RESUMO
Resistin-like molecule α (RELMα) is a highly secreted protein in type 2 (Th2) cytokine-induced inflammation including helminth infection and allergy. In infection with Nippostrongylus brasiliensis (Nb), RELMα dampens Th2 inflammatory responses. RELMα is expressed by immune cells, and by epithelial cells (EC); however, the functional impact of immune versus EC-derived RELMα is unknown. We generated bone marrow (BM) chimeras that were RELMα deficient (RELMα-/- ) in BM or non BM cells and infected them with Nb. Non BM RELMα-/- chimeras had comparable inflammatory responses and parasite burdens to RELMα+/+ mice. In contrast, both RELMα-/- and BM RELMα-/- mice exhibited increased Nb-induced lung and intestinal inflammation, correlated with elevated Th2 cytokines and Nb killing. CD11c+ lung macrophages were the dominant BM-derived source of RELMα and can mediate Nb killing. Therefore, we employed a macrophage-worm co-culture system to investigate whether RELMα regulates macrophage-mediated Nb killing. Compared to RELMα+/+ macrophages, RELMα-/- macrophages exhibited increased binding to Nb and functionally impaired Nb development. Supplementation with recombinant RELMα partially reversed this phenotype. Gene expression analysis revealed that RELMα decreased cell adhesion and Fc receptor signaling pathways, which are associated with macrophage-mediated helminth killing. Collectively, these studies demonstrate that BM-derived RELMα is necessary and sufficient to dampen Nb immune responses, and identify that one mechanism of action of RELMα is through inhibiting macrophage recruitment and interaction with Nb. Our findings suggest that RELMα acts as an immune brake that provides mutually beneficial effects for the host and parasite by limiting tissue damage and delaying parasite expulsion.
Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Infecções por Strongylida/imunologia , Trifosfato de Adenosina/metabolismo , Células Epiteliais Alveolares/metabolismo , Animais , Adesão Celular , Células Cultivadas , Técnicas de Cocultura , Células Dendríticas/metabolismo , Feminino , Regulação da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intercelular/deficiência , Peptídeos e Proteínas de Sinalização Intercelular/genética , Macrófagos Alveolares/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nippostrongylus/isolamento & purificação , Nippostrongylus/ultraestrutura , Quimera por Radiação , Proteínas Recombinantes/metabolismo , Infecções por Strongylida/parasitologia , Células Th2/imunologiaRESUMO
Lapses in the prevention of graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (HSCT) warrant novel approaches. Such approaches include, among others, the use of post-transplantation cyclophosphamide (PTC) and proteasome inhibitors. Although PTC alone consistently produces low rates of chronic GVHD, the incidence of acute GVHD remains significant. Inversely, prolonged post-transplantation administration of proteasome inhibitors carries a risk of paradoxical aggravation of GVHD. We examined whether the combination of cyclophosphamide and ixazomib addresses the limitations of each of these agents when used alone to prevent GVHD in mice subjected to allogeneic HSCT across MHC barriers. We chose ixazomib, an orally bioavailable proteasome inhibitor, because of its favorable physiochemical characteristics. The combination of cyclophosphamide and ixazomib improved overall survival of mice in comparison to an untreated control group and to groups receiving either cyclophosphamide alone or ixazomib alone. Furthermore, cyclophosphamide prevented the surge of IL-1ß, GVHD aggravation, and sudden death associated with prolonged administration of ixazomib after HSCT. Finally, we demonstrated that although ixazomib was administered before cyclophosphamide, it did not impair the preferential depletion of proliferating as opposed to resting donor T cells. Our data suggest that the combination of cyclophosphamide and ixazomib for the prevention of GVHD after allogeneic HSCT is promising and merits further investigation in clinical trials.
Assuntos
Compostos de Boro/uso terapêutico , Ciclofosfamida/uso terapêutico , Glicina/análogos & derivados , Doença Enxerto-Hospedeiro/prevenção & controle , Inibidores de Proteassoma/uso terapêutico , Animais , Transplante de Medula Óssea/efeitos adversos , Compostos de Boro/administração & dosagem , Compostos de Boro/farmacologia , Ciclofosfamida/administração & dosagem , Ciclofosfamida/farmacologia , Citocinas/sangue , Esquema de Medicação , Avaliação Pré-Clínica de Medicamentos , Sinergismo Farmacológico , Quimioterapia Combinada , Feminino , Glicina/administração & dosagem , Glicina/farmacologia , Glicina/uso terapêutico , Doença Enxerto-Hospedeiro/tratamento farmacológico , Doença Enxerto-Hospedeiro/patologia , Intestino Delgado/patologia , Depleção Linfocítica , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Inibidores de Proteassoma/administração & dosagem , Inibidores de Proteassoma/farmacologia , Quimera por Radiação , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/transplanteRESUMO
Diamond-Blackfan anaemia (DBA) is a rare congenital disease causing severe anaemia and progressive bone marrow failure. The majority of patients carry mutations in ribosomal proteins, which leads to depletion of erythroid progenitors in the bone marrow. As many as 40% of all DBA patients receive glucocorticoids to alleviate their anaemia. However, despite their use in DBA treatment for more than half a century, the therapeutic mechanisms of glucocorticoids remain largely unknown. Therefore we sought to study disease specific effects of glucocorticoid treatment using a ribosomal protein s19 (Rps19) deficient mouse model of DBA. This study determines for the first time that a mouse model of DBA can respond to glucocorticoid treatment, similar to DBA patients. Our results demonstrate that glucocorticoid treatment reduces apoptosis, rescues erythroid progenitor depletion and premature differentiation of erythroid cells. Furthermore, glucocorticoids prevent Trp53 activation in Rps19-deficient cells- in a disease-specific manner. Dissecting the therapeutic mechanisms behind glucocorticoid treatment of DBA provides indispensible insight into DBA pathogenesis. Identifying mechanisms important for DBA treatment also enables development of more disease-specific treatments of DBA.
