RESUMEN
As hematopoietic progenitors supply a large number of blood cells, therapeutic strategies targeting hematopoietic progenitors are potentially beneficial to eliminate unwanted blood cells, such as leukemic cells and immune cells causing diseases. However, due to their pluripotency, targeting those cells may impair the production of multiple cell lineages, leading to serious side effects such as anemia and increased susceptibility to infection. To minimize those side effects, it is important to identify monopotent progenitors that give rise to a particular cell lineage. Monocytes and monocyte-derived macrophages play important roles in the development of inflammatory diseases and tumors. Recently, we identified human monocyte-restricted progenitors, namely, common monocyte progenitors and pre-monocytes, both of which express high levels of CD64, a well-known monocyte marker. Here, we introduce a dimeric pyrrolobenzodiazepine (dPBD)-conjugated anti-CD64 antibody (anti-CD64-dPBD) that selectively induces the apoptosis of proliferating human monocyte-restricted progenitors but not non-proliferating mature monocytes. Treatment with anti-CD64-dPBD did not affect other types of hematopoietic cells including hematopoietic stem and progenitor cells, neutrophils, lymphocytes and platelets, suggesting that its off-target effects are negligible. In line with these findings, treatment with anti-CD64-dPBD directly killed proliferating monocytic leukemia cells and prevented monocytic leukemia cell generation from bone marrow progenitors of chronic myelomonocytic leukemia patients in a patient-derived xenograft model. Furthermore, by depleting the source of monocytes, treatment with anti-CD64-dPBD ultimately eliminated tumor-associated macrophages and significantly reduced tumor size in humanized mice bearing solid tumors. Given the selective action of anti-CD64-dPBD on proliferating monocyte progenitors and monocytic leukemia cells, it should be a promising tool to target cancers and other monocyte-related inflammatory disorders with minimal side effects on other cell lineages.
Asunto(s)
Antineoplásicos Inmunológicos/farmacología , Inmunoconjugados/farmacología , Células Precursoras de Monocitos y Macrófagos/efectos de los fármacos , Animales , Antineoplásicos Inmunológicos/uso terapéutico , Humanos , Inmunoconjugados/uso terapéutico , Inmunofenotipificación , Ratones , Ratones Noqueados , Ratones Transgénicos , Células Precursoras de Monocitos y Macrófagos/metabolismo , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Células THP-1 , Macrófagos Asociados a Tumores/efectos de los fármacos , Macrófagos Asociados a Tumores/metabolismoRESUMEN
In this study, we developed an analytical method using LC-MS/MS for the simultaneous determination of five bile acids (BAs) that have been recently reported as candidate diagnostic biomarkers for Alzheimer's disease (AD) or AD related factors in the brain. The measurement of BAs in the brains of healthy mice led to the determination of candidate diagnostic markers for AD, such as cholic acid and deoxycholic acid, and other bile acids, such as chenodeoxycholic acid noted for the ameliorating effect on the symptoms of AD. Significant positive correlations were observed between the brain and plasma concentrations of four BAs in healthy young mice. These results indicate that the BA level in the brain may be estimated by the corresponding BA level in the plasma. Thus, our study suggested that the proposed method for the analysis of the five bile acids would aid in the diagnosis of AD or in studies that use AD model mice.
Asunto(s)
Enfermedad de Alzheimer , Ácidos y Sales Biliares , Enfermedad de Alzheimer/diagnóstico , Animales , Biomarcadores , Encéfalo , Cromatografía Liquida , Ratones , Espectrometría de Masas en TándemRESUMEN
Tumor-stromal interaction is implicated in tumor progression. Although CCR1 expression in myeloid cells could be associated with pro-tumor activity, it remains elusive whether disruption of CCR1-mediated myeloid cell accumulation can suppress tumor progression. Here, we investigated the role of CCR1 depletion in myeloid cells in two syngeneic colorectal cancer mouse models: MC38, a transplanted tumor model and CMT93, a liver metastasis model. Both cells induced tumor accumulation of CCR1+ myeloid cells that express MMP2, MMP9, iNOS, and VEGF. Lack of the Ccr1 gene in host mice dramatically reduced MC38 tumor growth as well as CMT93 liver metastasis. To delineate the contribution of CCR1+ myeloid cells, we performed bone marrow (BM) transfer experiments in which sub-lethally irradiated wild-type mice were reconstituted with BM from either wild-type or Ccr1-/- mice. Mice reconstituted with Ccr1-/- BM exhibited marked suppression of MC38 tumor growth and CMT93 liver metastasis, compared with control mice. Consistent with these results, administration of a neutralizing anti-CCR1 monoclonal antibody, KM5908, significantly suppressed MC38 tumor growth and CMT93 liver metastases. Our findings highlight the importance of the application of CCR1 blockade as a therapeutic strategy.
