RESUMEN
Chromatographic separation serves as "a workhorse" for downstream process development and plays a key role in the removal of product-related, host-cell-related, and process-related impurities. Complex and poorly characterized raw materials and feed material, low feed concentration, product instability, and poor mechanistic understanding of the processes are some of the critical challenges that are faced during the development of a chromatographic step. Traditional process development is performed as a trial-and-error-based evaluation and often leads to a suboptimal process. A high-throughput process development (HTPD) platform involves the integration of miniaturization, automation, and parallelization and provides a systematic approach for time- and resource-efficient chromatographic process development. Creation of such platforms requires the integration of mechanistic knowledge of the process with various statistical tools for data analysis. The relevance of such a platform is high in view of the constraints with respect to time and resources that the biopharma industry faces today.This protocol describes the steps involved in performing the HTPD of chromatography steps. It describes the operation of a commercially available device (PreDictor™ plates from GE Healthcare). This device is available in 96-well format with 2 or 6 µL well size. We also discuss the challenges that one faces when performing such experiments as well as possible solutions to alleviate them. Besides describing the operation of the device, the protocol also presents an approach for statistical analysis of the data that are gathered from such a platform. A case study involving the use of the protocol for examining ion exchange chromatography of the Granulocyte Colony Stimulating Factor (GCSF), a therapeutic product, is briefly discussed. This is intended to demonstrate the usefulness of this protocol in generating data that are representative of the data obtained at the traditional lab scale. The agreement in the data is indeed very significant (regression coefficient 0.93). We think that this protocol will be of significant value to those involved in performing the high-throughput process development of the chromatography process.
Asunto(s)
Cromatografía por Intercambio Iónico/métodos , Animales , Cromatografía por Intercambio Iónico/instrumentación , Diseño de Equipo , Ensayos Analíticos de Alto Rendimiento/instrumentación , Ensayos Analíticos de Alto Rendimiento/métodos , Humanos , Receptores de Factor Estimulante de Colonias de Granulocito/análisis , Receptores de Factor Estimulante de Colonias de Granulocito/aislamiento & purificaciónRESUMEN
BACKGROUND Chemotherapy for advanced gastric cancer (GC) patients has been the mainstay of therapy for many years. Although adding anti-angiogenic drugs to chemotherapy improves patient survival slightly, identifying anti-angiogenic therapy-sensitive patients remains challenging for oncologists. Granulocyte colony-stimulating factor (G-CSF) promotes tumor growth and angiogenesis, which can be minimized with the anti-G-CSF antibody. Thus, G-CSF might be a potential tumor marker. However, the effects of G-CSF and G-CSFR expression on GC patient survival remain unclear. MATERIAL AND METHODS Seventy GC tissue samples were collected for G-CSF and G-CSFR detection by immunohistochemistry. A total of 40 paired GC tissues and matched adjacent mucosa were used to measure the G-CSF and G-CSFR levels by ELISA. Correlations between G-CSF/G-CSFR and clinical characteristics, VEGF-A levels and overall survival were analyzed. Biological function and underlying mechanistic investigations were carried out using SGC7901 cell lines, and the effects of G-CSF on tumor proliferation, migration, and tube formation were examined. RESULTS The levels of G-CSFR were upregulated in GC tissues compared to normal mucosa tissues. Higher G-CSF expression was associated with later tumor stages and higher tumor VEGF-A and serum CA724 levels, whereas higher G-CSFR expression was associated with lymph node metastasis. Patients with higher G-CSF expression had shorter overall survival times. In vitro, G-CSF stimulated SGC7901 proliferation and migration through the JAK2/STAT3 pathway and accelerated HUVEC tube formation. CONCLUSIONS These data suggest that increased G-CSF and G-CSFR in tumors leads to unfavorable outcomes for GC patients by stimulating tumor proliferation, migration, and angiogenesis, indicating that these factors are potential tumor targets for cancer treatment.
