RESUMO
Aminoglycoside antibiotics have been in use since 1944 with the discovery of streptomycin. The aim of this study was to derive a new, highly resistant multicopy neo(R) transgenic mouse strain, named TgN3Ems, by random insertion of the plasmid, pPGKneobpA, and compare the level of drug resistance of wild-type and transgenic mice in vivo and corresponding primary mouse embryonic fibroblasts (MEFs) in vitro to a model neomycin analog, G418. The expression neoR in transgenic animals caused a 5-fold increase in the approximate lethal dose of G418, compared to wild type. No adverse pathological changes were found for the transgenic mice treated with G418, as they all died within minutes after injection. In contrast, the G418 treatment of wild-type mice resulted in a marked liver and kidney toxicity detected microscopically and via increases of serum biomarkers for liver and kidney damage. In addition, there was a mild bone marrow and lymphoid depletion. In in vitro studies, the transgenic MEFs survived 20-fold higher G418 levels, compared to the wild-type MEF cells. Therefore, TgN3Ems transgenic mice could be used as a source of G418-resistant feeder cells for gene targeting. Since the expression of drug-resistance genes in transgenic animals confers resistance to toxicity, the TgN3Ems mice might serve as a tool applicable in drug design.
Assuntos
Resistência a Medicamentos/genética , Células Alimentadoras/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Marcação de Genes , Gentamicinas/toxicidade , Canamicina Quinase/genética , Animais , Southern Blotting , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Células Cultivadas , Células Alimentadoras/citologia , Células Alimentadoras/enzimologia , Fibroblastos/citologia , Fibroblastos/enzimologia , Gentamicinas/farmacologia , Dose Letal Mediana , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Plasmídeos , Regiões Promotoras Genéticas , Testes de Toxicidade Aguda , TransgenesRESUMO
Myostatin is a secreted protein that normally functions as a negative regulator of muscle growth. Agents capable of blocking the myostatin signaling pathway could have important applications for treating human muscle degenerative diseases as well as for enhancing livestock production. Here we describe a potent myostatin inhibitor, a soluble form of the activin type IIB receptor (ACVR2B), which can cause dramatic increases in muscle mass (up to 60% in 2 weeks) when injected into wild-type mice. Furthermore, we show that the effect of the soluble receptor is attenuated but not eliminated in Mstn(-/-) mice, suggesting that at least one other ligand in addition to myostatin normally functions to limit muscle growth. Finally, we provide genetic evidence that these ligands signal through both activin type II receptors, ACVR2 and ACVR2B, to regulate muscle growth in vivo.
Assuntos
Receptores de Activinas Tipo II/metabolismo , Músculo Esquelético/crescimento & desenvolvimento , Transdução de Sinais/fisiologia , Fator de Crescimento Transformador beta/antagonistas & inibidores , Receptores de Activinas Tipo II/genética , Receptores de Activinas Tipo II/farmacologia , Animais , Ligantes , Camundongos , Camundongos Knockout , Músculo Esquelético/efeitos dos fármacos , Miostatina , Tamanho do ÓrgãoRESUMO
PURPOSE: CMC-544 is a CD22-targeted immunoconjugate of calicheamicin and exerts a potent cytotoxic effect against CD22+ B-cell lymphoma. This study evaluated antitumor efficacy of CMC-544 against systemically disseminated B-cell lymphoma. EXPERIMENTAL DESIGN: Scid mice received i.v. injections of CD22+ Ramos B-cell lymphoma cells for their systemic dissemination. CMC-544, G5/44, CD33-targeted CMA-676 (control conjugate) or rituximab were given i.p. 3, 9, 15, or 21 days after B-cell lymphoma dissemination. Diseased mice were monitored daily for hind-limb paralysis and death. Histopathological examination of CMC-544-treated and vehicle-treated diseased mice was also performed. RESULTS: Mice with disseminated B-cell lymphoma developed hind-limb paralysis within 35 days. When given up to 15 days after B-cell lymphoma dissemination, CMC-544 extended survival of the diseased mice to >100 days, and these mice were considered cured. CMC-544 was efficacious when given during both the early initiation phase and the late established phase of the disease. A single dose of CMC-544 was effective in delaying the occurrence of hind-limb paralysis. In contrast, neither CMA-676 nor unconjugated G5/44 was effective. Rituximab was effective when given early in the disease process but not when the disease was established. Histopathological analysis revealed B-cell lymphoma infiltration in brain, spinal cord, bone marrow, and kidney in vehicle-treated but not in CMC-544-treated diseased mice. Consistent with its efficacy against the disseminated B-cell lymphoma, CMC-544 also caused regression of established Ramos B-cell lymphoma xenografts in scid mice. CONCLUSIONS: CMC-544 confers strong therapeutic activity against systemic disseminated B-cell lymphoma and protects mice from hind-limb paralysis and death. These results support clinical evaluation of CMC-544 in the treatment of CD22+ lymphoid malignancies.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos B/metabolismo , Moléculas de Adesão Celular/metabolismo , Membro Posterior , Imunoconjugados/uso terapêutico , Lectinas/metabolismo , Linfoma de Células B/terapia , Paralisia/etiologia , Animais , Anticorpos Monoclonais Humanizados , Anticorpos Monoclonais Murinos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Humanos , Imunoglobulina G/metabolismo , Imunoterapia/métodos , Inotuzumab Ozogamicina , Linfoma de Células B/metabolismo , Linfoma de Células B/patologia , Masculino , Camundongos , Camundongos SCID , Rituximab , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico , Taxa de Sobrevida , Transplante HeterólogoRESUMO
