RESUMO
Most of the tropical areas have sanitation problems to contend with. The French system of vertical-flow treatment wetlands (FS-VFTW) fed with raw wastewater could be a good water and sludge management solution. The purpose-adapted tropical design can reduce area requirement to below 1â¯m2/population equivalents (p.e.). The Taupinière FS-VFTW on Martinique Island was built according to this design, with one stage but with a saturated layer at the bottom of the filter and a simplified trickling filter (TF) added for further treatment to meet the high performances targeted. Unsaturated/saturated vertical-flow filters (US/S FS-VFTW) have shown improved performances on total nitrogen, carbon and suspended solids removal in temperate climates, but the performances in tropical conditions remain unknown. Here, we report on real-world-operation in the French Overseas Territories (FOT), the reliability and performances of this VFCW tropical-design. The system experienced loading conditions ranging from 30% to 165% of nominal carbonaceous biological oxygen demand (BOD5), as well as tropical rainstorms that brought over 7 times the nominal hydraulic load. Over a period of 3â¯years, 29 campaigns collected 24-h flow-proportional samples at each treatment stage (raw wastewater, FS-VFTW outlet, TF outlet). When applied loads were close to nominal values, the US/S FS-VFTW itself guarantees 85/90/60/50% removal and 125/25/40/50â¯mg/L at the outlet for chemical oxygen demand (COD)/total suspended solids (TSS)/total Kjeldahl nitrogen (TKN)/total nitrogen (TN), respectively. By comparison with US/S systems in mainland France, it appears that the warmer tropical-climate temperatures facilitate both nitrification and denitrification kinetics. Performances in overload conditions confirm that the US/S FS-VFTW remains robust and reliable although COD and TKN removal are impacted, especially after strong tropical rain events. By adding a simple compact trickling filter to a US/S FS-VFTW, the treatment system delivers high-level performances (>95% removal for BOD5, COD, TSS and TKN) at less than 1â¯m2/p.e.
RESUMO
OBJECTIVES: During the reproductive cycle, altered calcium homeostasis is observed due to variable demand for mineral requirements. This results in increased bone resorption during the time period leading up to parturition and subsequent lactation. During lactation, women will lose 1-3% of bone mineral density per month, which is comparable to the loss experienced on an annual basis post-menopausal. The purpose of this study was to determine the effect of parity on bone formation in middle-aged mice. METHODS: Mice were mated and grouped by number of parity and compared with age matched nulliparous controls. Measurements were taken of femoral trabecular and cortical bone. Calcium, protein and alkaline phosphatase levels were also measured. RESULTS: An increase in trabecular bone mineral density was observed when comparing mice that had undergone parity once to the nulliparous control. An overall decrease in trabecular bone mineral density was observed as parity increased from 1 to 5 pregnancies. No alteration was seen in cortical bone formation. No difference was observed when calcium, protein and alkaline phosphatase levels were assessed. CONCLUSIONS: This study demonstrates that number of parity has an impact on trabecular bone formation in middle-aged mice, with substantial changes in bone density seen among the parous groups.
Assuntos
Densidade Óssea/fisiologia , Osso e Ossos/fisiologia , Paridade , Animais , Feminino , Camundongos , Camundongos Endogâmicos C57BL , GravidezRESUMO
Plants are essential in the functioning of constructed wetlands. When setting up systems in tropical areas, Phragmites australis is not always a good choice because of its invasiveness. In vertical-flow constructed wetlands (VFCWs) fed with raw wastewater, the main role of plants is their mechanical action, which helps prevent clogging of the deposited organic matter. Various species have already been used in some tropical climate studies, but generally not for such systems, and no attempt has been made to screen large numbers of alternative species. Here we describe a method to select species among a hundred studied, along with promising plants tested in batches, and at full scale. Species of the order Zingiberales showed good adaptation to the main stresses generated by VFCWs. They have long vegetative cycles, which may require weed growth control after plantation, but low harvesting frequency. Root systems with long rhizomes such as Heliconia psittacorum should take priority to ensure even growth and avoid clumps. To limit the phytosanitary risk with Musaceæ (banana tree), Canna indica or Canna glauca are preferable. Species of the genus Cyperus also demonstrate good adaptation, and could be of interest, especially when a high stem density is required (e.g. planted sludge drying beds).