Asunto(s)
Factor Estimulante de Colonias de Granulocitos/análisis , Receptores de Factor Estimulante de Colonias de Granulocito/análisis , Neoplasias Gástricas/metabolismo , Anciano , Anciano de 80 o más Años , Inductores de la Angiogénesis/metabolismo , Biomarcadores de Tumor/sangre , Línea Celular Tumoral , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Factor Estimulante de Colonias de Granulocitos/metabolismo , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Neovascularización Patológica , Pronóstico , Receptores de Factor Estimulante de Colonias de Granulocito/metabolismo , Transducción de Señal/efectos de los fármacos , Neoplasias Gástricas/mortalidad , Análisis de Supervivencia , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Granulocyte colony-stimulating factor (G-CSF) is required for proliferation, differentiation, and survival of cells. It is also a biomarker of human oocyte developmental competence for embryo implantation. In humans, the G-CSF concentration peaks during the ovulatory phase of the ovarian cycle. In this study, the expressions of G-CSF and its receptor were analyzed by polymerase chain reaction in granulosa cells (GCs), CL, cumulus cells (CCs), and oocytes. Cumulus-oocyte complexes were aspirated from antral follicles of 1 to 3 mm (small follicles) and 4 to 6 mm (medium follicles). Cumulus-oocyte complexes from two kinds of follicles were matured in protein-free maturation medium supplemented with various concentrations of G-CSF (0, 10, and 100 ng/mL). By real-time polymerase chain reaction, the expressions of G-CSF and its receptor were detected in GCs, CL, CCs, and oocytes. Interestingly, the G-CSF transcript levels were significantly lower in oocytes than in the other cell types, whereas the G-CSF receptor transcript levels in oocytes were similar to those in GCs. After 44 hours of IVM, no differences in the rate of nuclear maturation were detected; however, the intracellular reactive oxygen species levels in oocytes from both groups of follicles matured with 10 ng/mL of human recombinant G-CSF (hrG-CSF) groups were significantly lower (P < 0.05). After parthenogenetic activation, the cleavage rates were significantly (P < 0.05) higher in 100 ng/mL hrG-CSF-treated small (63.3%) follicles than in 0, 10 ng/mL hrG-CSF-treated small (38.6% and 49.0%, respectively) follicles and 0 ng/mL hrG-CSF-treated medium (52.1%) follicles, and the cleavage rates were significantly (P < 0.05) higher in 10 ng/mL hrG-CSF-treated medium (76.3%) follicles than in all other groups. The blastocyst formation rates were significantly (P < 0.05) higher in 100 ng/mL hrG-CSF-treated small (31.2%) follicles than in 0 and 10 ng/mL hrG-CSF small (10.4% and 15.6%, respectively) follicles, and the 10 ng/mL hrG-CSF medium (45.7%) follicle was significantly (P < 0.05) higher than in all other groups. The total cell number in blastocysts from the 10 ng/mL hrG-CSF medium (106.5) follicles was significantly (P < 0.05) increased compared to 0, 10, 100 ng/mL hrG-CSF small (55.0, 73.7 and 59.5, respectively) follicles and 0, 100 ng/mL hrG-CSF-treated medium (82.5 and 93.5, respectively) follicles. After IVF, the blastocysts stage was significantly (P < 0.05) increased in 10 ng/mL hrG-CSF-treated medium (36.4%) follicles. Fertilization efficiency was significantly high in 100 ng/mL of small (29.1%) and 10 ng/mL of medium (44.0%) follicles. We also examined the Bcl2 and ERK2 transcript levels and found that they were significantly higher in the small and medium follicle treatment groups. In conclusion, these results indicate that hrG-CSF improve the viability of porcine embryos.
Asunto(s)
Desarrollo Embrionario/efectos de los fármacos , Factor Estimulante de Colonias de Granulocitos/farmacología , Técnicas de Maduración In Vitro de los Oocitos/veterinaria , Porcinos , Animales , Blastocisto/fisiología , Células del Cúmulo/química , Femenino , Expresión Génica , Factor Estimulante de Colonias de Granulocitos/análisis , Factor Estimulante de Colonias de Granulocitos/genética , Células de la Granulosa/química , Humanos , Oocitos/química , ARN Mensajero/análisis , Especies Reactivas de Oxígeno/análisis , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de Factor Estimulante de Colonias de Granulocito/análisis , Receptores de Factor Estimulante de Colonias de Granulocito/genética , Proteínas RecombinantesRESUMEN
The enteric nervous system (ENS) controls and modulates gut motility and responds to food intake and to internal and external stimuli such as toxins or inflammation. Its plasticity is maintained throughout life by neural progenitor cells within the enteric stem cell niche. Granulocyte-colony stimulating factor (G-CSF) is known to act not only on cells of the immune system but also on neurons and neural progenitors in the central nervous system (CNS). Here, we demonstrate, for the first time, that G-CSF receptor is present on enteric neurons and progenitors and that G-CSF plays a role in the expansion and differentiation of enteric neural progenitor cells. Cultured mouse ENS-neurospheres show increased expansion with increased G-CSF concentrations, in contrast to CNS-derived spheres. In cultures from differentiated ENS- and CNS-neurospheres, neurite outgrowth density is enhanced depending on the amount of G-CSF in the culture. G-CSF might be an important factor in the regeneration and differentiation of the ENS and might be a useful tool for the investigation and treatment of ENS disorders.
