RESUMO
Dendritic cells (DC) are extremely efficient at generating both prophylactic and therapeutic anti-tumour immunity. We aimed to analyse the respective roles of humoral and cellular immune responses generated in mice vaccinated with bone marrow (BM)-derived DC in terms of in vivo anti-leukaemia effect. We used the murine L1210 B lymphocytic leukaemia genetically modified to express on the cell surface of human CD4 (hCD4) (L1210/hCD4) as a model tumour-associated antigen (TAA). DC cultures were loaded with either purified soluble hCD4 (shCD4) protein or unfractionated L1210/hCD4 extracts and injected as vaccine into mice. The efficacy of these vaccinations was compared with that of vaccination with shCD4 protein emulsified in Freund's adjuvant (FA). We evaluated the immune responses generated after these vaccinal protocols and the survival rate of vaccinated mice subsequently challenged with a lethal injection of L1210/hCD4 cells. Our results demonstrated that vaccination with shCD4 protein or tumour extract-loaded DC mainly generated an hCD4 antigen-specific cell-mediated cytotoxic immune response that was associated with a specific protection against leukaemia. In contrast, vaccination with the protein emulsified in FA only generated potent humoral immune responses that were not protective against leukaemia. Altogether, our results indicate that the unique property of loaded DC to trigger an anti-leukaemia protective effect is mainly associated with cellular immune responses.
Assuntos
Antígenos CD4/imunologia , Vacinas Anticâncer/administração & dosagem , Células Dendríticas/imunologia , Imunoterapia Adotiva/métodos , Leucemia Experimental/prevenção & controle , Linfócitos T Citotóxicos/imunologia , Animais , Antígenos CD4/administração & dosagem , Vacinas Anticâncer/imunologia , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Antígenos de Histocompatibilidade Classe I/imunologia , Leucemia Experimental/imunologia , Camundongos , Camundongos Endogâmicos DBA , Organismos Livres de Patógenos Específicos , Células Tumorais CultivadasRESUMO
Dendritic cells (DC) are professional antigen-presenting cells that can be used as immune adjuvant for anti-tumoural therapies. This approach requires the generation of large quantities of DC that are fully characterized on the immunophenotypical and functional levels. In a murine model, we analysed the in vitro effects of granulocyte-macrophage colony-stimulating factor (GM-CSF) alone or combined with interleukin-4 (IL-4) or Flt3 ligand (Flt3-L) on the number, immunophenotype and functions of bone marrow-derived DC. In GM-CSF cultures, we have identified two populations based on their level of expression of major histocompatibility complex (MHC) class II molecules: MHC-IIhi cells, exhibiting the typical morphology and immunophenotype of myeloid DC (CD11c+ 33D1+ DEC-205+ F4/80+), and MHC-IIlo cells, heterogeneous for DC markers (30% CD11c+; 50% 33D1+; DEC-205-; F4/80+). The addition of Flt3-L to GM-CSF induced a twofold increase in MHC-IIhi DC number; besides, the MHC-IIlo cells lost all DC markers. In contrast, after addition of IL-4 to GM-CSF, the two populations displayed a very similar phenotype (CD11c+ 33D1- DEC-205+ F4/80-), differing only in their expression levels of MHC class II and costimulatory molecules, and showed similar stimulatory activity in mixed leucocyte reaction. We next analysed the migration of these cultured cells after fluorescent labelling. Twenty-four hours after injection into the footpads of mice, fluorescent cells were detected in the draining popliteal lymph nodes, with an enhanced migration when cells were cultured with GM-CSF+Flt3-L. Finally, we showed that MHC-IIhi were more efficient than MHC-IIlo cells in an anti-tumoral vaccination protocol. Altogether, our data highlight the importance of characterizing in vitro-generated DC before use in immunotherapy.