Assuntos
Anemia de Diamond-Blackfan/tratamento farmacológico , Eritropoese/efeitos dos fármacos , Prednisolona/uso terapêutico , Proteínas Ribossômicas/deficiência , Proteína Supressora de Tumor p53/fisiologia , Adolescente , Anemia de Diamond-Blackfan/sangue , Animais , Apoptose/efeitos dos fármacos , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p21/biossíntese , Inibidor de Quinase Dependente de Ciclina p21/genética , Dexametasona/farmacologia , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Células Precursoras Eritroides/efeitos dos fármacos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Prednisolona/farmacologia , Quimera por Radiação , Proteínas Ribossômicas/genética , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Regulação para Cima/efeitos dos fármacos , Proteína X Associada a bcl-2/biossíntese , Proteína X Associada a bcl-2/genéticaAssuntos
Fator 15 de Diferenciação de Crescimento/farmacologia , Neutrófilos/fisiologia , Adesividade Plaquetária/fisiologia , Lesão Pulmonar Induzida por Ventilação Mecânica/prevenção & controle , Animais , Plaquetas/efeitos dos fármacos , Plaquetas/fisiologia , Permeabilidade Capilar , Adesão Celular , Quimiotaxia de Leucócito/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Armadilhas Extracelulares/efeitos dos fármacos , Fator 15 de Diferenciação de Crescimento/deficiência , Fator 15 de Diferenciação de Crescimento/genética , Fator 15 de Diferenciação de Crescimento/uso terapêutico , Pulmão/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/patologia , Neutrófilos/efeitos dos fármacos , Adesividade Plaquetária/efeitos dos fármacos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/antagonistas & inibidores , Transfusão de Plaquetas , Edema Pulmonar/fisiopatologia , Edema Pulmonar/prevenção & controle , Troca Gasosa Pulmonar , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Quimera por Radiação , Tirofibana , Tirosina/análogos & derivados , Tirosina/farmacologia , Tirosina/uso terapêutico , Lesão Pulmonar Induzida por Ventilação Mecânica/genética , Lesão Pulmonar Induzida por Ventilação Mecânica/fisiopatologiaRESUMO
Iron, an essential nutrient for cellular growth and proliferation, enters cells via clathrin-mediated endocytosis. The clathrin assembly lymphoid myeloid (CALM) protein plays an essential role in the cellular import of iron by clathrin-mediated endocytosis. CALM-AF10 leukemias harbor a single copy of the normal CALM gene and therefore may be more sensitive to the growth-inhibitory effect of iron restriction compared with normal hematopoietic cells. We found that CALM heterozygous (CALM(HET)) murine fibroblasts exhibit signs of iron deficiency, with increased surface transferrin receptor levels and reduced growth rates. CALM(HET) hematopoietic cells are more sensitive in vitro to iron chelators than their wild type counterparts. Iron chelation also displayed toxicity toward cultured CALM(HET)CALM-AF10 leukemia cells, and this effect was additive to that of chemotherapy. In mice transplanted with CALM(HET)CALM-AF10 leukemia, we found that dietary iron restriction reduced tumor burden in the spleen. However, dietary iron restriction, used alone or in conjunction with chemotherapy, did not increase survival of mice with CALM(HET)CALM-AF10 leukemia. In summary, although CALM heterozygosity results in iron deficiency and increased sensitivity to iron chelation in vitro, our data in mice do not suggest that iron depletion strategies would be beneficial for the therapy of CALM-AF10 leukemia patients.
Assuntos
Ferro da Dieta/administração & dosagem , Ferro/metabolismo , Leucemia Experimental/terapia , Proteínas de Fusão Oncogênica/metabolismo , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Benzoatos/farmacologia , Benzoatos/uso terapêutico , Transporte Biológico , Linhagem Celular Tumoral , Terapia por Quelação , Terapia Combinada , Deferasirox , Desferroxamina/farmacologia , Desferroxamina/uso terapêutico , Fibroblastos/metabolismo , Células-Tronco Hematopoéticas/efeitos dos fármacos , Heterozigoto , Humanos , Quelantes de Ferro/farmacologia , Quelantes de Ferro/uso terapêutico , Ferro da Dieta/farmacocinética , Leucemia Experimental/metabolismo , Camundongos , Camundongos Knockout , Proteínas Monoméricas de Montagem de Clatrina/deficiência , Proteínas Monoméricas de Montagem de Clatrina/genética , Quimera por Radiação , Baço/patologia , Triazóis/farmacologia , Triazóis/uso terapêutico , Carga TumoralRESUMO
Indoleamine 2,3-dioxygenase (IDO) is a rate-limiting enzyme in tryptophan catabolism that plays an important role in the induction of immune tolerance. Its role in graft-versus-tumor effect after allogeneic stem cell transplantation (allo-SCT) remains unclear. Using a murine graft-versus-tumor model of reduced-intensity allo-HSCT followed by donor leukocyte infusion (DLI), we examined the role of IDO inhibition. Two stereoisomers of 1-methyl tryptophan (1-MT), a small-molecule inhibitor of IDO, reduced the growth of inoculated tumor in the mice that received DLI and had higher expression of IDO1 and IFNγ. However, L-1MT, but not D-1MT, mitigated tumor growth in mice that did not receive DLI and did not express IDO1 and IFNγ. Accordingly, both stereoisomers reduced plasma kynurenine concentrations early after DLI and enhanced in vitro cytotoxic lymphocyte function after allogeneic mixed lymphocyte reaction. Furthermore, L-1MT was more efficient in causing direct cytotoxic effects than D-1MT. Our results suggest that IDO inhibition can benefit anti-tumor therapy in the setting of reduced-intensity allo-SCT using DLI.