Asunto(s)
Sistema Nervioso Entérico/citología , Sistema Nervioso Entérico/metabolismo , Factor Estimulante de Colonias de Granulocitos/metabolismo , Células-Madre Neurales/citología , Neurogénesis , Neuronas/citología , Animales , Células Cultivadas , Femenino , Factor Estimulante de Colonias de Granulocitos/análisis , Granulocitos/patología , Enfermedad de Hirschsprung/patología , Humanos , Masculino , Ratones , Células-Madre Neurales/metabolismo , Neuronas/metabolismo , Receptores de Factor Estimulante de Colonias de Granulocito/análisis , Receptores de Factor Estimulante de Colonias de Granulocito/metabolismoRESUMEN
After skeletal muscle injury, neutrophils, monocytes, and macrophages infiltrate the damaged area; this is followed by rapid proliferation of myoblasts derived from muscle stem cells (also called satellite cells). Although it is known that inflammation triggers skeletal muscle regeneration, the underlying molecular mechanisms remain incompletely understood. In this study, we show that granulocyte colony-stimulating factor (G-CSF) receptor (G-CSFR) is expressed in developing somites. G-CSFR and G-CSF were expressed in myoblasts of mouse embryos during the midgestational stage but not in mature myocytes. Furthermore, G-CSFR was specifically but transiently expressed in regenerating myocytes present in injured adult mouse skeletal muscle. Neutralization of endogenous G-CSF with a blocking antibody impaired the regeneration process, whereas exogenous G-CSF supported muscle regeneration by promoting the proliferation of regenerating myoblasts. Furthermore, muscle regeneration was markedly impaired in G-CSFR-knockout mice. These findings indicate that G-CSF is crucial for skeletal myocyte development and regeneration and demonstrate the importance of inflammation-mediated induction of muscle regeneration.
Asunto(s)
Proliferación Celular , Factor Estimulante de Colonias de Granulocitos/fisiología , Músculo Esquelético/embriología , Mioblastos/fisiología , Regeneración/fisiología , Animales , Factor Estimulante de Colonias de Granulocitos/análisis , Ratones , Músculo Esquelético/fisiología , Mioblastos/citología , Receptores de Factor Estimulante de Colonias de Granulocito/análisis , Receptores de Factor Estimulante de Colonias de Granulocito/fisiologíaRESUMEN
The recombinant monoclonal antibody trastuzumab has antiproliferative effect on breast cancer (BC) cells with ErbB2 overexpression. We postulated that a mechanism able to modify ErbB2 expression enhances the antitumor effect of trastuzumab. We analyzed whether granulocyte-colony stimulating factor (G-CSF), widely used in adjuvant cancer therapy to alleviate chemotherapy-induced myelotoxicity, could influence ErbB2 expression in BC cells and patients. The expression of ErbB2 (Herceptest) was analyzed in four BC cell lines (BT474, SKBR3, ZR75.1, and T47D) treated with G-CSF and in five samples biopsies from BC patients subjected to G-CSF rescue after chemotherapy. The effects of G-CSF and trastuzumab alone or their combination on cell growth and apoptosis were investigated. G-CSF receptor was detected on all cell lines and BC patients. G-CSF induced upregulation of ErbB2 in SKBR3, ZR75, and T47D cells. This modulation was not associated with an increase in tumor cell growth in vitro. Trastuzumab alone inhibited colony formation in soft agar but did not induce apoptosis on BC cells with no or low ErbB2 genomic amplification. The combination of trastuzumab and G-CSF enhanced the inhibition of tumor colony formation and induced apoptosis on these cells. This effect was further increased by G-CSF pretreatment. Five of nine BC patients showed an increase of Herceptest score after G-CSF administration. G-CSF treatment increases ErbB2 expression in vitro and in vivo enhancing the activity of trastuzumab on BC cell lines inducing apoptosis of BC cells with low or no ErbB2 genomic amplification.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Genes erbB-2 , Factor Estimulante de Colonias de Granulocitos/farmacología , Anticuerpos Monoclonales Humanizados , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Resistencia a Antineoplásicos , Humanos , Receptores de Factor Estimulante de Colonias de Granulocito/análisis , Trastuzumab , Regulación hacia ArribaRESUMEN
AIM: Granulocyte colony-stimulating factor (G-CSF) has been shown to exert protective effects in various tissues and experimental models of ischaemia-induced injury. However, the mechanism of renoprotective action in ischaemia/reperfusion (I/R) renal injury of G-CSF was unknown. METHODS: Male C57BL/6J mice, subjected to renal ischaemia for 45 min, 48 h and 7 days reperfusion, were administered either saline, wortmannin, G-CSF, and G-CSF plus wortmannin 3 days prior to I/R. Saline-treated group served as the control. At 48 h and 7 days of reperfusion, the mice were killed. RESULTS: Significantly, renal dysfunction and morphological injury were identified at 48 h and 7 days after I/R. Wortmannin pretreatment worsened the renal injury significantly. However, G-CSF pretreatment significantly attenuated renal injury, reduced the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positive ratio of renal tubular epithelial cells and inflammation cytokine expression in the kidney. Moreover, G-CSF pretreatment inhibited the expression of Bax and increased the expression of bcl-2 and p-Akt in the kidney. Wortmannin blunted the beneficial effects of G-CSF. CONCLUSION: The cytoprotective action of G-CSF against I/R injury seems to be associated with its anti-apoptotic action mediated by upregulation of p-Akt signal pathway.