Assuntos
Células da Medula Óssea/imunologia , Vacinas Anticâncer/uso terapêutico , Citocinas/imunologia , Células Dendríticas/imunologia , Animais , Diferenciação Celular/imunologia , Feminino , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Imunofenotipagem , Interleucina-4/imunologia , Leucemia L1210/prevenção & controle , Linfonodos/imunologia , Teste de Cultura Mista de Linfócitos , Masculino , Proteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Células Tumorais Cultivadas , VacinaçãoRESUMO
Dendritic cells (DCs), the most potent antigen-presenting cells, can be generated from CD34+ hematopoietic stem cells and used for generating therapeutic immune responses. To develop immunotherapy protocols based on genetically modified DCs, we have investigated the conditions for high-level transduction of a large amount of CD34+-derived DCs. Thus, we have used an efficient and clinically applicable protocol for the retroviral transduction of cord blood (CB) or mobilized peripheral blood (MPB) CD34+ cells based on infection with gibbon ape leukemia virus (GALV)-pseudotyped retroviral vectors carrying the nls-LacZ reporter gene. Infected cells have been subsequently cultured under conditions allowing their dendritic differentiation. The results show that using a growth factor combination including granulocyte-macrophage colony-stimulating factor plus tumor necrosis factor alpha plus interleukin 4 plus stem cell factor plus Flt3 ligand, more than 70% of DCs derived from CB or MPB CD34+ cells can be transduced. Semiquantitative PCR indicates that at least two proviral copies per cell were detected. Transduced DCs retain normal immunophenotype and potent T cell stimulatory capacity. Finally, by using a semisolid methylcellulose assay for dendritic progenitors (CFU-DCs), we show that more than 90% of CFU-DCs can be transduced. Such a highly efficient retrovirus-mediated gene transfer into CD34+-derived DCs makes it possible to envision the use of this methodology in clinical trials.
Assuntos
Antígenos CD34/imunologia , Células Dendríticas/metabolismo , Sangue Fetal/citologia , Técnicas de Transferência de Genes , Retroviridae/genética , Sequência de Bases , Divisão Celular , Linhagem Celular , Primers do DNA , Células Dendríticas/imunologia , Mobilização de Células-Tronco Hematopoéticas , Humanos , Imunofenotipagem , Linfócitos T/citologia , Transdução GenéticaRESUMO
The tetrapeptide Acetyl-Ser-Asp-Lys-Pro (AcSDKP) has been described as an inhibitor of CFU-S entry into DNA synthesis; as a result, its administration can protect mice against lethal doses of cytosine arabinoside (Ara-C). In the present study, we tested the protective effect of AcSDKP on CFU-MK and CFU-GM progenitor cells in mice treated at lower doses of Ara-C more relevant to human clinical situations. Firstly, we report for the first time that in vitro pre-incubation of murine BM MNC with AcSDKP at concentrations of 10(-10) and 10(-9) M for 48 h decreased CFU-MK, in parallel to CFU-GM, progenitor growth. This resulted in an increase of recovery of these progenitors after exposure to Ara-C. Secondly, we tested the effect of AcSDKP on progenitor cells in vivo in different conditions in Ara-C treated mice. We show that the administration of AcSDKP before starting Ara-C treatment resulted in a significant increase in progenitor CFU-GM, CFU-MK and mature MK numbers, 6 and 8 days after the first Ara-C injection. Interestingly, no difference was observed whether AcSDKP was started 24 or 48 h before Ara-C. In a protocol in which AcSDKP was administered for 8 days starting 48 h before Ara-C treatment, the dose did not appear to be critical at least within the range tested (4 vs. 40 micrograms/injection). In addition, the administration of AcSDKP at 64 micrograms/kg per injection for 5 days and stopping it 3 days before the end of Ara-C treatment, i.e. five instead of eight applications, further increased its protective effect. Thus our results demonstrate protective effect of AcSDKP for progenitors during a fractionated protocol of Ara-C treatment and indicates an importance of the dose and the schedule of administration of AcSDKP in designing future clinical trials.
Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Citarabina/farmacologia , Inibidores do Crescimento/farmacologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Oligopeptídeos/farmacologia , Animais , Contagem de Células/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células-Tronco Hematopoéticas/citologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Oligopeptídeos/administração & dosagem , Fatores de TempoRESUMO
We have developed a murine model to study the involvement of dendritic cells (DC) in human immunodeficiency virus (HIV) routing from an inoculation site to the lymph nodes (LN). Murine bone marrow-derived DC migrate to the draining LN within 24 h after subcutaneous injection. After incubation of these cells with heat-inactivated (Hi) HIV type 1 (HIV-1), HIV RNA sequences were detected in the draining LN only. Upon injection of DC pulsed with infectious HIV, the virus recovered in the draining LN was still able to productively infect human T cells. After a vaginal challenge with Hi HIV-1, the virus could be detected in the iliac and sacral draining LN at 24 h after injection. After an intravenous challenge, the virus could be detected in peripheral LN as soon as 30 min after injection. The specific depletion of a myeloid-related LN DC population, previously shown to take up blood macromolecules and to translocate them into the LN, prevented HIV transport to LN. Together, our data demonstrate the critical role of DC for HIV routing to LN after either a vaginal or an intravenous challenge, which does not require their infection. Therefore, despite the fact that the mouse is not infectable by HIV, this small animal model might be useful to test preventive strategies against HIV.
Assuntos
Células Dendríticas/imunologia , HIV-1/patogenicidade , Linfonodos/virologia , Vagina , Animais , Sequência de Bases , Linhagem Celular , Técnicas de Cocultura , Primers do DNA , Feminino , HIV-1/imunologia , Humanos , Injeções Intravenosas , Injeções Subcutâneas , Camundongos , Camundongos Endogâmicos , Camundongos Transgênicos , Modelos BiológicosRESUMO
The mechanisms were investigated of haematopoietic progenitor growth inhibition, observed after in vitro infection of cord blood mononuclear cells (CBMNC) by a clinical isolate of dengue 3 (29-56DSS). The level of virus replication was not different when CBMNC were inoculated with 29-56DSS compared with a prototype strain of dengue 3 (H-87) which had no inhibitory effect. An inhibitory effect was also observed when cell-free and heat-inactivated supernatants from 29-56DSS cultures, but not from H-87 cultures, were added to cultures of normal CBMNC, suggesting an indirect mechanism via the release of soluble suppressive factor(s). Macrophage inflammatory protein-1alpha (MIP-1alpha) was detected at a significantly higher level in 29-56DSS cultures than in controls. Blocking experiments with anti-MIP-1alpha antibody demonstrated that the inhibitory effect was related at least partly to high MIP-1alpha levels. To our knowledge, this is the first report suggesting an indirect effect of dengue infection on haematopoiesis mediated by a suppressive cytokine.
Assuntos
Vírus da Dengue/fisiologia , Células-Tronco Hematopoéticas/virologia , Proteínas Inflamatórias de Macrófagos/metabolismo , Divisão Celular , Sobrevivência Celular , Quimiocina CCL3 , Quimiocina CCL4 , Células-Tronco Hematopoéticas/metabolismo , Humanos , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/virologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The best methods for transducing hematopoietic progenitor cells usually involve either direct co-cultivation with virus-producing cells or human stromal supportive cells. However, these methods cannot be safely or easily applied to clinical use. Therefore, we aimed at improving retrovirus-mediated gene transfer into hematopoietic progenitors derived from cord blood CD34+ cells using viral supernatant to levels achieved at least with direct co-cultivation and under conditions that are suitable for clinical applications. In a first set of experiments, CD34+ cells were infected with supernatant containing amphotropic retroviral particles carrying the nls-lacZ reporter gene and the effects of centrifugation, cell adhesion to fibronectin, and Polybrene on the transduction of both clonogenic progenitors (CFC) and long-term culture initiating cells (LTC-IC) were studied. Transduction efficiency was evaluated on the percentage and total number of progenitors expressing the beta-galactosidase activity. Results show that a 48-hr infection of CD34+ cells with viral supernatant combining centrifugation at 1000 x g for 3 hr followed by adhesion to fibronectin allows transduction levels for both CFC and LTC-IC to be reached that are as good as using direct co-cultivation. In a second set of experiments, CD34+ cells were infected using this optimized protocol with pseudotyped retroviral particles carrying the gibbon ape leukemia virus (GALV) envelope protein. Under these conditions, between 50 and 100% of CFC and LTC-IC were transduced. Thus, we have developed a protocol capable of highly transducing cord blood progenitors under conditions suitable for a therapeutical use.