Assuntos
Efeito Enxerto vs Tumor/fisiologia , Fatores Imunológicos/farmacologia , Indolamina-Pirrol 2,3,-Dioxigenase/antagonistas & inibidores , Mastocitoma/terapia , Proteínas de Neoplasias/antagonistas & inibidores , Triptofano/análogos & derivados , Aloenxertos , Animais , Transplante de Medula Óssea , Linhagem Celular Tumoral , Citotoxicidade Imunológica , Avaliação Pré-Clínica de Medicamentos , Indução Enzimática , Efeito Enxerto vs Tumor/efeitos dos fármacos , Fatores Imunológicos/uso terapêutico , Indolamina-Pirrol 2,3,-Dioxigenase/genética , Indolamina-Pirrol 2,3,-Dioxigenase/fisiologia , Interferon gama/biossíntese , Cinurenina/biossíntese , Cinurenina/sangue , Transfusão de Leucócitos , Linfonodos/enzimologia , Teste de Cultura Mista de Linfócitos , Mastocitoma/tratamento farmacológico , Mastocitoma/enzimologia , Mastocitoma/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas de Neoplasias/fisiologia , Quimera por Radiação , Baço/enzimologia , Estereoisomerismo , Fatores de Tempo , Quimeras de Transplante , Triptofano/química , Triptofano/metabolismo , Triptofano/farmacologia , Triptofano/uso terapêuticoRESUMO
Local treatment with selected TLR ligands or bacteria such as bacillus Calmette-Guérin increases antitumor immune responses and delays tumor growth. It is thought that these treatments may act by activating tumor-associated dendritic cells (DCs), thereby supporting the induction of antitumor immune responses. However, common parameters of successful immune activation have not been identified. We used mouse models to compare treatments with different immune-activating agents for the ability to delay tumor growth, improve priming of tumor-specific T cells, and induce early cytokine production and DC activation. Treatment with polyinosinic-polycytidylic acid or a combination of monosodium urate crystals and Mycobacterium smegmatis was effective at delaying the growth of s.c. B16 melanomas, orthotopic 4T1 mammary carcinomas, and reducing 4T1 lung metastases. In contrast, LPS, monosodium urate crystals, or M. smegmatis alone had no activity. Effective treatments required both NK1.1(+) and CD8(+) cells, and resulted in increased T cell priming and the infiltration of NK cells and CD8(+) T cells in tumors. Unexpectedly, both effective and ineffective treatments increased DC numbers and the expression of costimulatory molecules in the tumor-draining lymph node. However, only effective treatments induced the rapid appearance of a population of monocyte-derived DCs in the draining lymph node, early release of IL-12p70 and IFN-γ, and low IL-10 in the serum. These results suggest that the activation of existing DC subsets is not sufficient for the induction of antitumor immune responses, whereas early induction of Th1 cytokines and monocyte-derived DCs are features of successful activation of antitumor immunity.