Asunto(s)
Factor Estimulante de Colonias de Granulocitos/uso terapéutico , Isquemia/tratamiento farmacológico , Riñón/irrigación sanguínea , Fosfatidilinositol 3-Quinasas/fisiología , Proteínas Proto-Oncogénicas c-akt/fisiología , Daño por Reperfusión/prevención & control , Transducción de Señal/fisiología , Animales , Apoptosis/efectos de los fármacos , Nitrógeno de la Urea Sanguínea , Proliferación Celular/efectos de los fármacos , Citocinas/biosíntesis , Factor Estimulante de Colonias de Granulocitos/farmacología , Riñón/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas Proto-Oncogénicas c-bcl-2/análisis , Receptores de Factor Estimulante de Colonias de Granulocito/análisis , Proteína X Asociada a bcl-2/análisisAsunto(s)
Crisis Blástica/patología , Factor Estimulante de Colonias de Granulocitos/análisis , Interleucina-6/análisis , Síndromes Mielodisplásicos/patología , Receptores de Factor Estimulante de Colonias de Granulocito/análisis , Comunicación Autocrina , Examen de la Médula Ósea , Progresión de la Enfermedad , Factor Estimulante de Colonias de Granulocitos/biosíntesis , Factor Estimulante de Colonias de Granulocitos/fisiología , Humanos , Inmunohistoquímica , Interleucina-6/biosíntesis , Masculino , Persona de Mediana EdadRESUMEN
BACKGROUND: Ewing sarcoma (ES) is a highly vascular malignancy. It has been demonstrated that both angiogenesis and vasculogenesis contribute to the growth of ES tumors. Granulocyte-colony-stimulating factor (G-CSF), a cytokine known to stimulate bone marrow (BM) stem cell production and angiogenesis, is routinely administered to ES patients after chemotherapy. Whether ES cells and patient tumor samples express G-CSF and its receptor (G-CSFR) and whether treatment with this factor enhances tumor growth was examined. METHODS: Human ES cell lines were analyzed for expression of G-CSF and G-CSFR in vitro and in vivo. Sixty-eight paraffin-embedded and 15 frozen tumor specimens from patients with ES were also evaluated for the presence of G-CSF and G-CSFR. The in vivo effect of G-CSF on angiogenesis and BM cell migration was determined. Using a TC/7-1 human ES mouse model, the effect of G-CSF administration on ES tumors was investigated. RESULTS: G-CSF and G-CSFR protein and RNA expression was identified in all ES cell lines and patient samples analyzed. In addition, G-CSF was found to stimulate angiogenesis and BM cell migration in vivo. Tumor growth was found to be significantly increased in mice treated with G-CSF. The average tumor volume for the group treated with G-CSF was 1218 mm(3) compared with 577 mm(3) for the control group (P = .006). CONCLUSIONS: The findings that ES cells and patient tumors expressed both G-CSF and its receptor in vitro and in vivo and that the administration of G-CSF promoted tumor growth in vivo suggest that the potential consequences of G-CSF administration should be investigated further.
Asunto(s)
Neoplasias Óseas/química , Factor Estimulante de Colonias de Granulocitos/administración & dosificación , Factor Estimulante de Colonias de Granulocitos/análisis , Receptores de Factor Estimulante de Colonias de Granulocito/análisis , Sarcoma de Ewing/química , Neoplasias Óseas/irrigación sanguínea , Neoplasias Óseas/patología , Línea Celular Tumoral , Quimiotaxis , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunohistoquímica , Neovascularización Patológica , ARN/análisis , Receptores de Factor Estimulante de Colonias de Granulocito/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sarcoma de Ewing/irrigación sanguínea , Sarcoma de Ewing/patologíaRESUMEN
Signals induced by granulocyte colony-stimulating factor (G-CSF), the major cytokine involved in neutrophil development, are tightly controlled by ligand-induced receptor internalization. Truncated G-CSF receptors (G-CSF-Rs) that fail to internalize show sustained proliferation and defective differentiation signaling. Steady-state forward routing also determines cell surface levels of cytokine receptors, but mechanisms controlling this are poorly understood. Here, we show that WD40 and suppressor of cytokine signaling (SOCS) box protein-2 (Wsb-2), an SOCS box-containing WD40 protein with currently unknown function, binds to the COOH-terminal region of G-CSF-R. Removal of this region did not affect internalization, yet resulted in increased membrane expression of G-CSF-R and enhanced proliferation signaling at the expense of differentiation induction. Conversely, Wsb-2 binding to the G-CSF-R reduced its cell surface expression and inhibited proliferation signaling. These effects depended on the SOCS box involved in ubiquitylation and on cytosolic lysines of G-CSF-R and imply a major role for ubiquitylation through the G-CSF-R C-terminus in forward routing of the receptor. Importantly, the Wsb-2 gene is commonly disrupted by virus integrations in mouse leukemia. We conclude that control of forward routing of G-CSF-R is essential for a balanced response of myeloid progenitors to G-CSF and suggest that disturbance of this balance may contribute to myeloid leukemia.