Assuntos
Desenvolvimento Vegetal , Águas Residuárias/análise , França , Esgotos/análise , Clima Tropical , Eliminação de Resíduos Líquidos/métodos , Purificação da Água , Áreas AlagadasRESUMO
The inhibitor of apoptosis protein Survivin regulates hematopoiesis, although its mechanisms of regulation of hematopoietic stem cells (HSCs) remain largely unknown. While investigating conditional Survivin deletion in mice, we found that Survivin was highly expressed in phenotypically defined HSCs, and Survivin deletion in mice resulted in significantly reduced total marrow HSCs and hematopoietic progenitor cells. Transcriptional analysis of Survivin(-/-) HSCs revealed altered expression of multiple genes not previously linked to Survivin activity. In particular, Survivin deletion significantly reduced expression of the Evi-1 transcription factor indispensable for HSC function, and the downstream Evi-1 target genes Gata2, Pbx1 and Sall2. The loss of HSCs following Survivin deletion and impaired long-term HSC repopulating function could be partially rescued by ectopic Evi-1 expression in Survivin -/- HSCs. These data demonstrate that Survivin partially regulates HSC function by modulating the Evi-1 transcription factor and its downstream targets and identify new genetic pathways in HSCs regulated by Survivin.
Assuntos
Proteínas de Ligação a DNA/genética , Regulação Leucêmica da Expressão Gênica , Células-Tronco Hematopoéticas/citologia , Proteínas Inibidoras de Apoptose/genética , Proto-Oncogenes/genética , Proteínas Repressoras/genética , Fatores de Transcrição/genética , Alelos , Animais , Células da Medula Óssea/citologia , Ciclo Celular , Proliferação de Células , Proteínas de Ligação a DNA/metabolismo , Feminino , Fator de Transcrição GATA2/metabolismo , Deleção de Genes , Hematopoese , Proteínas de Homeodomínio/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteína do Locus do Complexo MDS1 e EVI1 , Camundongos , Camundongos Endogâmicos C57BL , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Fator de Transcrição 1 de Leucemia de Células Pré-B , Retroviridae/genética , Survivina , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
Drug resistance in acute lymphoblastic leukemia (ALL) remains a major problem warranting new treatment strategies. Wnt/catenin signaling is critical for the self-renewal of normal hematopoietic progenitor cells. Deregulated Wnt signaling is evident in chronic and acute myeloid leukemia; however, little is known about ALL. Differential interaction of catenin with either the Kat3 coactivator CREBBP (CREB-binding protein (CBP)) or the highly homologous EP300 (p300) is critical to determine divergent cellular responses and provides a rationale for the regulation of both proliferation and differentiation by the Wnt signaling pathway. Usage of the coactivator CBP by catenin leads to transcriptional activation of cassettes of genes that are involved in maintenance of progenitor cell self-renewal. However, the use of the coactivator p300 leads to activation of genes involved in the initiation of differentiation. ICG-001 is a novel small-molecule modulator of Wnt/catenin signaling, which specifically binds to the N-terminus of CBP and not p300, within amino acids 1-110, thereby disrupting the interaction between CBP and catenin. Here, we report that selective disruption of the CBP/ß- and γ-catenin interactions using ICG-001 leads to differentiation of pre-B ALL cells and loss of self-renewal capacity. Survivin, an inhibitor-of-apoptosis protein, was also downregulated in primary ALL after treatment with ICG-001. Using chromatin immunoprecipitation assay, we demonstrate occupancy of the survivin promoter by CBP that is decreased by ICG-001 in primary ALL. CBP mutations have been recently identified in a significant percentage of ALL patients, however, almost all of the identified mutations reported occur C-terminal to the binding site for ICG-001. Importantly, ICG-001, regardless of CBP mutational status and chromosomal aberration, leads to eradication of drug-resistant primary leukemia in combination with conventional therapy in vitro and significantly prolongs the survival of NOD/SCID mice engrafted with primary ALL. Therefore, specifically inhibiting CBP/catenin transcription represents a novel approach to overcome relapse in ALL.