Assuntos
Sangue Fetal/virologia , Técnicas de Transferência de Genes , Vetores Genéticos/uso terapêutico , Vírus da Leucemia do Macaco Gibão/genética , Células-Tronco/virologia , Antígenos CD34/análise , Linhagem Celular , Centrifugação , Técnicas de Cocultura , Meios de Cultura , Sangue Fetal/citologia , Sangue Fetal/efeitos dos fármacos , Fibronectinas/farmacologia , Brometo de Hexadimetrina/farmacologia , Humanos , Vírus da Leucemia do Macaco Gibão/crescimento & desenvolvimento , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismoRESUMO
Culturing cord blood CD34+ cells with granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor (TNF)-alpha for 12 days, and stem cell factor (SCF) for 5 days, resulted in a 40- +/- 26-fold expansion in cell numbers, with 38 +/- 20% dendritic cells (DCs). Interleukin (IL)-4 and IL-13, which share properties, were examined first. Adding either one to the former baseline condition beginning on day 0 halved cell growth while the percentage of DCs increased to 60-70%, resulting in unchanged DC yields. Delaying use of IL-4 or IL-13 to day 5 led to 25-fold cell expansion with approximately 80% DC, the yield of which was then twofold over that of baseline control cultures, while numbers of other cells decreased. IL-4 and IL-13 had no additive or antagonistic effect on DC generation. The effect of Flt3 ligand (FL), known to enhance proliferation of hematopoietic progenitors induced by other growth factors, was examined next. FL added alone induced DC in the same manner as SCF. Using both FL and SCF throughout the culture period enhanced total cell recovery fourfold above that of baseline control cultures on day 12 compared with > or =2.5-fold if either one was stopped on day 5. When both FL and SCF were used for 12 days, DC recovery was fivefold that of control cultures, whereas it was to three- to 3.5-fold when either one was stopped on day 5. A similar trend was noted for CD15+ cells, and, to a lesser extent, for CD14+ cells. Finally, using SCF and FL for 12 days, with IL-4 or IL-13 added from day 5 onwards, led to comparably enhanced cell yields relative to control cultures with approximately 60% DC. These data underline the need to use appropriate cytokine combinations and schedules to optimize generation of DCs from CD34+ progenitors. Associated with GM-CSF and TNF-alpha, IL-4 or IL-13 promotes differentiation and maturation of DCs over other myeloid cells. Under the same baseline conditions, FL appears to potentiate SCF throughout the culture period, inducing proliferation and development of DC as well as of other myeloid cells.
Assuntos
Células Dendríticas/citologia , Sangue Fetal/citologia , Células-Tronco Hematopoéticas/citologia , Interleucina-13/farmacologia , Interleucina-4/farmacologia , Antígenos CD34 , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Feminino , Citometria de Fluxo , Células-Tronco Hematopoéticas/efeitos dos fármacos , HumanosRESUMO
Dengue disease, whether it be classical dengue fever (DF), dengue hemorrhagic fever (DHF), or dengue shock syndrome (DSS), is frequently associated with hematologic disorders. The underlying cause of these abnormalities is unknown. To determine if an inhibitory effect on human hematopoietic progenitor growth can be observed, normal cord blood mononuclear cells were exposed to low-passaged clinical isolates from DF, DHF, and DSS patients and to the prototype strain of dengue-3 virus (H-87). In primary methylcellulose cultures, there was no inhibition of colony formation. After an initial 8-day liquid culture, inhibition was observed with the isolates, but strain H-87 had no effect. Furthermore, isolates from patients with DSS showed a more potent inhibitory effect. These data represent the first documented study of in vitro impaired progenitor cell growth by dengue virus and suggest that this inhibition could be dependent upon the isolate tested.