Asunto(s)
Proteínas Portadoras/metabolismo , Factor Estimulante de Colonias de Granulocitos/metabolismo , Leucemia Mieloide/etiología , Receptores de Factor Estimulante de Colonias de Granulocito/metabolismo , Proteínas Supresoras de la Señalización de Citocinas/metabolismo , Animales , Proteínas Portadoras/análisis , Proteínas Portadoras/genética , Diferenciación Celular , Membrana Celular/química , Membrana Celular/metabolismo , Proliferación Celular , Humanos , Leucemia Mieloide/genética , Leucemia Mieloide/metabolismo , Ratones , Mapeo de Interacción de Proteínas , Receptores de Factor Estimulante de Colonias de Granulocito/análisis , Transducción de Señal , Proteínas Supresoras de la Señalización de Citocinas/análisis , Proteínas Supresoras de la Señalización de Citocinas/genética , Técnicas del Sistema de Dos Híbridos , Ubiquitina/metabolismoRESUMEN
We have experienced a number of cases of AML1/ETO+ acute myelogenous leukemia that showed remission based on bone marrow (BM) morphological criteria, but that revealed clonal abnormalities in most cells by fluorescence in situ hybridization (FISH). Interestingly, most of these cases had AML with AML1/ETO rearrangement. The malignant cells were differentiated and considered mature cells after granulocyte-colony stimulating factor (G-CSF) treatment. To clarify the possible mechanisms underlying this phenomenon, we investigated the expression levels of G-CSFR in AML cells with AML1/ETO rearrangement by flow cytometry and real-time polymerase chain reaction (PCR). The number of AML1/ETO+ cells expressing G-CSFR at baseline was significantly higher than that of AML1/ETO- AML cells (2673 vs 522). In addition, the G-CSFR gene was more highly expressed in AML1/ETO+ cells than in AML1/ETO- cells by real-time PCR. This study reveals that cases showing remission after treatment with G-CSF mostly had leukemia with AML1/ETO rearrangement. This finding might be explained by the higher expression of G-CSF receptor in AML1/ETO+ cells than in AML1/ETO- cells. We recommend that remission should be confirmed by FISH, because malignant clones can be differentiated and masked in morphological examination or chromosome test, especially for AML with AML1/ETO rearrangement.
Asunto(s)
Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Reordenamiento Génico , Factor Estimulante de Colonias de Granulocitos/uso terapéutico , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Adolescente , Adulto , Niño , Femenino , Citometría de Flujo , Humanos , Masculino , Persona de Mediana Edad , Neoplasia Residual , Reacción en Cadena de la Polimerasa , Receptores de Factor Estimulante de Colonias de Granulocito/análisis , Receptores de Factor Estimulante de Colonias de Granulocito/genéticaRESUMEN
PURPOSE: One of the major reasons for failure of radiotherapeutic cancer treatment is the limitation in dose that can be applied to the tumor because of coirradiation of the normal healthy tissue. Late radiation-induced damage reduces the quality of life of the patient and may even be life threatening. Replacement of the radiation-sterilized stem cells with unirradiated autologous stem cells may restore the tissue function. Here, we assessed the potential of granulocyte colony-stimulating factor (G-CSF)-mobilized bone marrow-derived cells (BMC) to regenerate and functionally restore irradiated salivary glands used as a model for normal tissue damage. EXPERIMENTAL DESIGN: Male-eGFP+ bone marrow chimeric female C57BL/6 mice were treated with G-CSF, 10 to 60 days after local salivary gland irradiation. Four months after irradiation, salivary gland morphology and flow rate were assessed. RESULTS: G-CSF treatment induced homing of large number of labeled BMCs to the submandibular glands after irradiation. These animals showed significant increased gland weight, number of acinar cells, and salivary flow rates. Donor cells expressed surface markers specific for hematopoietic or endothelial/mesenchymal cells. However, salivary gland acinar cells neither express the G-CSF receptor nor contained the GFP/Y chromosome donor cell label. CONCLUSIONS: The results show that BMCs home to damaged salivary glands after mobilization and induce repair processes, which improve function and morphology. This process does not involve transdifferentiation of BMCs to salivary gland cells. Mobilization of BMCs could become a promising modality to ameliorate radiation-induced complications after radiotherapy.
