HIV-1 infection in T cells possibly by inducing antiviral gene expression, reducing the viral co-receptor CCR5 and, at least at the highest cholecalciferol concentration, by promoting an HIV-1-restrictive CD38HLA-DR immunophenotype.
Assuntos
Fármacos Anti-HIV/farmacologia , Calcifediol/farmacologia , Colecalciferol/farmacologia , HIV-1/efeitos dos fármacos , HIV-1/crescimento & desenvolvimento , Fatores Imunológicos/farmacologia , ADP-Ribosil Ciclase 1/análise , Células Cultivadas , Colômbia , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Perfilação da Expressão Gênica , Proteína do Núcleo p24 do HIV/análise , Antígenos HLA-DR/análise , Humanos , Imunidade Inata/efeitos dos fármacos , Itália , Leucócitos Mononucleares/química , Leucócitos Mononucleares/virologia , Glicoproteínas de Membrana/análise , Reação em Cadeia da Polimerase em Tempo RealRESUMO
HIV Nef-mediated up-regulation of invariant chain (Ii chain, also CD74) is presumed to play an active role in HIV immunopathogenesis. However, this has not been definitely ascertained. In order to help elucidate this hypothesis, Ii chain, CD4, HLA-DR and HLA-ABC expression was analyzed ex vivo in monocyte-derived macrophages (MDMs) from HIV(+) subjects. Viral load, CD4(+) T cell count and immune activation were also determined in enrolled subjects. Correlations between these parameters and the modulation of cell surface molecules in infected cells were studied. Ii chain expression was found to be up-regulated in infected MDMs derived from all patients but one (median fold up-regulation 2.47±1.82 (range 0.87-7.36)). Moreover, the magnitude of Ii chain up-regulation significantly correlated with higher activation of B and CD4(+) T cells (studied by HLA-DR and CD38 expression). On the other hand, lower HLA-ABC (i.e. stronger down-regulation) in infected MDMs was associated with higher CD4 counts. No correlation was observed between the magnitude of Ii chain up-regulation and the other Nef functions studied here. This is the first study reporting that Ii chain up-regulation occurs on naturally infected antigen presenting cells obtained directly from HIV(+) subjects. Moreover, it is also shown that the magnitude of this up-regulation correlates with immune activation. This allows postulating an alternative hypothesis regarding the contribution of Ii chain up-regulation to HIV-mediated immune damage.
Assuntos
Síndrome da Imunodeficiência Adquirida/imunologia , Síndrome da Imunodeficiência Adquirida/patologia , Antígenos de Diferenciação de Linfócitos B/biossíntese , HIV/imunologia , HIV/patogenicidade , Antígenos de Histocompatibilidade Classe II/biossíntese , ADP-Ribosil Ciclase 1/análise , Síndrome da Imunodeficiência Adquirida/virologia , Linfócitos B/química , Linfócitos B/imunologia , Contagem de Linfócito CD4 , Linfócitos T CD4-Positivos/química , Linfócitos T CD4-Positivos/imunologia , Células Cultivadas , Perfilação da Expressão Gênica , Antígenos HLA-DR/análise , Humanos , Macrófagos/imunologia , Macrófagos/virologia , Glicoproteínas de Membrana/análise , Regulação para Cima , Carga ViralRESUMO
In trying to contribute to our knowledge on the role of Notch and its ligands within the human hematopoietic system, we have assessed the effects of the OP9 stroma cell line - naturally expressing Jagged-1 - transduced with either the Delta-1 gene (OP9-DL1 cells) or with vector alone (OP9-V), on the in vitro growth of two different hematopoietic cell populations. Primitive (CD34(+) CD38(-) Lin(-)) and intermediate (CD34(+) CD38(+) Lin(-)) CD34(+) cell subsets from human cord blood were cultured in the presence of 7 stimulatory cytokines under four different conditions: cytokines alone (control); cytokines and mesenchymal stromal cells; cytokines and OP9-V cells; cytokines and OP9-DL1 cells. Proliferation and expansion were determined after 7days of culture. Culture of CD34(+) CD38(-) Lin(-) cells in the presence of OP9-V or OP9-DL1 cells resulted in a significant increase in the production of new CD34(+) CD38(-) Lin(-) cells (expansion), which expressed increased levels of Notch-1; in contrast, production of total nucleated cells (proliferation) was reduced, as compared to control conditions. In cultures of CD34(+) CD38(+) Lin(-) cells established in the presence of OP9-V or OP9-DL1 cells, expansion was similar to that observed in control conditions, whereas proliferation was also reduced. Interestingly, in these latter cultures we observed production of CD34(+) CD38(-) Lin(-) cells. Our results indicate that, as compared to MSC, OP9 cells were more efficient at inducing self-renewal and/or de novo generation of primitive (CD34(+) CD38(-) Lin(-)) cells, and suggest that such effects were due, at least in part, to the presence of Jagged-1 and DL1.