Assuntos
Vírus da Dengue/fisiologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/virologia , Antígenos Virais/análise , Divisão Celular , Células Cultivadas , Criança , Dengue/sangue , Vírus da Dengue/imunologia , Sangue Fetal , Humanos , Leucócitos Mononucleares , RNA Viral/análise , Inoculações Seriadas , Replicação ViralAssuntos
Pestanas/patologia , Neoplasias Palpebrais/patologia , Hidrocistoma/patologia , Neoplasias das Glândulas Sudoríparas/patologia , Neoplasias das Glândulas Suprarrenais/patologia , Feminino , Seguimentos , Humanos , Queratinas/análise , Pessoa de Meia-Idade , Mucina-1/análise , Neoplasias Primárias Múltiplas/patologia , Prolactinoma/patologiaRESUMO
The cycling status of cord blood progenitors and the culture conditions triggering their activation into S-phase have been studied using flow cytometry and a 3H-thymidine suicide assay. Mononuclear cells cultured either in Iscove's modified Dulbecco's medium (IMDM) +/- 10% fetal calf serum ([FCS]; IMDM + FCS) or in Dulbecco's modified Eagle's medium (DMEM) +/- 10% newborn bovine serum ([NBS]; DMEM + NBS) were stimulated by various growth factors (GFs). Results showed that CD34+ cells, clonogenic progenitors (colony forming cells [CFCs]) and long-term culture initiating cells (LTC-IC) present in freshly harvested cord blood were quiescent. CFC numbers were maintained without cycling after 48-h cultures in serum-containing media without GFs. Addition of interleukin 3 (IL-3) + IL-6 + stem cell factor stimulated into S-phase approximately 40% of CFCs within 24-48 h, without modifying their number except in DMEM + NBS where erythroid progenitors decreased. When cells were stimulated in IMDM + FCS by these three GFs + insulin-like growth factor I and basic fibroblast growth factor used at high concentration, more than 50% of CFCs were in S-phase and their total number was maintained. The latter culture conditions also recruited up to 66% of LTC-IC into S-phase. Our data underline the importance of the combination of GFs and culture media used for optimizing the cycling and maintenance of CFCs and LTC-IC within two days.
Assuntos
Sangue Fetal/citologia , Hormônio do Crescimento/farmacologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Antígenos CD34/análise , Proteínas Sanguíneas/farmacologia , Técnicas de Cultura de Células/métodos , Ciclo Celular/efeitos dos fármacos , Células Cultivadas , Feminino , Substâncias de Crescimento/farmacologia , Células-Tronco Hematopoéticas/química , Humanos , Fase S/efeitos dos fármacosAssuntos
Células Dendríticas/virologia , Infecções por HIV/etiologia , HIV/patogenicidade , Células-Tronco Hematopoéticas/virologia , Antígenos CD1/metabolismo , Antígenos CD13/metabolismo , Diferenciação Celular , Células Dendríticas/imunologia , Células Dendríticas/patologia , Infecções por HIV/imunologia , Infecções por HIV/patologia , Células-Tronco Hematopoéticas/imunologia , Células-Tronco Hematopoéticas/patologia , Humanos , Técnicas In VitroAssuntos
Células Dendríticas/virologia , HIV-1/patogenicidade , Linfonodos/virologia , Animais , Células da Medula Óssea/fisiologia , Células da Medula Óssea/virologia , Movimento Celular , Células Dendríticas/fisiologia , Infecções por HIV/etiologia , Infecções por HIV/virologia , Humanos , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos DBA , Camundongos TransgênicosRESUMO