Assuntos
Adjuvantes Imunológicos/uso terapêutico , Linfócitos T CD8-Positivos/imunologia , Carcinoma/terapia , Células Dendríticas/imunologia , Imunoterapia Adotiva , Imunoterapia , Células Matadoras Naturais/imunologia , Linfócitos do Interstício Tumoral/imunologia , Neoplasias Mamárias Experimentais/terapia , Melanoma Experimental/terapia , Monócitos/imunologia , Mycobacterium smegmatis/imunologia , Poli I-C/farmacologia , Subpopulações de Linfócitos T/imunologia , Ácido Úrico/farmacologia , Imunidade Adaptativa , Animais , Terapia Biológica , Carcinoma/imunologia , Carcinoma/secundário , Contagem de Células , Diferenciação Celular/efeitos dos fármacos , Citocinas/biossíntese , Citocinas/genética , Escherichia coli/imunologia , Feminino , Interferon gama/metabolismo , Interleucina-10/sangue , Interleucina-12/metabolismo , Neoplasias Pulmonares/prevenção & controle , Neoplasias Pulmonares/secundário , Linfonodos/patologia , Masculino , Neoplasias Mamárias Experimentais/imunologia , Neoplasias Mamárias Experimentais/patologia , Melanoma Experimental/imunologia , Melanoma Experimental/secundário , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Monócitos/efeitos dos fármacos , Ovalbumina/imunologia , Fragmentos de Peptídeos/imunologia , Poli I-C/uso terapêutico , Quimera por Radiação , Receptores de Antígenos de Linfócitos T/imunologia , Subpopulações de Linfócitos T/transplante , Células Th1/imunologia , Células Th1/metabolismo , Ácido Úrico/uso terapêuticoRESUMO
BACKGROUND: Platelet (PLT) transfusions can induce humoral and cellular alloimmunity. HLA antibodies can render patients refractory to subsequent transfusion, and both alloantibodies and cellular alloimmunity can contribute to subsequent bone marrow transplant (BMT) rejection. Currently, there are no approved therapeutic interventions to prevent alloimmunization to PLT transfusions other than leukoreduction. Targeted blockade of T-cell costimulation has shown great promise in inhibiting alloimmunity in the setting of transplantation, but has not been explored in the context of PLT transfusion. STUDY DESIGN AND METHODS: We tested the hypothesis that the costimulatory blockade reagent CTLA4-Ig would prevent alloreactivity against major and minor alloantigens on transfused PLTs. BALB/c (H-2(d)) mice and C57BL/6 (H-2(b)) mice were used as PLT donors and transfusion recipients, respectively. Alloantibodies were measured by indirect immunofluorescence using BALB/c PLTs and splenocytes as targets. BMTs were carried out under reduced-intensity conditioning using BALB.B (H-2(b) ) donors and C57BL/6 (H-2(b)) recipients to model HLA-identical transplants. Experimental groups were given CTLA4-Ig (before or after PLT transfusion) with control groups receiving isotype-matched antibody. RESULTS: CTLA4-Ig abrogated both humoral alloimmunization (H-2(d) antibodies) and transfusion-induced BMT rejection. Whereas a single dose of CTLA4-Ig at time of transfusion prevented alloimmunization to subsequent PLT transfusions, administration of CTLA4-Ig after initial PLT transfusion was ineffective. Delaying treatment until after PLT transfusion failed to prevent BMT rejection. CONCLUSIONS: These findings demonstrate a novel strategy using an FDA-approved drug that has the potential to prevent the clinical sequelae of alloimmunization to PLT transfusions.
Assuntos
Transplante de Medula Óssea/imunologia , Facilitação Imunológica de Enxerto/métodos , Rejeição de Enxerto/prevenção & controle , Antígenos H-2/imunologia , Imunoconjugados/uso terapêutico , Isoanticorpos/biossíntese , Transfusão de Plaquetas , Abatacepte , Transferência Adotiva , Animais , Linfócitos T CD4-Positivos/transplante , Avaliação Pré-Clínica de Medicamentos , Feminino , Antígeno de Histocompatibilidade H-2D , Imunização , Imunoconjugados/farmacologia , Procedimentos de Redução de Leucócitos , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Quimera por RadiaçãoRESUMO
The goal of this study was to elucidate the role of α-tocopherol succinate (TS)- and AMD3100-mobilized progenitors in mitigating the ionizing-radiation-induced gastrointestinal syndrome in mice. We demonstrate the efficacy of a bridging therapy that will allow the lymphohematopoietic system of severely immunocompromised victims exposed to ionizing radiation to recover from high doses of radiation. CD2F1 mice were irradiated with a high dose of radiation causing gastrointestinal syndrome (11 Gy, cobalt-60 γ-radiation) and then transfused intravenously (retro-orbital sinus) with whole blood or peripheral blood mononuclear cells (PBMC) from TS- and AMD3100-injected mice 2, 24, or 48 hours post irradiation and monitored for 30-day survival. Jejunum sections were analyzed for tissue area, surviving crypts, villi, mitotic figures, and basal lamina enterocytes. Our results demonstrate that infusion of whole blood or PBMC from TS- and AMD3100-injected mice significantly improved survival of mice receiving a high dose of radiation. Histopathology and immunostaining of jejunum from irradiated and TS- and AMD3100-mobilized PBMC-transfused mice reveal significant protection of gastrointestinal tissue from radiation injury. We demonstrate that TS and AMD3100 mobilize progenitors into peripheral circulation and that the infusion of mobilized progenitor-containing blood or PBMC acts as a bridging therapy for immune-system recovery in mice exposed to high, potentially fatal, doses of ionizing radiation.