The c-Jun NH(2)-terminal kinase (JNK) group of mitogen-activated protein kinases is activated in response to a wide array of cellular stresses and proinflammatory cytokines. Roles for JNK in the developing nervous system and T-cell-mediated immunity have been established by detailed studies of mice with compound mutations in the Jnk genes. However, little is known concerning the roles of JNK in other mammalian tissues. Mice lacking both of the ubiquitously expressed isoforms (JNK1 and -2) die during midgestation with neural tube closure defects and brain abnormalities. Here we show that JNK-deficient mice exhibit delayed epithelial development in the epidermis, intestines, and lungs. In addition, JNK-deficient mice exhibit an eyelid closure defect associated with markedly reduced epidermal growth factor (EGF) receptor function, and loss of expression of the ligand EGF. We further demonstrate that adult mice lacking either JNK1 or -2 display striking differences in epidermal proliferation and differentiation, indicative of distinct roles for these kinases in the skin. We conclude that JNK is necessary for epithelial morphogenesis and is an essential regulator of signal transduction by the EGF receptor in the epidermis.
Assuntos
Fator de Crescimento Epidérmico/biossíntese , Proteínas Quinases JNK Ativadas por Mitógeno/fisiologia , Pele/embriologia , Animais , Fator de Crescimento Epidérmico/genética , Pálpebras/anormalidades , Pálpebras/embriologia , Pálpebras/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Imuno-Histoquímica , Hibridização In Situ , Intestinos/anormalidades , Intestinos/embriologia , Intestinos/ultraestrutura , Isoenzimas , Proteínas Quinases JNK Ativadas por Mitógeno/deficiência , Pulmão/anormalidades , Pulmão/embriologia , Pulmão/ultraestrutura , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Morfogênese , Sondas de Oligonucleotídeos/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Pele/enzimologia , Língua/anormalidades , Língua/embriologia , Língua/ultraestruturaRESUMO
Pathogenic mechanisms relevant to rheumatoid arthritis occur in the mouse model of collagen-induced arthritis (CIA). Cytosolic phospholipase A2alpha (cPLA2alpha) releases arachidonic acid from cell membranes to initiate the production of prostaglandins and leukotrienes. These inflammatory mediators have been implicated in the development of CIA. To test the hypothesis that cPLA2alpha plays a key role in the development of CIA, we backcrossed cPLA2alpha-deficient mice on the DBA/1LacJ background that is susceptible to CIA. The disease severity scores and the incidence of disease were markedly reduced in cPLA2alpha-deficient mice compared with wild-type littermates. At completion of the study, >90% of the wild-type mice had developed disease whereas none of the cPLA2alpha-deficient mice had more than one digit inflamed. Furthermore, visual disease scores correlated with severity of disease determined histologically. Pannus formation, articular fibrillation, and ankylosis were all dramatically reduced in the cPLA2alpha-deficient mice. Although the disease scores differed significantly between cPLA2alpha mutant and wild-type mice, anti-collagen antibody levels were similar in the wild-type mice and mutant littermates. These data demonstrate the critical role of cPLA2alpha in the pathogenesis of CIA.
Assuntos
Artrite/etiologia , Colágeno/imunologia , Citosol/enzimologia , Fosfolipases A/fisiologia , Proteínas Ativadoras de 5-Lipoxigenase , Animais , Artrite/prevenção & controle , Proteínas de Transporte/fisiologia , Ciclo-Oxigenase 2 , Fosfolipases A2 do Grupo IV , Imunização , Imunoglobulina G/sangue , Imunoglobulina G/classificação , Isoenzimas/fisiologia , Proteínas de Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Fosfolipases A/deficiência , Prostaglandina-Endoperóxido Sintases/fisiologia , Receptores de Prostaglandina E/fisiologia , Receptores de Prostaglandina E Subtipo EP4RESUMO
The c-Jun NH(2)-terminal kinase (JNK) group of mitogen-activated protein kinases is stimulated in response to a wide array of cellular stresses and proinflammatory cytokines. Mice lacking individual members of the Jnk family (Jnk1, Jnk2, and Jnk3) are viable and survive without overt structural abnormalities. Here we show that mice with a compound deficiency in Jnk expression can survive to birth, but fail to close the optic fissure (retinal coloboma). We demonstrate that JNK initiates a cytokine cascade of bone morphogenetic protein-4 (BMP4) and sonic hedgehog (Shh) that induces the expression of the paired-like homeobox transcription factor Pax2 and closure of the optic fissure. Interestingly, the role of JNK to regulate BMP4 expression during optic fissure closure is conserved in Drosophila during dorsal closure, a related morphogenetic process that requires JNK-regulated expression of the BMP4 ortholog Decapentaplegic (Dpp).