Asunto(s)
Células de la Médula Ósea/efectos de los fármacos , Trasplante de Médula Ósea , Factor Estimulante de Colonias de Granulocitos/farmacología , Glándulas Salivales/efectos de los fármacos , Animales , Células de la Médula Ósea/química , Células de la Médula Ósea/metabolismo , Femenino , Expresión Génica/efectos de los fármacos , Factor Estimulante de Colonias de Granulocitos/administración & dosificación , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Movilización de Célula Madre Hematopoyética , Inmunohistoquímica , Hibridación in Situ , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Receptores de Factor Estimulante de Colonias de Granulocito/análisis , Receptores de Factor Estimulante de Colonias de Granulocito/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Glándulas Salivales/patología , Glándulas Salivales/efectos de la radiación , Factores de TiempoRESUMEN
Recently we reported that macrophage colony-stimulating factor (M-CSF) can mobilize endothelial progenitor cells (EPCs) from the bone marrow into the peripheral blood, resulting in an increase in the number of blood vessels and augmentation of blood flow in the ischemia-induced legs. M-CSF accelerates neovascularization of ischemic lesions resulting from the mobilization of EPCs. In the present paper, we analyze the mechanisms underling the mobilization of EPCs by M-CSF. M-CSF augments the production of vascular endothelial growth factor (VEGF) from the bone marrow cells, especially from myeloid lineage cells. In vivo administration of anti-VEGF antibody abrogates both the acceleration of the recovery of blood flow in the ischemia-induced limbs by M-CSF and the augmentation of the mobilization of EPCs induced by M-CSF. These results suggest that the M-CSF contributes to rapid recovery of blood flow in ischemic lesions by mobilization of EPCs from the bone marrow through augmentation of VEGF production in the bone marrow and that the VEGF is mainly produced by myeloid lineage cells.
Asunto(s)
Células Endoteliales/citología , Endotelio Vascular/citología , Miembro Posterior/irrigación sanguínea , Isquemia/tratamiento farmacológico , Factor Estimulante de Colonias de Macrófagos/fisiología , Neovascularización Patológica , Animales , Células de la Médula Ósea/química , Células Cultivadas , Modelos Animales de Enfermedad , Células Endoteliales/efectos de los fármacos , Células Endoteliales/fisiología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/fisiología , Factor Estimulante de Colonias de Granulocitos/genética , Factor Estimulante de Colonias de Granulocitos/farmacología , Miembro Posterior/fisiopatología , Humanos , Isquemia/fisiopatología , Factor Estimulante de Colonias de Macrófagos/genética , Factor Estimulante de Colonias de Macrófagos/farmacología , Ratones , Ratones Endogámicos C57BL , Receptores de Factor Estimulante de Colonias de Granulocito/análisis , Receptores de Factor Estimulante de Colonias de Granulocito/metabolismo , Proteínas Recombinantes/metabolismo , Flujo Sanguíneo Regional/efectos de los fármacosRESUMEN
BACKGROUND: Our previous study showed that granulocyte-colony stimulating factor (G-CSF) enhanced bone-marrow-cell migration into the injured heart and that bone-marrow cells differentiated into cardiomyocytes. However, the number of bone-marrow-derived cardiomyocytes seems too small to have a direct, positive impact on pump function. Therefore, we hypothesized that G-CSF directly could affect the host myocardium through G-CSF receptors (G-CSFRs). METHODS: In experiment 1, we cultured normal mouse heart cells with G-CSF at concentrations of 0, 1, 10, 50, and 100 ng/ml. In experiment 2, we cultured heart cells derived from a recipient with idiopathic cardiomyopathy (IDCM) after heart transplantation. We compared the total number of heart cells and Ki67- and troponin I (TnI)-positive cells with/without G-CSF at 50 ng/ml. We also performed immunochemical staining of the heart specimen from a recipient with IDCM using a rabbit polyclonal anti-G-CSFR antibody. RESULTS: In experiment 1, mouse heart cells with G-CSF (50 ng/ml) proliferated maximally. In experiment 2, the total numbers of heart cells, Ki67-positive cells. TnI-positive cells, Ki67- and TnI-double-positive cells in the G-CSF group were greater than those in the non-G-CSF group at Days 14 and 28 (p <0.05). In the IDCM heart, G-CSFRs on cardiomyocytes were expressed heterogeneously and widely. CONCLUSIONS: Granulocyte-colony stimulating factor directly enhanced the proliferation of TnI-positive cells derived from a recipient with IDCM through the G-CSFR.