Assuntos
Antineoplásicos/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Fragmentos de Peptídeos/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Pirimidinonas/farmacologia , Sialoglicoproteínas/metabolismo , beta Catenina/metabolismo , Animais , Asparaginase/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Dexametasona/farmacologia , Regulação para Baixo/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Sinergismo Farmacológico , Humanos , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Mutação , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/genética , Sialoglicoproteínas/antagonistas & inibidores , Sialoglicoproteínas/genética , Survivina , Vincristina/farmacologia , Via de Sinalização Wnt , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Proliferation and differentiation of hematopoietic stem/progenitor cells (HSPC) within bone marrow (BM) niches are regulated by adhesion molecules and cytokines produced by mesenchymal stem/progenitor cells (MPC) and osteoblasts (OB). HSPCs that egresses to peripheral blood are widely used for transplant and granulocyte-colony stimulating factor (G-CSF) is used clinically to induce mobilization. The mechanisms, through which G-CSF regulates HSPC trafficking, however, are not completely understood. Herein we show that G-CSF-driven neutrophil expansion alters the BM niche that leads to HSPC mobilization. Alcam(-)Sca-1(+)MPC and Alcam(+)Sca-1(-) OB are reduced coincident with mobilization, which correlates inversely with BM neutrophil expansion. In mice made neutropenic by the neutrophil-specific anti-Ly6G antibody, G-CSF-mediated reduction in MPC and OB is attenuated and mobilization reduced without an effect on monocytes/macrophages. Neutrophils, expanded in response to G-CSF-induced MPC and OB apoptosis leading to reduced production of BM HSPC retention factors, including stromal cell-derived factor-1, stem cell factor and vascular cell adhesion molecule-1. Blockade of neutrophil reactive oxygen species attenuates G-CSF-mediated MPC and OB apoptosis. These data show that the expansion of BM neutrophils by G-CSF contributes to the transient degradation of retention mechanisms within the BM niche, facilitating enhanced HSPC egress/mobilization.
Assuntos
Apoptose/fisiologia , Células da Medula Óssea/imunologia , Fator Estimulador de Colônias de Granulócitos/administração & dosagem , Células-Tronco Hematopoéticas/imunologia , Neutrófilos/citologia , Animais , Sequência de Bases , Linhagem da Célula , Técnicas de Cocultura , Primers do DNA , Camundongos , Dados de Sequência Molecular , Reação em Cadeia da PolimeraseRESUMO
OBJECTIVES: The aim of this study was to evaluate the effects of prostaglandin E(2) (PGE(2) ) on haematopoietic stem cell (HSC) function and determine its mechanism of action. MATERIALS AND METHODS: HSC were exposed to PGE(2) for 2 h and effects on their homing, engraftment and self-renewal evaluated in vivo. Effects of PGE(2) on HSC cell cycle, CXCR4 expression and migration to SDF-1α were analysed in vitro. Apoptosis was evaluated by examination of survivin expression and active caspase-3 levels. RESULTS: Equivalent haematopoietic reconstitution was demonstrated using 4-fold fewer PGE(2) -treated cells compared to controls. Multilineage reconstitution was stable on secondary transplantation, indicating that PGE(2) affects long-term repopulating HSC (LT-HSC) and that enhanced chimaerism of PGE(2) -pulsed cells results from their initial treatment. PGE(2) increased CXCR4 expression on mouse and human HSC, increased their migration to SDF-1αin vitro and enhanced in vivo marrow homing 2-fold, which was blocked by a CXCR4 receptor antagonist. PGE(2) pulse exposure reduced apoptosis of mouse and human HSC, with increase in endogenous caspase inhibitor survivin, and concomitant decrease in active caspase-3. Two-fold more HSC entered the cell cycle and proliferated within 24 h after PGE(2) pulse exposure. CONCLUSIONS: These studies demonstrate that short-term PGE(2) exposure enhances HSC function and supports the concept of utility of PGE(2) as an ex vivo strategy to improve function of haematopoietic grafts, particularly those where HSC numbers are limited.
Assuntos
Dinoprostona/administração & dosagem , Dinoprostona/farmacologia , Transplante de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/citologia , Animais , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Quimiocina CXCL12/metabolismo , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Receptores CXCR4/metabolismoRESUMO
Hematopoietic stem cell (HSC) transplantation is a potentially curative treatment for numerous hematological malignancies. The transplant procedure as performed today takes advantage of HSC trafficking; either egress of HSC from the bone marrow to the peripheral blood, that is, mobilization, for acquisition of the hematopoietic graft, and/or trafficking of HSC from the peripheral blood to bone marrow niches in the recipient patient, that is HSC homing. Numerous studies, many of which are reviewed herein, have defined hematopoietic regulatory mechanisms mediated by the 20-carbon lipid family of eicosanoids, and recent evidence strongly supports a role for eicosanoids in regulation of hematopoietic trafficking, adding a new role whereby eicosanoids regulate hematopoiesis. Short-term exposure of HSC to the eicosanoid prostaglandin E(2) increases CXCR4 receptor expression, migration and in vivo homing of HSC. In contrast, cannabinoids reduce hematopoietic progenitor cell (HPC) CXCR4 expression and induce HPC mobilization when administered in vivo. Leukotrienes have been shown to alter CD34(+) cell adhesion, migration and regulate HSC proliferation, suggesting that eicosanoids have both opposing and complimentary roles in the regulation of hematopoiesis. As numerous FDA approved compounds regulate eicosanoid signaling or biosynthesis, the utility of eicosanoid-based therapeutic strategies to improve hematopoietic transplantation can be rapidly evaluated.