Assuntos
Antioxidantes/uso terapêutico , Células Sanguíneas/transplante , Gastroenteropatias/etiologia , Mobilização de Células-Tronco Hematopoéticas/métodos , Transplante de Células-Tronco Hematopoéticas/métodos , Compostos Heterocíclicos/uso terapêutico , Leucócitos Mononucleares/transplante , Lesões por Radiação/cirurgia , Protetores contra Radiação/uso terapêutico , alfa-Tocoferol/uso terapêutico , Animais , Antioxidantes/farmacologia , Benzilaminas , Ciclamos , Filgrastim , Raios gama/efeitos adversos , Gastroenteropatias/cirurgia , Fator Estimulador de Colônias de Granulócitos/uso terapêutico , Compostos Heterocíclicos/farmacologia , Mucosa Intestinal/efeitos da radiação , Mucosa Intestinal/ultraestrutura , Jejuno/patologia , Jejuno/efeitos da radiação , Masculino , Camundongos , Quimera por Radiação , Protetores contra Radiação/farmacologia , Proteínas Recombinantes/uso terapêutico , alfa-Tocoferol/farmacologiaRESUMO
Polycomb group (PcG) proteins are transcriptional repressors with a central role in the establishment and maintenance of gene expression patterns during development. We have investigated the role of polycomb repressive complexes (PRCs) in hematopoietic stem cells (HSCs) and progenitor populations. We show that mice with loss of function mutations in PRC2 components display enhanced HSC/progenitor population activity, whereas mutations that disrupt PRC1 or pleiohomeotic repressive complex are associated with HSC/progenitor cell defects. Because the hierarchical model of PRC action would predict synergistic effects of PRC1 and PRC2 mutation, these opposing effects suggest this model does not hold true in HSC/progenitor cells. To investigate the molecular targets of each complex in HSC/progenitor cells, we measured genome-wide expression changes associated with PRC deficiency, and identified transcriptional networks that are differentially regulated by PRC1 and PRC2. These studies provide new insights into the mechanistic interplay between distinct PRCs and have important implications for approaching PcG proteins as therapeutic targets.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Hematopoéticas/citologia , Proteínas Repressoras/fisiologia , Células-Tronco/citologia , Animais , Transplante de Medula Óssea , Proteína Potenciadora do Homólogo 2 de Zeste , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Células-Tronco Hematopoéticas/metabolismo , Histona-Lisina N-Metiltransferase/deficiência , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Complexos Multiproteicos , Mutação , Fenótipo , Complexo Repressor Polycomb 2 , Proteínas do Grupo Polycomb , Interferência de RNA , Quimera por Radiação , Proteínas Repressoras/deficiência , Proteínas Repressoras/genética , Células-Tronco/metabolismo , Transcrição Gênica , Fator de Transcrição YY1/fisiologiaRESUMO
While bone marrow or stem cell transplantation can rescue bone marrow aplasia in patients accidentally exposed to a lethal radiation dose, radiation-induced irreversible gastrointestinal damage (GI syndrome) is fatal. We investigated the effects of ascorbic acid on radiation-induced GI syndrome in mice. Ascorbic acid (150 mg/kg/day) was orally administered to mice for 3 days, and then the mice underwent whole body irradiation (WBI). Bone marrow transplantation (BMT) 24 h after irradiation rescued mice receiving a WBI dose of less than 12 Gy. No mice receiving 14 Gy-WBI survived, because of radiation-induced GI syndrome, even if they received BMT. However, pretreatment with ascorbic acid significantly suppressed radiation-induced DNA damage in the crypt cells and prevented denudation of intestinal mucosa; therefore, ascorbic acid in combination with BMT rescued mice after 14 Gy-WBI. DNA microarray analysis demonstrated that irradiation up-regulated expressions of apoptosis-related genes in the small intestine, including those related to the caspase-9-mediated intrinsic pathway as well as the caspase-8-mediated extrinsic pathway, and down-regulated expressions of these genes in ascorbic acid-pretreated mice. Thus, pretreatment with ascorbic acid may effectively prevent radiation-induced GI syndrome.
Assuntos
Ácido Ascórbico/uso terapêutico , Diarreia/prevenção & controle , Hemorragia Gastrointestinal/prevenção & controle , Pré-Medicação , Lesões Experimentais por Radiação/prevenção & controle , Protetores contra Radiação/uso terapêutico , Animais , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Proteínas Reguladoras de Apoptose/biossíntese , Proteínas Reguladoras de Apoptose/genética , Ácido Ascórbico/análise , Ácido Ascórbico/farmacologia , Medula Óssea/patologia , Medula Óssea/efeitos da radiação , Transplante de Medula Óssea , Caspases/metabolismo , Dano ao DNA/efeitos dos fármacos , Diarreia/etiologia , Avaliação Pré-Clínica de Medicamentos , Radicais Livres/sangue , Hemorragia Gastrointestinal/etiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos da radiação , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/efeitos da radiação , Mucosa Intestinal/ultraestrutura , Intestino Delgado/patologia , Intestino Delgado/efeitos da radiação , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Quimera por Radiação , Lesões Experimentais por Radiação/etiologia , Protetores contra Radiação/análise , Protetores contra Radiação/farmacologia , Irradiação Corporal Total/efeitos adversosRESUMO
OBJECTIVE: The signaling by thrombopoietin (TPO) via its receptor, c-MPL, plays a crucial role in the maintenance of hematopoietic stem cells (HSCs). Small-molecule c-MPL agonists have recently been shown to be beneficial in the treatment of thrombocytopenia. However, their effects on HSCs have not yet been explored. In this study, we evaluated the effects of NR-101, a novel small-molecule c-MPL agonist, on the ex vivo expansion of human cord blood (hCB) HSCs. MATERIALS AND METHODS: hCB CD34(+) or CD34(+)CD38(-) hematopoietic stem and progenitor cells were cultured for 7 days in the presence of thrombopoietin (TPO) or NR-101, and then subjected to flow cytometric analyses, colony-forming cell assays, and severe combined immunodeficiency-repopulating cell assays. RESULTS: During a 7-day culture of CD34(+) or CD34(+)CD38(-) hematopoietic stem and progenitor cells, NR-101 efficiently increased their numbers, with a greater than twofold increase compared to TPO, although its effect on megakaryocytopoiesis was comparable to that of TPO. Correspondingly, severe combined immunodeficiency-repopulating cells were increased 2.9-fold during a 7-day culture with NR-101 compared to freshly isolated CD34(+) cells, and 2.3-fold compared to that with TPO. Of note, NR-101 persistently activated signal transducer and activator of transcription (STAT) 5 but not signal transducer and activator of transcription 3. Furthermore, NR-101 induced a long-term accumulation of hypoxia-inducible factor-1alpha protein and enhanced activation of its downstream target genes. CONCLUSION: This is the first time that a small-molecule c-MPL agonist has been demonstrated to promote net expansion of HSCs. NR-101 is more efficient in ex vivo expansion of HSCs than TPO. NR-101 could be a useful tool for the therapeutic manipulation of human HSCs.