Asunto(s)
Tratamiento Basado en Trasplante de Células y Tejidos , Neoplasias Colorrectales/genética , Neoplasias Hepáticas/genética , Proteínas Serina-Treonina Quinasas/genética , Animales , Médula Ósea/metabolismo , Médula Ósea/patología , Línea Celular Tumoral , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/terapia , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Regulación Neoplásica de la Expresión Génica/genética , Xenoinjertos , Humanos , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/secundario , Neoplasias Hepáticas/terapia , Metaloproteinasa 2 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/genética , Ratones , Células Mieloides/metabolismo , Células Mieloides/patología , Metástasis de la Neoplasia , Óxido Nítrico Sintasa de Tipo II/genética , Proteínas Serina-Treonina Quinasas/uso terapéutico , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
BACKGROUND: Tks5 regulates invadopodium formation, but the precise timing during invadopodium lifetime (initiation, stabilization, maturation) when Tks5 plays a role is not known. RESULTS: We report new findings based on high-resolution spatiotemporal live-cell imaging of invadopodium precursor assembly. Cortactin, N-WASP, cofilin, and actin arrive together to form the invadopodium precursor, followed by Tks5 recruitment. Tks5 is not required for precursor initiation but is needed for precursor stabilization, which requires the interaction of the phox homology (PX) domain of Tks5 with PI(3,4)P2. During precursor formation, PI(3,4)P2 is uniformly distributed but subsequently starts accumulating at the precursor core 3-4 min after core initiation, and conversely, PI(3,4,5)P3 gets enriched in a ring around the precursor core. SHIP2, a 5'-inositol phosphatase, localizes at the invadopodium core and regulates PI(3,4)P2 levels locally at the invadopodium. The timing of SHIP2 arrival at the invadopodium precursor coincides with the onset of PI(3,4)P2 accumulation. Consistent with its late arrival, we found that SHIP2 inhibition does not affect precursor formation but does cause decreases in mature invadopodia and matrix degradation, whereas SHIP2 overexpression increases matrix degradation. CONCLUSIONS: Together, these findings lead us to propose a new sequential model that provides novel insights into molecular mechanisms underlying invadopodium precursor initiation, stabilization, and maturation into a functional invadopodium.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Movimiento Celular , Metástasis de la Neoplasia/patología , Monoéster Fosfórico Hidrolasas/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/química , Proteínas Adaptadoras del Transporte Vesicular/genética , Técnicas Biosensibles , Extensiones de la Superficie Celular/metabolismo , Femenino , Humanos , Inositol Polifosfato 5-Fosfatasas , Proteínas de Microfilamentos/metabolismo , Monoéster Fosfórico Hidrolasas/química , Monoéster Fosfórico Hidrolasas/genéticaRESUMEN
In a previous study, we generated novel antithrombopoietin receptor agonist antibodies as therapeutic candidates. In this report, we investigated the in vivo effects of one of these antibodies, MA01G4344U, on primary human hematopoietic cells using xenotransplantation. NOD/Shi-scid, IL-2Rgamma(null) (NOG) mice were pretreated by total-body irradiation and received a transplant of human cord blood-derived CD34(+) cells. Weekly intraperitoneal injection of MA01G4344U (100 microg/mouse per week) or Peg-rhMGDF (5 microg/mouse per week) or phosphate-buffered saline (PBS) was performed. Human cells in peripheral blood were analyzed by flow cytometry and bone marrow cells were analyzed by flow cytometry and colony assay. MA01G4344U successfully increased the number of human CD41(+) platelets and human CD45(+) cells in peripheral blood. In the bone marrow, MA01G4344U increased the number of human CD45(+)/CD34(+) cells, which resulted in more multilineage progenitor cells. The efficacy of MA01G4344U in promoting primary human hematopoietic cells in vivo suggests its therapeutic potential for thrombocytopenic and pancytopenic disorders.