Assuntos
Eicosanoides/fisiologia , Hematopoese , Células-Tronco Hematopoéticas/fisiologia , Animais , Canabinoides/farmacologia , Movimento Celular/efeitos dos fármacos , Dinoprostona/fisiologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Leucotrienos/fisiologia , Yin-YangRESUMO
Stem cell research is currently focused on totipotent stem cells and their therapeutic potential, however adult stem cells, while restricted to differentiation within their tissue or origin, also have therapeutic utility. Transplantation with bone marrow hematopoietic stem cells (HSC) has been used for curative therapy for decades. More recently, alternative sources of HSC, particularly those induced to exit marrow or mobilize to peripheral blood by G-CSF, have become the most widely used hematopoietic graft and show significant superiority to marrow HSC. The chemokine/chemokine receptor axis also mobilizes HSC that occurs more rapidly than with G-CSF. In mice, the HSC and progenitor cells (HPC) mobilized by the CXCR2 receptor agonist GRObeta can be harvested within minutes of administration and show significantly lower levels of apoptosis, enhanced homing to marrow, expression of more activated integrin receptors and superior repopulation kinetics and more competitive engraftment than the equivalent cells mobilized by G-CSF. These characteristics suggest that chemokine axis-mobilized HSC represent a population of adult stem cells distinct from those mobilized by G-CSF, with superior therapeutic potential. It remains to be determined if the chemokine mobilization axis can be harnessed to mobilize other populations of unique adult stem cells with clinical utility.
Assuntos
Células-Tronco Adultas/efeitos dos fármacos , Quimiocinas/farmacologia , Mobilização de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Receptores de Quimiocinas/agonistas , Células-Tronco Adultas/classificação , Células-Tronco Adultas/citologia , Animais , Quimiocina CXCL12/fisiologia , Quimiocina CXCL2/farmacologia , Fator Estimulador de Colônias de Granulócitos/farmacologia , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/classificação , Células-Tronco Hematopoéticas/citologia , Humanos , Imunofenotipagem , Camundongos , Camundongos Congênicos , Camundongos Endogâmicos C57BL , Transplante de Células-Tronco de Sangue Periférico , Quimera por Radiação , Receptores de Quimiocinas/fisiologia , Receptores de Interleucina-8B/agonistas , Receptores de Interleucina-8B/efeitos dos fármacosRESUMO
The inhibitor-of-apoptosis protein survivin is expressed in most cancers and leukemias and during fetal development, but not in most normal adult tissues. Survivin expression was analyzed in umbilical cord blood (UCB) and adult bone marrow CD34(+) cells and in the factor-dependent MO7e cell line; also investigated was whether survivin expression was regulated by hematopoietic growth factors. Survivin messsenger RNA (mRNA) and protein were expressed in fresh UCB and marrow CD34(+) cells. The combination of thrombopoietin, Flt3 ligand, and stem cell factor upregulated survivin expression in CD34(+) cells within 24 hours; survivin expression was cell-cycle related and highest during G2/M, whereas growth-factor withdrawal resulted in decreased survivin expression. Cell-cycle fractionation of UCB CD34(+) with Hoechst-33342/pyronin-Y demonstrated that survivin message was undetectable in freshly isolated G0 cells, but present in G1 cells. After cytokine stimulation, survivin mRNA and protein expression were observed in both G0 and G1 CD34(+) cells as well as in cells that had progressed to S and G2/M phase, indicating that survivin expression is regulated in all phases of the cell cycle. This contrasts with the expression of survivin predominantly during G2/M in cancer cells. In CD34(+) cells and MO7e cells, growth factor-mediated upregulation of survivin was associated with inhibition of apoptosis, and downregulation of survivin was coincident with increased apoptosis. Furthermore, an inverse correlation between survivin and active caspase-3 was observed in CD34(+) cells. These findings demonstrate that survivin is not a cancer-specific antiapoptotic protein and plays a regulatory role in normal adult hematopoiesis.