Assuntos
Células-Tronco Hematopoéticas/efeitos dos fármacos , Receptores de Trombopoetina/agonistas , Trombopoese/efeitos dos fármacos , Animais , Hipóxia Celular/efeitos dos fármacos , Hipóxia Celular/genética , Linhagem Celular Tumoral/citologia , Linhagem Celular Tumoral/efeitos dos fármacos , Células Cultivadas/citologia , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/transplante , Transplante de Células-Tronco de Sangue do Cordão Umbilical , Subunidade beta Comum dos Receptores de Citocinas/genética , Subunidade beta Comum dos Receptores de Citocinas/fisiologia , DNA Complementar/genética , Avaliação Pré-Clínica de Medicamentos , Sangue Fetal/citologia , Regulação da Expressão Gênica/efeitos dos fármacos , Células-Tronco Hematopoéticas/citologia , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/biossíntese , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa de Receptor de Interleucina-3/genética , Subunidade alfa de Receptor de Interleucina-3/fisiologia , Leucemia Mieloide/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Quimera por Radiação , Receptores da Eritropoetina/genética , Receptores da Eritropoetina/fisiologia , Receptores de Trombopoetina/genética , Receptores de Trombopoetina/fisiologia , Proteínas Recombinantes de Fusão/fisiologia , Transdução de Sinais/efeitos dos fármacos , Trombopoetina/farmacologiaRESUMO
Cytopenia represents a significant complication after chemotherapy, irradiation before bone marrow (BM) transplantation or as a therapy for cancer. The mechanisms that determine the pace of BM recovery are not fully understood. During the recovery phase after chemotherapy or irradiation, the signals for retention of white blood cells within the BM increase significantly. This leads to a delay in the release of WBC, which can be overcome by targeting the CXCR4 axis with the antagonist 4F-benzoyl-TN14003 (T140). The delay in the release of WBC is also accompanied by suppression in the production of progenitor cells and mature cells by the BM stroma. Administration of T140 to mice transplanted with BM cells stimulates the production of all types of progenitors and mature cells, and increases the exit of mature cells to the periphery. Moreover, addition of T140, but not AMD3100, to BM stromal cultures stimulates the production of mature cells and progenitors from all lineages. The unique ability of the CXCR4 antagonist, T140 to stimulate the production and exit of WBC cells may be used as a novel therapeutic approach to overcome cytopenia associated with treatments for cancer and BM transplantation.
Assuntos
Medula Óssea/efeitos dos fármacos , Peptídeos/uso terapêutico , Receptores CXCR4/antagonistas & inibidores , Animais , Benzilaminas , Medula Óssea/efeitos da radiação , Divisão Celular/efeitos dos fármacos , Técnicas de Cocultura , Ensaio de Unidades Formadoras de Colônias , Ciclamos , Ciclofosfamida/farmacologia , Avaliação Pré-Clínica de Medicamentos , Feminino , Sobrevivência de Enxerto/efeitos dos fármacos , Mobilização de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Compostos Heterocíclicos/farmacologia , Compostos Heterocíclicos/uso terapêutico , Integrina alfa4beta1/biossíntese , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Neutropenia/tratamento farmacológico , Neutropenia/etiologia , Peptídeos/farmacologia , Quimera por Radiação , Receptores CXCR4/biossíntese , Receptores CXCR4/fisiologia , Recuperação de Função Fisiológica/efeitos dos fármacos , Organismos Livres de Patógenos Específicos , Células Estromais/fisiologiaRESUMO
The recent explosion in the understanding of the cellular and molecular mechanisms underlying hematopoietic stem and progenitor cell (HSPC) mobilization has facilitated development of novel therapeutic agents, targeted at improving mobilization kinetics as well as HSPC yield. With the development of new agents comes the challenge of choosing efficient and relevant preclinical studies for the testing of the HSPC mobilization efficacy of these agents. This article reviews the use of the mouse as a convenient small animal model of HSPC mobilization and transplantation, and outlines the range of murine assays that can be applied to assess novel HSPC mobilizing agents. Techniques to demonstrate murine HSPC mobilization are discussed, as well as the role of murine assays to confirm human HSPC mobilization, and techniques to investigate the biologic phenotype of HSPC mobilized by these novel agents. Technical aspects regarding mobilization regimens and control arms, and choice of experimental animals are also discussed.