Assuntos
Antígenos CD34/análise , Proteínas de Ligação ao Cálcio/metabolismo , Sangue Fetal/citologia , Células-Tronco Hematopoéticas/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , ADP-Ribosil Ciclase 1/análise , Adolescente , Adulto , Linhagem Celular , Proliferação de Células , Células Cultivadas , Células-Tronco Hematopoéticas/citologia , Humanos , Proteína Jagged-1 , Ligantes , Receptores Notch/metabolismo , Proteínas Serrate-Jagged , Células Estromais/metabolismo , Adulto JovemRESUMO
BACKGROUND: Concomitant infections may influence HIV progression by causing chronic activation leading to decline in T-cell function. In the Americas, visceral (AVL) and tegumentary leishmaniasis (ATL) have emerged as important opportunistic infections in HIV-AIDS patients and both of those diseases have been implicated as potentially important co-factors in disease progression. We investigated whether leishmaniasis increases lymphocyte activation in HIV-1 co-infected patients. This might contribute to impaired cellular immune function. METHODS: To address this issue we analyzed CD4+ T absolute counts and the proportion of CD8+ T cells expressing CD38 in Leishmania/HIV co-infected patients that recovered after anti-leishmanial therapy. RESULTS: We found that, despite clinical remission of leishmaniasis, AVL co-infected patients presented a more severe immunossupression as suggested by CD4+ T cell counts under 200 cells/mm3, differing from ATL/HIV-AIDS cases that tends to show higher lymphocytes levels (over 350 cells/mm3). Furthermore, five out of nine, AVL/HIV-AIDS presented low CD4+ T cell counts in spite of low or undetectable viral load. Expression of CD38 on CD8+ T lymphocytes was significantly higher in AVL or ATL/HIV-AIDS cases compared to HIV/AIDS patients without leishmaniasis or healthy subjects. CONCLUSIONS: Leishmania infection can increase the degree of immune system activation in individuals concomitantly infected with HIV. In addition, AVL/HIV-AIDS patients can present low CD4+ T cell counts and higher proportion of activated T lymphocytes even when HIV viral load is suppressed under HAART. This fact can cause a misinterpretation of these laboratorial markers in co-infected patients.
Assuntos
Infecções por HIV/complicações , Infecções por HIV/imunologia , Leishmaniose/complicações , Leishmaniose/imunologia , Linfócitos T/imunologia , Carga Viral , ADP-Ribosil Ciclase 1/análise , Adulto , América , Contagem de Linfócito CD4 , Relação CD4-CD8 , Linfócitos T CD8-Positivos/química , Feminino , Infecções por HIV/virologia , HIV-1 , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
CONTEXT AND OBJECTIVE: Counting and separating hematopoietic stem cells from different sources has importance for research and clinical assays. Our aims here were to characterize and quantify hematopoietic cell populations in marrow donors and to evaluate CD34 expression and relate this to engraftment. DESIGN AND SETTING: Cross-sectional study on hematopoietic stem cell assays, using flow cytometry on donor bone marrow samples, for allogenic transplantation patients at two hospitals in São Paulo. METHODS: Immunophenotyping of marrow cells was performed in accordance with positive findings of CD34FITC, CD117PE, CD38PE, CD7FITC, CD33PE, CD10FITC, CD19PE, CD14FITC, CD13PE, CD11cPE, CD15FITIC, CD22PE, CD61FITC and CD56PE monoclonal antibodies in CD45PerCP+ cells, searching for differentiation and maturation regions. CD34+ sorting cells were analyzed for CD38 and CD117. Rh-123 retention was done before and after sorting. Antigen expression and CD34+ cells were correlated with engraftment. RESULTS: In region R1, 0.1% to 2.8% of cells were CD34+/CD45+ and 1.1%, CD34+/CD45-. The main coexpressions of CD45+ cells were CD38, CD22, CD19 and CD56 in R2 and CD33, CD11c, CD14, CD15 and CD61 in R3 and R4. After sorting, 2.2x10(6) CD34+ cells were equivalent to 4.9% of total cells. Coexpression of CD34+/CD38+ and CD34+/CD117+ occurred in 94.9% and 82% of events, respectively. There was a positive relationship between CD34+ cells and engraftment. More than 80% of marrow cells expressed high Rh-123. CD34+ cell sorting showed that cells in regions of more differentiated lineages retained Rh-123 more intensively than in primitive lineage regions. CONCLUSION: We advocate that true stem cells are CD34+/CD45-/CD38-/low-Rh-123 accumulations.