We have compared the effects of AcSDKP, Thymosin beta4 (Tbeta4), MIP1alpha and TGFbeta on acute myeloid leukemia (AML) and B-lineage acute lymphoid leukemia (B-ALL) cells using liquid cultures in the presence of GM-CSF, IL-3 and SCF for AML cells and IL-3 and IL-7 for ALL cells. Each molecule was added daily and cell proliferation was evaluated on day 3 by thymidine incorporation. Whereas TGFbeta was found inhibitory in all the AML and B-ALL cases studied, MIP1alpha was inhibitory in 6/12 AML cases and had no effect on B-ALL cells. AcSDKP and Tbeta4 showed an inhibitory effect in a few cases but only at high doses which were inactive on normal cells. Thus, our study not only confirms the effect of TGFbeta, MIP1alpha and AcSDKP on AML cells but also provides new data concerning their effect on B-ALL and the possible inhibitory effect of AcSDKP at high doses. Furthermore, we show for the first time the effect of Tbeta4 on leukemic cells. Altogether, our data indicate differences of sensitivity of leukemic cells to negative regulators, some leukemias being inhibited by one or several of these molecules whereas others were unresponsive to all used. The clinical relevance of these observations still remains to be determined.
Assuntos
Linfoma de Burkitt/patologia , Divisão Celular/efeitos dos fármacos , Leucemia Mieloide Aguda/patologia , Proteínas Inflamatórias de Macrófagos/farmacologia , Oligopeptídeos/farmacologia , Timosina/farmacologia , Fator de Crescimento Transformador beta/farmacologia , Adolescente , Adulto , Idoso , Quimiocina CCL4 , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
CD1a+ dendritic cells (DC) differentiate from a major population of nonadherent CD13(hi)lin- cells that appear when human cord blood CD34+ hematopoietic progenitor cells are cultured with stem-cell factor, granulocyte/macrophage (MA) colony-stimulating factor, and tumor necrosis factor-alpha (TNF-alpha) for 5 days. CD13hilin- cells, which also comprise MA and granulocyte precursors, are CD4+ and can thus be targets of human immunodeficiency virus (HIV). Low replication was noted when these day 5 cells were infected with lymphotropic HIV-1LA1 (p24: < or = 4 ng/mL on day 8 postinfection [PI]), while high virus production occurred with MA-tropic HIV-1Ba-L, HIV-1Ada, or HIV-1-m-n. (p24: 50 to > or = 1,000 ng/mL). Strong cytopathicity (CPE) was then observed in nonadherent cells as in adherent MA. However, FACS analysis on day 7 PI showed that HIV did not affect differentiation of DC that survived CPE: apart from CD4 downmodulation related to HIV production, overall expression of CD40, CD80, and CD86 costimulatory molecules, and of HLA-DR, was unchanged relative to controls. At that time, the capacity of DC from HIV-infected cultures to stimulate the mixed leukocyte reaction was only altered less than 10-fold. Immunocytochemistry on day 7 PI showed that most HIV-infected cells were included in syncytia that were stained by anti-CD1a, anti-S100, and anti-CD14 antibodies, indicating that syncytia consisted of DC and cells of the MA lineage. Polymerase chain reaction analysis of FACS-sorted CD1a+ cells confirmed that they harbored then HIV DNA. Viral DNA was also detected in CD1a+ DC from noninfected cultures that had been exposed to HIV only after sorting. Therefore, we examined whether in infected cultures DC precursors were infected at the onset or if virus spread later from other infected cells to differentiated DC. This was answered by showing that, 24 hours postexposure to HIV, viral DNA was preferentially detected in day 5 sorted CD13hilin- versus CD13hilin- cells, and that it was found in the CD1a+ progeny of CD13(hi)lin- cells 48 hours later. In addition, HIV replication did not affect myeloid clonogenic progenitors in day 0 to day 7 PI cultures, although viral DNA was detected in colony-forming unit-granulocyte/macrophage (CFU-GM)/CFU-M colonies derived from day 3 and 7 PI cultures. Thus, precursors of DC and their progeny are susceptible to HIV in vitro, but, apart from CPE, the effect of virus production on DC differentiation or function is limited.