Assuntos
Antígenos CD34 , Células da Medula Óssea/metabolismo , Proteínas Cromossômicas não Histona/efeitos dos fármacos , Proteínas Cromossômicas não Histona/fisiologia , Inibidores de Cisteína Proteinase/fisiologia , Sangue Fetal/metabolismo , Fatores de Crescimento de Células Hematopoéticas/farmacologia , Proteínas Associadas aos Microtúbulos , Apoptose/efeitos dos fármacos , Células da Medula Óssea/citologia , Ciclo Celular , Proteínas Cromossômicas não Histona/metabolismo , Inibidores de Cisteína Proteinase/metabolismo , Sangue Fetal/citologia , Citometria de Fluxo , Regulação da Expressão Gênica , Hematopoese/efeitos dos fármacos , Fatores de Crescimento de Células Hematopoéticas/fisiologia , Humanos , Proteínas Inibidoras de Apoptose , Proteínas de Neoplasias , Survivina , Linfócitos T/metabolismo , Células Tumorais CultivadasRESUMO
SB-251353 is an N-terminal truncated form of the human CXC chemokine GRObeta. Recombinant SB-251353 was profiled in murine and rhesus monkey peripheral blood stem cell mobilization and transplantation models. SB-251353 rapidly and transiently mobilized hematopoietic stem cells and neutrophils into the peripheral blood after a single subcutaneous injection. Transplantation of equivalent numbers of hematopoietic stem cells mobilized by SB-251353 into lethally irradiated mice resulted in faster neutrophil and platelet recovery than stem cells mobilized by granulocyte colony-stimulating factor (G-CSF). A single injection of SB-251353 in combination with 4 days of G-CSF administration resulted in augmented stem and progenitor cell mobilization 5-fold greater than G-CSF alone. Augmented stem cell mobilization could also be demonstrated in mice when a single injection of SB-251353 was administered with only one-day treatment with G-CSF. In addition, SB-251353, when used as a single agent or in combination with G-CSF, mobilized long-term repopulating stem cells capable of hematopoietic reconstitution of lethally irradiated mice. In rhesus monkeys, a single injection of SB-251353 induced rapid increases in peripheral blood hematopoietic progenitor cells at a 50-fold lower dose than in mice, which indicates a shift in potency. These studies provide evidence that the use of SB-251353 alone or in combination with G-CSF mobilizes hematopoietic stem cells with long-term repopulating ability. In addition, this treatment may (1) reduce the number of apheresis sessions and/or amount of G-CSF required to collect adequate numbers of hematopoietic stem cells for successful peripheral blood cell transplantation and (2) improve hematopoietic recovery after transplantation.
Assuntos
Quimiocinas CXC/farmacologia , Fatores Quimiotáticos/farmacologia , Substâncias de Crescimento/farmacologia , Mobilização de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intercelular , Animais , Plaquetas/citologia , Plaquetas/efeitos dos fármacos , Quimiocina CXCL1 , Quimiocina CXCL2 , Quimiocinas CXC/administração & dosagem , Quimiocinas CXC/fisiologia , Quimiocinas CXC/uso terapêutico , Fatores Quimiotáticos/administração & dosagem , Fatores Quimiotáticos/fisiologia , Fatores Quimiotáticos/uso terapêutico , Quimioterapia Combinada , Fator Estimulador de Colônias de Granulócitos/uso terapêutico , Substâncias de Crescimento/fisiologia , Substâncias de Crescimento/uso terapêutico , Hematopoese/efeitos dos fármacos , Mobilização de Células-Tronco Hematopoéticas/normas , Transplante de Células-Tronco Hematopoéticas/métodos , Transplante de Células-Tronco Hematopoéticas/normas , Humanos , Macaca mulatta , Masculino , Metaloproteinase 9 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , Proteínas de Neoplasias/farmacologia , Proteínas de Neoplasias/fisiologia , Proteínas de Neoplasias/uso terapêutico , Neutrófilos/citologia , Neutrófilos/efeitos dos fármacos , Especificidade da EspécieRESUMO
UT-7/Epo cells are human factor-dependent erythroleukemic cells, requiring erythropoietin (Epo) for long-term growth. Stem cell factor (SCF) stimulates proliferation of UT-7/Epo only transiently, for three to five days. An investigation of the signal transduction pathways activated by these cytokines in UT-7/Epo cells may identify those signals specifically required for sustained growth. Proliferation assays demonstrate that SCF generates a substantial growth response in UT-7/Epo cells; however, the cells do not multiply or survive past five to seven days. While Epo induces the activation of JAK2 and STAT5, SCF stimulation shows no activation of JAK2 or STATs 1, 3, or 5. The activation of MAPK (p42/44) by SCF was transient, lasting only 30 min, in contrast to Epo, which stimulated phosphorylation of p42/44 for up to 2 h. The expression of the early response genes c-fos, egr1, and cytokine-inducible SH2 protein (CIS) in response to SCF or Epo stimulation demonstrated that the transient expression of p42/44 correlated with the transient expression of c-fos and egr1. In addition, CIS was activated by Epo but not SCF. These data indicate that EpoR, JAK2, and STAT5 activation are not required for the initiation of proliferation of these erythroid cells, that the transient activation of p42/44 correlates with the transient gene expression of c-fos and egr1, and sustained expression of c-fos and egr1 as seen in UT-7/Epo cells continuously grown in Epo may be necessary for long-term proliferation.