Asunto(s)
Anticuerpos/farmacología , Células Sanguíneas/efectos de los fármacos , Células de la Médula Ósea/efectos de los fármacos , Receptores de Trombopoyetina/agonistas , Animales , Antígenos CD34/metabolismo , Células Sanguíneas/metabolismo , Células de la Médula Ósea/metabolismo , Citometría de Flujo , Humanos , Antígenos Comunes de Leucocito/metabolismo , Masculino , Ratones , Glicoproteína IIb de Membrana Plaquetaria/metabolismo , Receptores de Trombopoyetina/inmunologíaRESUMEN
We enhanced the activities of two agonist antibodies specific for the thrombopoietin receptor (c-MPL) by switching domains within their constant regions to those of different antibody isotypes. Our results suggest the importance of the hinge region in modulating agonist activity. The antibodies' thrombopoietin-like activity in vitro and in vivo, as well as the desirable pharmacokinetic profile conferred by retaining the whole-IgG structure, suggests that they provide a valuable option for treating thrombocytopenia.
Asunto(s)
Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Mejoramiento Genético/métodos , Ingeniería de Proteínas/métodos , Receptores de Trombopoyetina/genética , Receptores de Trombopoyetina/inmunología , Anticuerpos Monoclonales/química , Estructura Terciaria de Proteína , Receptores de Trombopoyetina/química , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunología , Relación Estructura-ActividadRESUMEN
Previous reports have revealed that estrogen agonists or anti-androgenic chemicals induce abnormal spermiogenesis in rodents. In the seminiferous epithelium, the apical ectoplasmic specialization (ES) is an actin-based (cell-cell) junctional structure developing between the Sertoli cells and spermatids as is the basal ES also--although it is located between adjoining Sertoli cells. In the apical and basal ES are several adhesion complex proteins that control the spermatid developing process. Cortactin, an actin-binding protein, is one of the ES adhesion proteins, combining with several cell-cell adhesions associating proteins. In the present study, 17beta-estradiol (E2, 1.2 microg/kg), bisphenol A (BPA, 2.4 microg/kg), and diethylstilbestrol (DES, 2.5 microg/kg) were subcutaneously injected in ICR 12-week-old male mice. Mice testes were observed for the expression of cortactin protein after E2, BPA, and DES treatments by Western blot analysis, immunohistochemical analysis, and immunoelectron microscopic analysis. Observations showed that the immunoreactivity of the treated testes was significantly decreased. The immunohistochemical reactivity of cortactin in the apical ES was decreased in the treated testis. In immunoelectron microscopic observations, ultrastructural immunolocalizations of cortactin protein in the apical ES by both E2 and BPA were decreased, and the immuno-gold particles of apical and basal ES by DES were much less than the control. In the toxicological field, cortactin may be considered to be one of the indicator proteins of abnormal spermiogenesis which is affected by exogenous chemicals, such as endocrine disrupting chemicals. In summary, this study helps toward understanding the cortactin protein expression underlying the histological abnormalities of spermatogenesis induced by exogenous hormonal chemical treatment.