Assuntos
Células Dendríticas/imunologia , Infecções por HIV/imunologia , HIV-1/fisiologia , Antígenos CD/biossíntese , Antígenos CD1/análise , Antígenos CD34/análise , Antígeno B7-1/biossíntese , Antígeno B7-2 , Antígenos CD13/análise , Antígenos CD40/biossíntese , Diferenciação Celular/efeitos dos fármacos , Células Clonais , Ensaio de Unidades Formadoras de Colônias , Efeito Citopatogênico Viral , DNA Viral/análise , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/virologia , Sangue Fetal/citologia , HIV-1/isolamento & purificação , Humanos , Teste de Cultura Mista de Linfócitos , Glicoproteínas de Membrana/biossíntese , Reação em Cadeia da Polimerase , Provírus/isolamento & purificação , Replicação ViralRESUMO
In vivo effects of platelet factor 4 (PF4) and tetrapeptide N-acetyl-Ser-Asp-Lys-Pro (AcSDKP) on haemopoietic progenitors were studied in mice treated with 5-fluorouracil (5-FU). The mice were injected with PF4 (40 micrograms/kg) or AcSDKP (4 micrograms/kg) twice at 6 h intervals, and 20 h after the second injection they were given one injection of 5-FU (150 mg/kg). 6, 8 and 13 d later the high proliferative potential-colony forming cell (HPP-CFC), burst-forming unit erythroid (BFU-E), colony forming unit granulocyte-macrophage (CFU-GM) colony forming unit megakaryocyte (CFU-MK), and megakaryocytes (MK) were examined. The results showed that the administration of PF4 or AcSKDP resulted in a significant increase in the number of HPP-CFC on days 6-8 and BFU-E and CFU-GM on day 8 when compared to 5-FU alone. Furthermore, PF4 was found to increase significantly the number of CFU-MK and MK on day 8, which was not observed with AcSDKP. However, both molecules had no obvious effect on peripheral blood cells. These data indicate that PF4 or AcSDKP accelerate the recovery in vivo of HPP-CFC, CFU-GM and BFU-E after 5-FU treatment but their effect may be different on megakaryocytic progenitors and suggests that both molecules may have a haemoprotective effect against chemotherapeutic agents.
Assuntos
Fluoruracila/farmacologia , Hematopoese/efeitos dos fármacos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Oligopeptídeos , Fator Plaquetário 4/farmacologia , Animais , Hemoglobinas/metabolismo , Contagem de Leucócitos , Masculino , Megacariócitos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Oligopeptídeos/farmacologia , Trombocitopenia/patologiaRESUMO
Thymosin beta4 (Tbeta4), isolated from the calf thymus fraction 5, has a ubiquitous localization and plays a pleiotropic role in both the immune and nonimmune systems. Because it contains at its N-terminal end the sequence of a known inhibitor of hematopoiesis, the acetylated tetrapeptide Ac-N-Ser-Asp-Lys-Pro (AcSDKP, Goralatide), we have assayed Tbeta4 on human hematopoietic cells. We demonstrate that it inhibits normal bone marrow progenitor cell growth; indeed, it decreased the growth of both granulo-macrophagic and erythroid progenitors and reduces their percentage in S phase. Furthermore, we show that Tbeta4 reduces both the clonogenicity and the cell proliferation of purified CD34+ cells induced by a combination of seven growth factors. Although Tbeta4's inhibitory effect is very similar to that of AcSDKP, we demonstrate, using neutralizing antibodies and a truncated form of Tbeta4 devoid of the AcSDKP sequence, that the inhibitory effect of Tbeta4 is not mediated by the sequence AcSDKP. These data indicate that Tbeta4 is a novel inhibitor for human normal hematopoietic progenitors.