Assuntos
Eritropoetina/farmacologia , Proteínas Imediatamente Precoces , Proteínas do Leite , Proteínas Proto-Oncogênicas , Transdução de Sinais/fisiologia , Fator de Células-Tronco/farmacologia , Divisão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteína 1 de Resposta de Crescimento Precoce , Ativação Enzimática , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genes Precoces/efeitos dos fármacos , Genes fos/efeitos dos fármacos , Humanos , Janus Quinase 2 , Leucemia Eritroblástica Aguda , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Recombinantes/farmacologia , Fator de Transcrição STAT5 , Transdução de Sinais/efeitos dos fármacos , Transativadores/metabolismo , Fatores de Transcrição/genética , Células Tumorais CultivadasRESUMO
We have investigated the interaction of the SH2-containing protein tyrosine phosphatase-1 (SHP-1) and Jak2 in an erythropoietin (Epo)-dependent human leukemia cell line, UT-7/Epo, using reciprocal immunoprecipitation and immunoblotting. The Epo-induced kinetics and dose response on phosphorylated Jak2 in anti-SHP-1 precipitates of UT-7/Epo cell lysates were similar to those in direct anti-Jak2 precipitates, suggesting that Jak2 coprecipitated with SHP-1. Furthermore, immunoblotting with anti-Jak2 and anti-SHP-1 antibodies indicated that SHP-1 appeared to be constitutively associated with non-tyrosine-phosphorylated Jak2 in UT-7/Epo cells in the absence of Epo and without phosphorylation of the Epo receptor (EpoR). Competition studies with C-terminal SHP-1 and Jak2 peptides decreased the amounts of SHP-1 and Jak2 detected in immunoprecipitates supporting the specific coprecipitation of SHP-1 and Jak2. In the presence of a recombinant GST-fusion protein containing both the N-terminal and C-terminal SH2 domains of SHP-1, anti-GST precipitated the fusion protein but not cellular Jak2. These studies suggest that SHP-1 and Jak2 are constitutively associated in UT-7/EPO cells. The association is not dependent upon Epo and is not mediated via SHP-1 SH2 binding. Sequential double immunoprecipitation demonstrated that only a small portion of intracellular Jak2 and SHP-1 molecules are constitutively associated. This partial association pattern may allow a more flexible and diverse regulation of Jak2 and SHP-1 activities. Whether Jak2 and SHP-1 are directly associated with each other or are part of a larger complex needs further investigation.
Assuntos
Proteínas Tirosina Fosfatases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas , Anticorpos , Ligação Competitiva , Relação Dose-Resposta a Droga , Eritropoetina/farmacologia , Humanos , Immunoblotting , Peptídeos e Proteínas de Sinalização Intracelular , Janus Quinase 2 , Cinética , Peptídeos , Fosforilação/efeitos dos fármacos , Testes de Precipitina , Ligação Proteica , Proteína Fosfatase 1 , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Proteína Tirosina Fosfatase não Receptora Tipo 6 , Proteínas Tirosina Fosfatases/imunologia , Proteínas Tirosina Quinases/imunologia , Receptores da Eritropoetina , Proteínas Tirosina Fosfatases Contendo o Domínio SH2 , Transdução de Sinais , Células Tumorais Cultivadas , Domínios de Homologia de srcRESUMO
The activity of a novel series of peptidomimetic hematoregulatory compounds, designed based on a pharmacophore model inferred from the structure activity relationships of a peptide SK&F 107647 (1), is reported. These compounds induce a hematopoietic synergistic factor (HSF) which in turn modulates host defense. The compounds may represent novel therapeutic agents in the area of hematoregulation.