Assuntos
Antígenos CD34 , Células da Medula Óssea , Transplante de Células-Tronco Hematopoéticas , Doadores de Tecidos , ADP-Ribosil Ciclase 1/análise , ADP-Ribosil Ciclase 1/imunologia , Antígenos CD34/análise , Antígenos CD34/imunologia , Células da Medula Óssea/citologia , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Diferenciação Celular , Estudos Transversais , Citometria de Fluxo , Células-Tronco Hematopoéticas/imunologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Imunofenotipagem , Antígenos Comuns de Leucócito/análise , Antígenos Comuns de Leucócito/imunologia , Proteínas Proto-Oncogênicas c-kit/análise , Proteínas Proto-Oncogênicas c-kit/imunologia , Rodamina 123/metabolismo , Transplante HomólogoRESUMO
The immunological response during early human Trypanosoma cruzi infection is not completely understood, despite its role in driving the development of distinct clinical manifestations of chronic infection. Herein we report the results of a descriptive flow cytometric immunophenotyping investigation of major and minor peripheral blood leucocyte subpopulations in T. cruzi-infected children, characterizing the early stages of the indeterminate clinical form of Chagas' disease. Our results indicated significant alterations by comparison with uninfected children, including increased values of pre-natural killer (NK)-cells (CD3- CD16+ CD56-), and higher values of proinflammatory monocytes (CD14+ CD16+ HLA-DR++). The higher values of activated B lymphocytes (CD19+ CD23+) contrasted with impaired T cell activation, indicated by lower values of CD4+ CD38+ and CD4+ HLA-DR+ lymphocytes, a lower frequency of CD8+ CD38+ and CD8+ HLA-DR+ cells; a decreased frequency of CD4+ CD25HIGH regulatory T cells was also observed. These findings reinforce the hypothesis that simultaneous activation of innate and adaptive immunity mechanisms in addition to suppression of adaptive cellular immune response occur during early events of Chagas' disease. Comparative cross-sectional analysis of these immunophenotypes with those exhibited by patients with late chronic indeterminate and cardiac forms of disease suggested that a shift toward high values of macrophage-like cells extended to basal levels of proinflammatory monocytes as well as high values of mature NK cells, NKT and regulatory T cells, may account for limited tissue damage during chronic infection favouring the establishment/maintenance of a lifelong indeterminate clinical form of the disease. On the other hand, development of an adaptive cell-mediated inflammatory immunoprofile characterized by high levels of activated CD8+ cells and basal levels of mature NK cells, NKT and CD4+ CD25HIGH cells might lead to late chronic pathologies associated with chagasic heart disease.
Assuntos
Doença de Chagas/imunologia , Células Matadoras Naturais/imunologia , Macrófagos/imunologia , Trypanosoma cruzi , ADP-Ribosil Ciclase 1/análise , Doença Aguda , Adolescente , Adulto , Idoso , Análise de Variância , Animais , Linfócitos B/imunologia , Biomarcadores/análise , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Estudos de Casos e Controles , Criança , Doença Crônica , Estudos Transversais , Progressão da Doença , Feminino , Citometria de Fluxo , Antígenos HLA-DR/análise , Humanos , Imunofenotipagem , Ativação Linfocitária , Contagem de Linfócitos , Masculino , Pessoa de Meia-Idade , Receptores de Interleucina-2/análiseRESUMO
Identification and enumeration of human hematopoietic stem cells remain problematic, since in vitro and in vivo stem cell assays have different outcomes. We determined if the altered expression of adhesion molecules during stem cell expansion could be a reason for the discrepancy. CD34+CD38- and CD34+CD38+ cells from umbilical cord blood were analyzed before and after culture with thrombopoietin (TPO), FLT-3 ligand (FL) and kit ligand (KL; or stem cell factor) in different combinations: TPO + FL + KL, TPO + FL and TPO, at concentrations of 50 ng/mL each. Cells were immunophenotyped by four-color fluorescence using antibodies against CD11c, CD31, CD49e, CD61, CD62L, CD117, and HLA-DR. Low-density cord blood contained 1.4 +/- 0.9% CD34+ cells, 2.6 +/- 2.1% of which were CD38-negative. CD34+ cells were isolated using immuno-magnetic beads and cultured for up to 7 days. The TPO + FL + KL combination presented the best condition for maintenance of stem cells. The total cell number increased 4.3 +/- 1.8-fold, but the number of viable CD34+ cells decreased by 46 +/- 25%. On the other hand, the fraction of CD34+CD38- cells became 52.0 +/- 29% of all CD34+ cells. The absolute number of CD34+CD38- cells was expanded on average 15 +/- 12-fold when CD34+ cells were cultured with TPO + FL + KL for 7 days. The expression of CD62L, HLA-DR and CD117 was modulated after culture, particularly with TPO + FL + KL, explaining differences between the adhesion and engraftment of primary and cultured candidate stem cells. We conclude that culture of CD34+ cells with TPO + FL + KL results in a significant increase in the number of candidate stem cells with the CD34+CD38- phenotype.