Asunto(s)
Cardiomiopatía Dilatada/patología , División Celular/efectos de los fármacos , Factor Estimulante de Colonias de Granulocitos/farmacología , Miocardio/química , Miocardio/patología , Receptores de Factor Estimulante de Colonias de Granulocito/análisis , Troponina I/análisis , Adulto , Animales , Cardiomiopatía Dilatada/cirugía , Células Cultivadas , Trasplante de Corazón , Humanos , Inmunohistoquímica , Antígeno Ki-67/análisis , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
The aim of this study was to evaluate G-CSF receptor (G-CSFr) expression on myeloid blasts, its prognostic significance and role in growth factor use and the safety and efficacy of G-CSF in the treatment of AML. Expression of G-CSFr, CD11a, CD11b, CD11c, CD13, CD33 and CD34 were analyzed with flow cytometry in 101 patients with AML aged 15-60 years. Results were reported as a percentage of positive cells. G-CSFr expression rate was found to be higher in M2 and M3 but lower in M5, M6 phenotypes, and in secondary leukemia. Patients were randomized for G-CSF use. Of 101 cases 51 received G-CSF. The overall remission rate was 68.7%. G-CSF use did not seem to have any effect on the remission rates. The median time to reach neutrophil counts > or = 1000/microliter in cases receiving G-CSF was 23 days, and 28 days in the control group (p < 0.01). G-CSF significantly reduced the number of febrile days (p < 0.01). Early and late relapses of 8 and 16 were observed during follow-up which was not effected by G-CSF use. In patients who were G-CSFr(+), G-CSF use did not alter overall survival rate. Univariate and multivariate analysis have revealed that not sex, G-CSF use or G-CSFr but age, FAB subtype and performance status at diagnosis were the important factors on both overall and disease free survival. We have demonstrated no beneficial effect of G-CSFr analysis on in vivo G-CSF use.
Asunto(s)
Factor Estimulante de Colonias de Granulocitos/uso terapéutico , Leucemia Mieloide/diagnóstico , Leucemia Mieloide/tratamiento farmacológico , Receptores de Factor Estimulante de Colonias de Granulocito/análisis , Enfermedad Aguda , Adulto , Anciano , Crisis Blástica/patología , Supervivencia sin Enfermedad , Femenino , Citometría de Flujo/métodos , Factor Estimulante de Colonias de Granulocitos/farmacología , Humanos , Inmunofenotipificación , Leucemia Mieloide/mortalidad , Masculino , Persona de Mediana Edad , Pronóstico , Tasa de Supervivencia , Resultado del TratamientoRESUMEN
CD34++ cells from 45 patients with myelodysplastic syndrome (MDS) and MDS-acute myeloid leukaemia (MDS-AML) were observed by flow cytometry for the expression of granulocyte colony-stimulating factor receptor (G-CSFR). Ten patients had a significantly reduced expression of G-CSFR. Late stages of disease showed a higher proportion of either high or low G-CSFR expression than earlier stages. In MDS refractory anaemia (RA), G-CSFR was inversely related to CD33 expression. Most patients (9/10) with low G-CSFR expression had neutropenia of the peripheral blood. Neutropenia was less common in the normal group, but also occurred in the high expression group. No neutrophil response was observed following G-CSF administration to MDS-AML patients (6/6) with low G-CSFR expression. In the high expression group, patients (3/3) showed a response to G-CSF while, in the normal group (1/2), the response was minor. In the normal- or high-receptor-expressing groups, the receptors were functionally active in terms of apoptosis but not proliferation and clonogenic growth, although no clear correlation to receptor expression was observed. The G-CSFR signal transduction pathway in the normal and high group was not deficient of messenger RNA for either janus kinases (Jaks) or signal transducers and activators of transcription (Stats). These findings suggest that the lowered expression of G-CSFR may cause neutropenia in MDS and MDS-AML patients and, therefore, may partially explain the neutropenia in myelodysplastic patients.
Asunto(s)
Antígenos CD34/inmunología , Leucemia Mieloide/inmunología , Síndromes Mielodisplásicos/inmunología , Receptores de Factor Estimulante de Colonias de Granulocito/metabolismo , Linfocitos T/inmunología , Enfermedad Aguda , Anciano , Anciano de 80 o más Años , Apoptosis , División Celular , Células Cultivadas , Femenino , Factor Estimulante de Colonias de Granulocitos/uso terapéutico , Granulocitos/efectos de los fármacos , Humanos , Masculino , Persona de Mediana Edad , Síndromes Mielodisplásicos/tratamiento farmacológico , Neutropenia/inmunología , Receptores de Factor Estimulante de Colonias de Granulocito/análisis , Proteínas Recombinantes , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
We examined the expression of granule constituent genes in myeloid progenitor cells during proliferation and differentiation in patients with severe congenital neutropenia (SCN). The heterozygous mutation of the neutrophil elastase gene was identified in two of four patients. The CD34+/granulocyte-colony stimulating factor receptor (G-CSFR)+ cells of SCN patients showed defective responsiveness to G-CSF in serum-deprived culture. The CD34+/G-CSFR+ cells expressed low levels of the granule constituent mRNAs. The transcription levels of primary granule enzyme genes in CD34+/G-CSFR+ cells were gradually enhanced and then decreased when cells were induced toward myeloid lineage with G-CSF in normal subjects. However, the primary up-regulation and the following down-regulation of these enzyme transcriptions were not clearly observed in SCN patients. No differences in expressions of the lactoferrin gene were seen between normal subjects and patients with SCN. We hypothesize that the abnormal regulation of the transcription in primary granule constituents might involve the defective proliferation and differentiation of myeloid cells in patients with SCN.