Assuntos
Fármacos Cardiovasculares/síntese química , Quimiocinas CXC , Peptídeos e Proteínas de Sinalização Intercelular , Oligopeptídeos/farmacologia , Aminoácidos/química , Animais , Candidíase/tratamento farmacológico , Fármacos Cardiovasculares/farmacologia , Fármacos Cardiovasculares/uso terapêutico , Linhagem Celular , Quimiocina CXCL1 , Fatores Quimiotáticos/metabolismo , Desenho de Fármacos , Fator Estimulador de Colônias de Granulócitos/biossíntese , Fator Estimulador de Colônias de Granulócitos e Macrófagos/biossíntese , Substâncias de Crescimento/metabolismo , Fator Estimulador de Colônias de Macrófagos/biossíntese , Camundongos , Oligopeptídeos/química , Receptores de Droga/química , Receptores de Droga/efeitos dos fármacosRESUMO
SK&F 107647, a previously described synthetic immunomodulatory peptide, indirectly stimulates bone marrow progenitor cells and phagocytic cells, and enhances host defense effector mechanisms in bacterial and fungal infection models in vivo. In vitro, SK&F 107647 induces the production of a soluble mediator that augments colony forming cell (CFU-GM) formation in the presence of CSFs. In this paper we purified and sequenced the stromal cell-derived hematopoietic synergistic factors (HSF) secreted from both murine and human cell lines stimulated with SK&F 107647. Murine HSF is an N-terminal 4-aa truncated form of the CXC chemokine, KC, while human HSF was identified as an N-terminal 4-aa truncated form of the CXC chemokine, GRO beta. In comparison to their full-length forms, truncated KC and truncated GRO beta were 10 million times more potent as synergistic growth stimulants for CFU-GM. Enhanced potency of these novel truncated chemokines relative to their full-length forms was also demonstrated in respiratory burst assays, CD11b Ag expression, and intracellular killing of the opportunistic pathogen, Candida albicans. Administration of truncated KC significantly enhanced survival of mice lethally infected with C. albicans. The results reported herein delineate the biological mechanism of action of SK&F 107647, which functions via the induction of unique specific truncated forms of the chemokines KC and GRO beta. To our knowledge, this represents the first example where any form of KC or GRO beta were purified from marrow stromal cells. Additionally, this is the first demonstration of in vivo efficacy of a CXC chemokine in an animal infectious fungal disease model.
Assuntos
Antifúngicos/isolamento & purificação , Quimiocinas CXC/isolamento & purificação , Fatores Quimiotáticos/isolamento & purificação , Substâncias de Crescimento/isolamento & purificação , Peptídeos e Proteínas de Sinalização Intercelular , Fragmentos de Peptídeos/isolamento & purificação , Adjuvantes Imunológicos/administração & dosagem , Adjuvantes Imunológicos/farmacologia , Sequência de Aminoácidos , Animais , Antifúngicos/sangue , Antifúngicos/imunologia , Células da Medula Óssea/química , Células da Medula Óssea/imunologia , Candidíase/imunologia , Candidíase/mortalidade , Candidíase/prevenção & controle , Linhagem Celular , Quimiocina CXCL1 , Quimiocinas CXC/sangue , Quimiocinas CXC/genética , Quimiocinas CXC/imunologia , Fatores Quimiotáticos/sangue , Fatores Quimiotáticos/genética , Fatores Quimiotáticos/imunologia , Sinergismo Farmacológico , Feminino , Substâncias de Crescimento/sangue , Substâncias de Crescimento/genética , Substâncias de Crescimento/imunologia , Humanos , Soros Imunes/farmacologia , Injeções Intraperitoneais , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Ativação de Neutrófilo/imunologia , Oligopeptídeos/administração & dosagem , Oligopeptídeos/farmacologia , Fragmentos de Peptídeos/sangue , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Proteínas Recombinantes/química , Células Estromais/química , Células Estromais/imunologiaRESUMO
Chemokines have been implicated in the regulation of stem/progenitor cell proliferation and movement. The purpose of the present study was to assess a number of new chemokines for suppressive activity and to delve further into SDF-1-mediated chemotaxis of progenitor cells. This report extends the list of chemokines that have suppressive activity against immature subsets of myeloid progenitors stimulated to proliferate by multiple growth factors to include: MCP-4/CK beta-10, MIP-4/CK beta-7, I-309, TECK, GCP-2, MIG and lymphotactin. The suppressive activity of a number of other chemokines was confirmed. Additionally, pretreatment of the active chemokines with an acetylnitrile solution enhanced specific activity of a number of these chemokines. The new chemokines found to be lacking suppressive activity include: MCP-2, MCP-3, eotaxin-1, MCIF/HCC-1/CK beta-1, TARC, MDC, MPIF-2/eotaxin-2/CK beta-6, SDF-1 and fractalkine/neurotactin. Overall, 19 chemokines, crossing the CC, CXC, and C subgroups, have now been found to be myelosuppressive, and 14 chemokines crossing the CC, CXC and CX3C subgroups have been found to lack myelosuppressive activity under the culture conditions of our assays. Because of the redundancy in chemokine/chemokine receptor interactions, it is not yet clear through which chemokine receptors many of these chemokines signal to elicit suppressive activities. It was also found that SDF-1-induced chemotaxis of progenitors can occur in the presence of fibronectin (FN) and extracellular matrix components and that FN effects involve activation of beta 1-, and possibly alpha 4-, integrins.