Assuntos
Mobilização de Células-Tronco Hematopoéticas/métodos , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/efeitos dos fármacos , Modelos Animais , Animais , Animais Congênicos , Técnicas de Cultura de Células/métodos , Linhagem da Célula , Células Cultivadas/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Feminino , Sobrevivência de Enxerto , Fatores de Crescimento de Células Hematopoéticas/farmacologia , Células-Tronco Hematopoéticas/classificação , Células-Tronco Hematopoéticas/citologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos Endogâmicos , Camundongos SCID , Agonistas Mieloablativos/farmacologia , Quimera por RadiaçãoRESUMO
There is no effective therapy for progressive hearing loss such as presbycusis, the causes of which remain poorly understood because of the difficulty of separating genetic and environmental contributions. In the present study, we show that the age-related dysfunctions of the systemic immune system in an animal model of accelerated presbycusis (SAMP1, senescence-accelerated mouse P1) can be corrected by allogeneic bone marrow transplantation (BMT). We also demonstrate that this presbycusis can be prevented; BMT protects the recipients from age-related hearing impairment and the degeneration of spiral ganglion cells (SGCs) as well as the dysfunctions of T lymphocytes, which have a close relation to immune senescence. No donor cells are infiltrated to the spiral ganglia, confirming that this experimental system using BMT is connected to the systemic immune system and does not contribute to transdifferentiation or fusion by donor hematopoietic stem cells (HSCs), or to the direct maintenance of ganglion cells by locally infiltrated donor immunocompetent cells. Therefore, another procedure which attempts to prevent the age-related dysfunctions of the recipient immune system is the inoculation of syngeneic splenocytes from young donors. These mice show no development of hearing loss, compared with the recipient mice with inoculation of saline or splenocytes from old donors. Our studies on the relationship between age-related systemic immune dysfunctions and neurodegeneration mechanisms open up new avenues of treatment for presbycusis, for which there is no effective therapy.
Assuntos
Envelhecimento/imunologia , Presbiacusia/imunologia , Presbiacusia/prevenção & controle , Estimulação Acústica/métodos , Fatores Etários , Animais , Limiar Auditivo/efeitos dos fármacos , Limiar Auditivo/fisiologia , Transplante de Medula Óssea/imunologia , Proliferação de Células/efeitos dos fármacos , Concanavalina A/farmacologia , Modelos Animais de Doenças , Potenciais Evocados Auditivos do Tronco Encefálico/fisiologia , Citometria de Fluxo , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mitógenos/farmacologia , Psicofísica , Quimera por Radiação , Gânglio Espiral da Cóclea/metabolismo , Gânglio Espiral da Cóclea/patologia , Linfócitos T/efeitos dos fármacosAssuntos
Modelos Animais de Doenças , Infecções por HIV/transmissão , Camundongos Endogâmicos , Adenina/análogos & derivados , Adenina/uso terapêutico , Administração Intravaginal , Animais , Desoxicitidina/análogos & derivados , Desoxicitidina/uso terapêutico , Suscetibilidade a Doenças , Avaliação Pré-Clínica de Medicamentos , Emtricitabina , Feminino , Infecções por HIV/prevenção & controle , HIV-1 , Transplante de Células-Tronco Hematopoéticas , Humanos , Transplante de Fígado , Macaca mulatta , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Organofosfonatos/uso terapêutico , Quimera por Radiação , Especificidade da Espécie , Tenofovir , Timo/transplante , Transplante HeterólogoRESUMO
BACKGROUND: Worldwide, vaginal transmission now accounts for more than half of newly acquired HIV-1 infections. Despite the urgency to develop and implement novel approaches capable of preventing HIV transmission, this process has been hindered by the lack of adequate small animal models for preclinical efficacy and safety testing. Given the importance of this route of transmission, we investigated the susceptibility of humanized mice to intravaginal HIV-1 infection. METHODS AND FINDINGS: We show that the female reproductive tract of humanized bone marrow-liver-thymus (BLT) mice is reconstituted with human CD4+ T and other relevant human cells, rendering these humanized mice susceptible to intravaginal infection by HIV-1. Effects of HIV-1 infection include CD4+ T cell depletion in gut-associated lymphoid tissue (GALT) that closely mimics what is observed in HIV-1-infected humans. We also show that pre-exposure prophylaxis with antiretroviral drugs is a highly effective method for preventing vaginal HIV-1 transmission. Whereas 88% (7/8) of BLT mice inoculated vaginally with HIV-1 became infected, none of the animals (0/5) given pre-exposure prophylaxis of emtricitabine (FTC)/tenofovir disoproxil fumarate (TDF) showed evidence of infection (Chi square = 7.5, df = 1, p = 0.006). CONCLUSIONS: The fact that humanized BLT mice are susceptible to intravaginal infection makes this system an excellent candidate for preclinical evaluation of both microbicides and pre-exposure prophylactic regimens. The utility of humanized mice to study intravaginal HIV-1 transmission is particularly highlighted by the demonstration that pre-exposure prophylaxis can prevent intravaginal HIV-1 transmission in the BLT mouse model.