Asunto(s)
Células de la Médula Ósea/metabolismo , Regulación de la Expresión Génica , Elastasa de Leucocito/genética , Neutropenia/congénito , Neutropenia/metabolismo , Transcripción Genética , Diferenciación Celular , División Celular , Células Cultivadas , Niño , Preescolar , Femenino , Humanos , Masculino , Peroxidasa/genética , Reacción en Cadena de la Polimerasa , Receptores de Factor Estimulante de Colonias de Granulocito/análisisRESUMEN
OBJECTIVES: Shwachman-Diamond syndrome (SDS) is characterized by varying degrees of marrow failure. Retrospective studies suggested a high propensity for malignant myeloid transformation into myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). The study's aims were to determine the cellular and molecular characteristics as well as the clinical course of malignant myeloid transformation and clonal marrow disease in patients with SDS. METHODS: This is a longitudinal prospective study of 14 patients recruited for annual hematological evaluations. Results of baseline and serial hematological assessments for up to 5 years are reported. RESULTS: Clonal marrow cytogenetic abnormalities (CMCA) were detected in 4 patients (29%) on first testing or at follow-up. The abnormalities were del(20q) in two patients, i(7q) in one, and combined del(20q) and i(7q) in one. The following tests did not distinguish patients with CMCA from other SDS patients: severity of peripheral cytopenia, fetal hemoglobin levels, percentage of marrow CD34+ cells, colony growth from marrow CD34+ cells, cluster-to-colony ratio, marrow stromal function, percentage of marrow apoptosis cells, and granulocyte colony-stimulating factor receptor expression. RAS and p53 mutation analysis and AML blast colony assays were uniformly negative. No patients showed progression into more advanced stages of MDS or into AML. In one patient, the abnormal clone became undetectable after 2 years of follow-up. CONCLUSIONS: We conclude that although CMCA in SDS is high, progression into advanced stages of MDS or to overt AML may be slow and difficult to predict. Treatment should be cautious since some abnormal clones can regress.
Asunto(s)
Células de la Médula Ósea/patología , Enfermedades de la Médula Ósea/patología , Enfermedad Aguda , Adolescente , Apoptosis , Recuento de Células Sanguíneas , Enfermedades de la Médula Ósea/sangre , Enfermedades de la Médula Ósea/genética , Transformación Celular Neoplásica , Niño , Preescolar , Aberraciones Cromosómicas , Células Clonales/patología , Ensayo de Unidades Formadoras de Colonias , Progresión de la Enfermedad , Insuficiencia Pancreática Exocrina/sangre , Insuficiencia Pancreática Exocrina/genética , Insuficiencia Pancreática Exocrina/patología , Femenino , Hemoglobina Fetal/análisis , Estudios de Seguimiento , Genes p53 , Genes ras , Trastornos del Crecimiento/sangre , Trastornos del Crecimiento/genética , Trastornos del Crecimiento/patología , Humanos , Lactante , Leucemia Mieloide/etiología , Masculino , Síndromes Mielodisplásicos/etiología , Estudios Prospectivos , Receptores de Factor Estimulante de Colonias de Granulocito/análisis , Síndrome , Receptor fas/análisisRESUMEN
We have studied the characterization of granulocyte colony-stimulating factor receptor (G-CSFR) in human lymphocytes. About one-third to one-quarter of the B lymphocytes from peripheral B-cell sources displayed G-CSF binding on the two-colour immunofluorescence study. The rate of G-CSFR-expressing (G-CSFR+) B cells was higher in bone marrow and cord blood than in peripheral blood, spleen and tonsil. G-CSFR expression was greater in the surface immunoglobulin D (IgD)-positive (sIgD+) B-cell population, but scarce in the sIgD- B-cell population. In tonsil, G-CSFR+ B cells were present among the cells with naive B and germinal-centre B phenotypes, but those with memory B phenotype were rarely found on triple-colour immunofluorescence analysis. Mitogen-activated, but not resting, T lymphocytes also showed G-CSF binding. Several continuous T- and B-cell lines expressed functional G-CSFR, because the addition of G-CSF enhanced the proliferative response of these cell lines. A sequence analysis of G-CSFR mRNA isoforms obtained from the T and B cells revealed that G-CSFR was derived from class I and class IV mRNA. Our results indicated that G-CSFR was constitutively expressed on the B-cell surface and was inducible in T cells.