Asunto(s)
Cortactina/metabolismo , Dietilestilbestrol/farmacología , Regulación hacia Abajo/efectos de los fármacos , Estradiol/farmacología , Estrógenos/agonistas , Fenoles/farmacología , Testículo/efectos de los fármacos , Animales , Compuestos de Bencidrilo , Western Blotting , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos ICR , Testículo/citología , Testículo/metabolismo , Testículo/ultraestructuraRESUMEN
Our previous study revealed that the ectoplasmic specialization (ES) was deleted by the treatment of anti-estrogen, ICI 182.780 (ICI), and anti-androgen, flutamide (FLUT) in mouse testis. Also, expression of cortactin, an F-actin-binding protein, was decreased by the treatment of FLUT in mouse testis. Cortactin has been suggested to promote actin polymerizer at the ES in the testis, and also actin depolymerization is induced by tyrosine phosphorylation of cortactin. The present study revealed that exogenous treatment of ICI and FLUT caused the deletion of the cortactin in the apical ES and the increase of tyrosine phosphorylated cortactin in mouse testis. These results suggest that the sex hormone antagonists', ICI and FLUT, induced actin depolymerization and tyrosine phosphorylation of cortactin in the mouse testis. Also, the present study may be a key to elucidate the adverse affect exogenous compounds that affect spermiation.
Asunto(s)
Antagonistas de Andrógenos/farmacología , Cortactina/metabolismo , Estradiol/análogos & derivados , Antagonistas de Estrógenos/farmacología , Flutamida/farmacología , Células de Sertoli/fisiología , Espermátides/fisiología , Tirosina/metabolismo , Animales , Estradiol/farmacología , Fulvestrant , Masculino , Ratones , Ratones Endogámicos ICR , Fosforilación , Células de Sertoli/ultraestructura , Espermátides/ultraestructura , Testículo/efectos de los fármacosRESUMEN
Flutamide (FLUT) has potent anti-androgenic activity and is used in the medical field and in a screening test to detect endocrinologically active compounds. Our previous study demonstrated that FLUT induced histological deformation of spermatids and ultrastructural defects of the apical ectoplasmic specialization (ES) in the mouse testis. The apical ES is an actin-based junctional structure between the Sertoli cells and germ cells. Cortactin, an actin-binding protein, is found in the actin layer of ES. The protein level of cortactin was decreased in FLUT-treated testes as shown by Western blot analysis. The detailed analysis indicated that the protein level was drastically decreased in FLUT-treated seminiferous tubules of stages from VI to IX. Immunohistochemistry and immunoelectron microscopy showed that FLUT depressed cortactin expression in the apical ES. In addition, the effect of FLUT on cortactin localization appeared between 12 h and 8 days (about 180 h) after a one-day treatment. These results suggest that FLUT depressed the expression of cortactin in the apical ES with stage specificity. Therefore, the initial target of FLUT may be the cell-cell interactions between the Sertoli and germ cells. To our knowledge, this study is the first to document the decrease of cortactin expression in the abnormal apical ES following treatment with FLUT.
Asunto(s)
Antagonistas de Andrógenos/farmacología , Cortactina/biosíntesis , Flutamida/farmacología , Espermátides/metabolismo , Testículo/metabolismo , Animales , Western Blotting , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos ICR , Microscopía Inmunoelectrónica , Espermátides/efectos de los fármacos , Testículo/citología , Testículo/efectos de los fármacosRESUMEN
Spermiogenesis is a complex process that is regulated by a plethora of genes and interactions between germ and somatic cells. Here we report a novel mutant mouse strain that carries a transgene insertional/translocational mutation and exhibits dominant male sterility. We named the mutation dominant spermiogenesis defect (Dspd). In the testes of Dspd mutant mice, spermatids detached from the seminiferous epithelium at different steps of the differentiation process before the completion of spermiogenesis. Microinsemination using spermatids collected from the mutant testes resulted in the birth of normal offspring. These observations indicate that the major cause of Dspd infertility is (are) a defect(s) in the Sertoli cell-spermatid interaction or communication in the seminiferous tubules. Fluorescent in situ hybridization (FISH) analysis revealed a translocation between chromosomes 7F and 14C at the transgene insertion site. The deletion of a genomic region of chromosome 7F greater than 1 megabase and containing at least six genes (Cttn, Fadd, Fgf3, Fgf4, Fgf15, and Ccnd1) was associated with the translocation. Cttn encodes the actin-binding protein cortactin. Immunohistochemical analysis revealed localization of cortactin beside elongated spermatids in wild-type testes; abnormality of cortactin localization was found in mutant testes. These data suggest an important role of cortactin in Sertoli cell-spermatid interactions and in the Dspd phenotype.