Assuntos
Adenina/análogos & derivados , Fármacos Anti-HIV/uso terapêutico , Desoxicitidina/análogos & derivados , Modelos Animais de Doenças , Infecções por HIV/prevenção & controle , Camundongos Endogâmicos , Organofosfonatos/uso terapêutico , Adenina/administração & dosagem , Adenina/uso terapêutico , Administração Intravaginal , Animais , Fármacos Anti-HIV/administração & dosagem , Desoxicitidina/administração & dosagem , Desoxicitidina/uso terapêutico , Avaliação Pré-Clínica de Medicamentos , Emtricitabina , Feminino , Transplante de Tecido Fetal , Genitália Feminina/imunologia , Genitália Feminina/virologia , Infecções por HIV/transmissão , HIV-1/isolamento & purificação , HIV-1/patogenicidade , Transplante de Células-Tronco Hematopoéticas , Humanos , Imunidade nas Mucosas , Transplante de Fígado , Camundongos , Camundongos SCID , Organofosfonatos/administração & dosagem , Quimera por Radiação , Especificidade da Espécie , Tenofovir , Timo/transplante , Transplante HeterólogoRESUMO
In the present work, the antitumor effect of fastuosain, a cysteine proteinase from Bromelia fastuosa, was investigated. In the intravenous model of lung colonization in C57Bl/6 mice, fastuosain and bromelain injected intraperitoneally were protective, and very few nodules of B16F10-Nex2 melanoma cells were detected. Tumor cells treated with fastuosain showed reduced expression of CD44 and decreased invasion through Matrigel, lost their cytoplasmic extensions and substrate adherence, and became round and detached, forming strongly bound cell clusters in suspension. Peritoneal cells recruited and activated by fastuosain treatment (mainly monocytic cells and lymphocytes) migrated to the lung, where pulmonary melanoma metastases grew. Adoptive transference of peritoneal cells recruited by fastuosain had no protective effect against lung metastases in recipient mice. Treatment of green fluorescent protein-chimeric animals with fastuosain did not change the number of cells that migrated to the lung, compared to PBS-injected control mice, but the number of positive major histocompatibility complex class II cells increased with fastuosain treatment. Murine antibodies against fastuosain, bromelain, and cathepsins B and L cross-reacted in ELISA and recognized surface and cytoplasmic components expressed on B16F10-Nex2 cells. Anti-fastuosain antibodies were cytotoxic/lytic to B16F10-Nex2 cells. Antitumor effects of fastuosain involve mainly the direct effect of the enzyme and elicitation of protective antibodies.
Assuntos
Antineoplásicos Fitogênicos/uso terapêutico , Cisteína Endopeptidases/uso terapêutico , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/secundário , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/secundário , Transferência Adotiva , Animais , Formação de Anticorpos , Antígenos de Neoplasias/biossíntese , Antígenos de Neoplasias/imunologia , Antineoplásicos Fitogênicos/imunologia , Antineoplásicos Fitogênicos/farmacologia , Bromelaínas/imunologia , Bromelaínas/farmacologia , Bromelaínas/uso terapêutico , Linhagem Celular Tumoral/efeitos dos fármacos , Quimiotaxia de Leucócito/efeitos dos fármacos , Cisteína Endopeptidases/imunologia , Cisteína Endopeptidases/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Neoplasias Pulmonares/imunologia , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/transplante , Masculino , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Papaína/imunologia , Papaína/farmacologia , Papaína/uso terapêutico , Quimera por RadiaçãoAssuntos
Células Apresentadoras de Antígenos/transplante , Transplante de Medula Óssea , Anergia Clonal/imunologia , Tolerância Imunológica/imunologia , Subpopulações de Linfócitos T/transplante , Animais , Células Apresentadoras de Antígenos/imunologia , Antígeno CD11b/análise , Células Dendríticas/classificação , Células Dendríticas/imunologia , Células Dendríticas/transplante , Sobrevivência de Enxerto , Doença Enxerto-Hospedeiro/etiologia , Doença Enxerto-Hospedeiro/imunologia , Doença Enxerto-Hospedeiro/prevenção & controle , Efeito Enxerto vs Leucemia , Histocompatibilidade , Memória Imunológica , Separação Imunomagnética , Interferon gama/sangue , Ativação Linfocitária , Depleção Linfocítica , Linfoma de Células T/imunologia , Linfoma de Células T/cirurgia , Camundongos , Camundongos Congênicos , Camundongos Endogâmicos C57BL , Quimera por Radiação , Baço/citologia , Baço/imunologia , Subpopulações de Linfócitos T/imunologia , Imunologia de Transplantes , Fator de Necrose Tumoral alfa/análiseRESUMO
T lineage commitment occurs in a discrete, stage-specific manner during thymic ontogeny. Intrathymic precursor transfer experiments and the identification of CD4(+)8+ double-positive (DP), V alpha 14J alpha 18 natural T (iNKT) cells suggest that commitment to this lineage might occur at the DP stage. Nevertheless, this matter remains contentious because others failed to detect V alpha 14J alpha 18-positive iNKT cells that are CD4(+)8+. In resolution to this issue, we demonstrate that retinoic acid receptor-related orphan receptor gamma (ROR gamma)0/0 thymi, which accumulate immature single-positive (ISP) thymocytes that precede the DP stage, do not rearrange V alpha 14-to-J alpha 18 gene segments, suggesting that this event occurs at a post-ISP stage. Mixed radiation bone marrow chimeras revealed that RORgamma functions in an iNKT cell lineage-specific manner. Further, introgression of a Bcl-x(L) transgene into ROR gamma(0/0) mice, which promotes survival and permits secondary rearrangements of distal V alpha and J alpha gene segments at the DP stage, rescues V alpha 14-to-J alpha 18 recombination. Similarly, introgression of a rearranged V alpha 14J alpha 18 transgene into ROR gamma(0/0) mice results in functional iNKT cells. Thus, our data support the "T cell receptor-instructive (mainstream precursor) model" of iNKT cell lineage specification where V alpha 14-to-J alpha 18 rearrangement, positive selection, and iNKT cell lineage commitment occur at or after the DP stage of ontogeny.