Assuntos
Quimiocinas CXC/farmacologia , Quimiocinas/farmacologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/fisiologia , Animais , Divisão Celular/efeitos dos fármacos , Quimiocina CXCL12 , Quimiocinas/fisiologia , Quimiocinas C/farmacologia , Quimiocinas CC/farmacologia , Quimiotaxia , Substâncias de Crescimento/farmacologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , HumanosRESUMO
Two human CC chemokines, SLC/6Ckine/Exodus2/TCA4 and CKbeta-11/MIP-3beta/ELC, are previously reported as efficacious chemoattractants for T- and B-cells and dendritic cells. SLC and CKbeta-11 share only 32% amino acid identity, but are ligands for the same chemokine receptor, CCR7. In this study, we examined chemotactic activity of SLC and CKbeta-11 for NK cells and lymphoid progenitors in bone marrow and thymus. It was found that these two CCR7 ligands are chemoattractants for neonatal cord blood and adult peripheral blood NK cells and cell lines. SLC and CKbeta-11 preferentially attract the CD56(+)CD16(-) NK cell subset over CD56(+)CD16(+) NK cells. SLC and CKbeta-11 also demonstrate selective chemotactic activity on late stage CD34(-)CD19(+)IgM- B-cell progenitors and CD4(+) and CD8(+) single-positive thymocytes, but not early stage progenitors. It was noted that SLC is an efficient desensitizer of CKbeta-11-dependent NK cell chemotaxis, while CKbeta-11 is a weak desensitizer of SLC-dependent chemotaxis. Taken together, these results suggest that SLC and CKbeta-11 have the potential to control trafficking of NK cell subsets and late stage lymphoid progenitors in bone marrow and thymus.
Assuntos
Quimiocinas CC/metabolismo , Quimiotaxia de Leucócito , Células-Tronco Hematopoéticas/fisiologia , Células Matadoras Naturais/fisiologia , Receptores de Quimiocinas/metabolismo , Antígeno CD56/isolamento & purificação , Polaridade Celular , Quimiocina CCL19 , Quimiocina CCL21 , Citotoxicidade Imunológica , Exocitose , Humanos , Ligantes , Subpopulações de Linfócitos/fisiologia , Ligação Proteica , Receptores CCR7 , Receptores de IgG/isolamento & purificação , Timo/citologiaRESUMO
Chemoattractants are potential factors influencing cell migration. Stromal cell-derived factor-1, a CXC chemokine, is the only chemokine reported to have chemotactic activity for hemopoietic progenitor cells (HPC). We report in this work another chemokine of the CC subfamily, which is chemotactic for HPC. Macrophage-inflammatory protein (MIP)-3 beta/EBI1-ligand chemokine/CK beta-11 attracted bone marrow and cord blood CD34+ cells. In contrast to stromal cell-derived factor-1, which attracts multiple types of HPC, MIP-3beta attracted mainly CFU granulocyte macrophage, but not other HPC such as burst-forming unit erythrocyte or CFU granulocyte, erythrocyte, macrophage, and megakaryocyte. Chemoattracted CD34+ cells formed CFU granulocyte macrophage-like colonies, which were morphologically determined as large macrophages. These progenitors were selectively responsive to stimulation by macrophage CSF, demonstrating that MIP-3 beta attracts macrophage progenitors. Expression of CCR7, the receptor for MIP-3 beta, was detected at a mRNA level in the attracted CD34+ cells as well as input CD34+HPC. Expression of MIP-3 beta mRNA was not constitutive, but was inducible in bone marrow stromal cells by inflammatory agents such as bacterial LPS, IFN-gamma, and TNF-alpha. Taken together, our findings suggest that MIP-3 beta is expressed in the bone marrow environment after induction with certain inflammatory cytokines and LPS, and may play a role in trafficking of macrophage progenitors in and out of the bone marrow in inflammatory conditions.