Assuntos
Inibidores do Crescimento/farmacologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Timosina/farmacologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Bovinos , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias , Células Precursoras Eritroides/citologia , Células Precursoras Eritroides/efeitos dos fármacos , Fatores de Crescimento de Células Hematopoéticas/farmacologia , Humanos , Dados de Sequência Molecular , Oligopeptídeos/antagonistas & inibidores , Oligopeptídeos/imunologia , Oligopeptídeos/farmacologia , Proteínas Recombinantes/farmacologia , Timosina/antagonistas & inibidores , Timosina/imunologiaRESUMO
The spontaneous proliferation and the effects of 8 various growth factors (GF) were evaluated on leukemic cells from 27 patients with B-lineage ALL. Two groups of ALLs were distinguished. ALLs from group I (21 patients) exhibited a low spontaneous proliferative rate and were stimulated by IL-3 + IL-7 +/- SCF and/or LIF, while ALLs from group II (6 patients) had a high spontaneous proliferative rate and did no longer require this combination of GFs for proliferation. No effect of bFGF, IGF-I, IL-10 and IL-11 alone or in combination, was observed. Such differences in the behaviour of B-ALLs indicated that the GF requirement of ALL blasts was not related to the presence of serum in the culture nor to the pattern of reactivity of ALL blasts for B lymphoid markers or CD34 antigen. Furthermore, we showed in 1/9 cases that high proliferation might be due to an overexpression of the bcl-2 proto-oncogene and to the acquisition of an autocrine secretion.
Assuntos
Linfoma de Burkitt/metabolismo , Linfoma de Burkitt/patologia , Citocinas/farmacologia , Substâncias de Crescimento/farmacologia , Proteínas Proto-Oncogênicas/biossíntese , Adulto , Apoptose/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-bcl-2 , RNA Mensageiro/metabolismoRESUMO
This paper will review the present knowledge on two small chemically unrelated peptides, the pentapeptide pGlu-Glu-Asp-Cys-Lys (pEEDCK) and the tetrapeptide acetyl-N-Ser-Asp-Lys-Pro (AcSDKP, Seraspenide) focusing on 1) their inhibitory effects on normal hematopoiesis, 2) their effect on malignant cells, especially leukemic cells, and 3) their potential clinical use as marrow protectors during cancer therapy.
Assuntos
Hematopoese/efeitos dos fármacos , Oligopeptídeos/farmacologia , Animais , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Ácido Pirrolidonocarboxílico/análogos & derivadosRESUMO
The aim of this study was to compare the inhibitory effect of the tetrapeptide AcSDKP, tumor necrosis factor-alpha (TNF-alpha), which contains the sequence of the peptide, transforming growth factor-beta (TGF-beta), and macrophage inflammatory protein-1 alpha (MIP-1 alpha) on sorted CD34+ cells using both proliferation and clonogenic assays. Although a short treatment with any of the molecules decreased the growth of colony-forming unit granulocyte/macrophage (CFU-GM) and burst-forming unit-erythroid (BFU-E) progenitors (except for TNF-alpha as it is a greater inhibitor for CFU-GM), further experiments using a 6-day liquid culture in the presence of a combination of growth factors (recombinant human interleukin-3 [rhIL-3], IL-6, IL-1 beta, GM colony-stimulating factor [GM-CSF], G-CSF, erythropoeitin [Epo], and stem cell factor [SCF]) allowed us to determine a number of differences between their effects: 1) TGF-beta and TNF-alpha induced a stronger decrease in the proliferation and clonogenicity of CD34+ subsets than MIP-1 alpha and AcSDKP, 2) the dose-response curves appeared different, and 3) contrary to TGF-beta and TNF-alpha, AcSDKP and MIP-1 alpha required repeated addition to induce inhibition. Therefore, our data clearly show that while the inhibitory effect of TNF-alpha and AcSDKP appeared to be different, there is a close similarity in the effect of AcSDKP and MIP-1 alpha on normal human progenitor response to the combination of growth factors used.