Assuntos
Coloboma/embriologia , Coloboma/metabolismo , Citocinas/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fatores de Transcrição/metabolismo , Animais , Proteína Morfogenética Óssea 4 , Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Drosophila/embriologia , Drosophila/metabolismo , Proteínas Hedgehog , Proteínas Quinases JNK Ativadas por Mitógeno , Cristalino/embriologia , Cristalino/metabolismo , Camundongos/embriologia , Fator de Transcrição PAX2 , Transdução de Sinais/fisiologia , Transativadores/genética , Transativadores/metabolismoRESUMO
Developing neurons must respond to a wide range of extracellular signals during the process of brain morphogenesis. One mechanism through which immature neurons respond to such signals is by altering cellular actin dynamics. A recently discovered link between extracellular signaling events and the actin cytoskeleton is the WASP/WAVE (Wiscott-Aldrich Syndrome protein/WASP-family verprolin-homologous protein) family of proteins. Through a direct interaction with the Arp2/3 (actin-related protein) complex, this family functions to regulate the actin cytoskeleton by mediating signals from cdc42 as well as other small GTPases. To evaluate the role of WASP/WAVE proteins in the process of neuronal morphogenesis, we used a retroviral gene trap to generate a line of mice bearing a disruption in the WAVE1 gene. Using a heterologous reporter gene, we found that WAVE1 expression becomes increasingly restricted to the CNS over the course of development. Homozygous disruption of the WAVE1 gene results in postnatal lethality. In addition, these animals have severe limb weakness, a resting tremor, and notable neuroanatomical malformations without overt histopathology of peripheral organs. We did not detect any alterations in neuronal morphology in vivo or the ability of embryonic neurons to form processes in vitro. Our data indicate that WAVE1, although important for the general development of the CNS, is not essential for the formation and extension of neuritic processes.
Assuntos
Sistema Nervoso Central/anormalidades , Proteínas dos Microfilamentos/deficiência , Malformações do Sistema Nervoso/genética , Anormalidades Múltiplas , Animais , Células Cultivadas , Sistema Nervoso Central/patologia , Córtex Cerebral/anormalidades , Córtex Cerebral/patologia , Regulação da Expressão Gênica no Desenvolvimento , Genes Letais , Genes Reporter , Heterozigoto , Homozigoto , Camundongos , Camundongos Knockout , Proteínas dos Microfilamentos/biossíntese , Proteínas dos Microfilamentos/genética , Morfogênese , Debilidade Muscular/genética , Malformações do Sistema Nervoso/patologia , Neurônios/citologia , Neurônios/patologia , Especificidade de Órgãos , Fenótipo , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismo , Tremor/genética , Família de Proteínas da Síndrome de Wiskott-Aldrich , beta-Galactosidase/biossíntese , beta-Galactosidase/genéticaRESUMO
Highly polarized type 2 cytokine responses can be harmful and even lethal to the host if they are too vigorous or persist too long. Therefore, it is important to elucidate the mechanisms that down-regulate these reactions. Interleukin (IL)-13 has emerged as a central mediator of T helper cell (Th)2-dominant immune responses, exhibiting a diverse array of functional activities including regulation of airway hyperreactivity, resistance to nematode parasites, and tissue remodeling and fibrosis. Here, we show that IL-13 receptor (R)alpha2 is a critical down-regulatory factor of IL-13-mediated tissue fibrosis induced by the parasitic helminth Schistosoma mansoni. IL-13Ralpha2 expression was induced after the onset of the fibrotic response, IL-10, IL-13, and Stat6 dependent, and inhibited by the Th1-inducing adjuvant IL-12. Strikingly, schistosome-infected C57BL/6 and BALB/c IL-13Ralpha2-deficient mice showed a marked exacerbation in hepatic fibrosis, despite displaying no change in granuloma size, tissue eosinophilia, or mastocytosis. Fibrosis increased despite the fact that IL-13 levels decreased significantly in the liver and serum. Importantly, pathology was prevented when IL-13Ralpha2-deficient mice were treated with a soluble IL-13Ralpha2-Fc construct, formally demonstrating that their exacerbated fibrotic response was due to heightened IL-13 activity. Together, these studies illustrate the central role played by the IL-13Ralpha2 in the down-regulation of a chronic and pathogenic Th2-mediated immune response.