RESUMEN
C2H2 zinc fingers are found in several key transcriptional regulators in the immune system. However, these proteins usually contain more fingers than are needed for sequence-specific DNA binding, which suggests that different fingers regulate different genes and functions. Here we found that mice lacking finger 1 or finger 4 of Ikaros exhibited distinct subsets of the hematological defects of Ikaros-null mice. Most notably, the two fingers controlled different stages of lymphopoiesis, and finger 4 was selectively required for tumor suppression. The distinct defects support the hypothesis that only a small number of genes that are targets of Ikaros are critical for each of its biological functions. The subcategorization of functions and target genes by mutagenesis of individual zinc fingers will facilitate efforts to understand how zinc-finger transcription factors regulate development, immunity and disease.
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Transformación Celular Neoplásica/genética , Regulación de la Expresión Génica , Factor de Transcripción Ikaros/genética , Leucemia/genética , Linfopoyesis/genética , Animales , Linfocitos B/citología , Linfocitos B/metabolismo , Secuencia de Bases , Sitios de Unión , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Inmunoprecipitación de Cromatina , Análisis por Conglomerados , Proteínas de Fusión bcr-abl/genética , Proteínas de Fusión bcr-abl/metabolismo , Perfilación de la Expresión Génica , Mutación de Línea Germinal , Secuenciación de Nucleótidos de Alto Rendimiento , Factor de Transcripción Ikaros/metabolismo , Inmunofenotipificación , Leucemia/metabolismo , Leucemia/mortalidad , Linfoma/genética , Linfoma/metabolismo , Linfoma/mortalidad , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Motivos de Nucleótidos , Fenotipo , Posición Específica de Matrices de Puntuación , Unión Proteica , Timocitos/metabolismoRESUMEN
INTRODUCTION: Although current techniques for accelerated tooth movement often involve invasive surgical procedures, micro-osteoperforations (MOPs) using mini-implants may facilitate orthodontic tooth movement without raising flaps, reduce surgical risks, and increase patient acceptance. In this study, we evaluated the effectiveness of mini-implant-facilitated MOPs in inducing accelerated tooth movement and investigated the potential risks for root resorption. METHODS: Five MOPs were placed on the left side around the maxillary first molars in 6 rats using an automated mini-implant driver, whereas the right side received no MOPs as the control. Closed-coiled springs were secured from incisors to first molars for orthodontic tooth movement. Tooth movement was measured, and samples underwent radiologic and histologic analyses. RESULTS: The MOP side exhibited a 1.86-fold increase in the rate of tooth movement with decreased bone density and bone volume around the first molars compared with the control side. Hematoxylin and eosin and tartrate-resistant acid phosphatase analyses showed increased numbers of osteoclasts as well as new bone formation. Three-dimensional volumetric analysis of all 5 roots of the maxillary first molars demonstrated no statistically significant difference in root volumes. CONCLUSIONS: Mini-implant-facilitated MOPs accelerated tooth movement without increased risk for root resorption and therefore may become a readily available and efficient treatment option to shorten orthodontic treatment time with improved patient acceptance.
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Alambres para Ortodoncia , Técnicas de Movimiento Dental/métodos , Animales , Masculino , Mandíbula/diagnóstico por imagen , Mandíbula/cirugía , Diente Molar/diagnóstico por imagen , Diente Molar/cirugía , Ratas , Ratas Sprague-Dawley , Resorción Radicular/diagnóstico por imagen , Resorción Radicular/etiología , Técnicas de Movimiento Dental/instrumentación , Microtomografía por Rayos XRESUMEN
OBJECTIVE: To investigate a topical local anesthesia technique as a means to prevent and/or diminish pain in mice in a laboratory setting associated with tail vein injections performed by personnel in training. STUDY DESIGN: Prospective, randomized experimental trial. ANIMALS: Thirty six adult female, 23-28 g CD-1 mice from an in-house training colony. They were acclimated to routine training and handling classes. METHODS: Eutectic mixture of local anesthetics (EMLA) cream (2.5% lidocaine/2.5% prilocaine) or a bland ointment control (n = 18) was applied on the tail prior to intravenous injection. The injections were performed by novices, who had never attempted the procedure, and experienced personnel. All participants were blinded to treatment groups. Three injection attempts were allowed per animal. The mice were observed and scored by blinded evaluators for behavioral and physiological changes, including respiratory rate, vocalization, tail flick, and escape behaviors, during and after the injection. RESULTS: This study demonstrates that aversive behaviors induced by lateral tail vein injection were not changed by the preemptive application of EMLA cream. The aversive behaviors associated with lateral tail vein injection were significantly affected by the number of injection attempts and the individual's experience level. CONCLUSIONS AND CLINICAL RELEVANCE: Topical EMLA cream did not reduce signs of aversive reaction to tail vein injection and thus we did not find support for its use in mouse training programs for tail vein injections.
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Anestésicos Combinados/administración & dosificación , Anestésicos Locales/administración & dosificación , Inyecciones Intravenosas/veterinaria , Lidocaína/administración & dosificación , Prilocaína/administración & dosificación , Animales , Femenino , Combinación Lidocaína y Prilocaína , Ratones , Pomadas/administración & dosificación , Cola (estructura animal)RESUMEN
ORAI1 is a pore subunit of Ca(2+) release-activated Ca(2+) channels that mediate TCR stimulation-induced Ca(2+) entry. A point mutation in ORAI1 (ORAI1(R91W)) causes SCID in human patients that is recapitulated in Orai1(-/-) mice, emphasizing its important role in the immune cells. In this study, we have characterized a novel function of ORAI1 in T cell death. CD4(+) T cells from Orai1(-/-) mice showed robust proliferation with repetitive stimulations and strong resistance to stimulation-induced cell death due to reduced mitochondrial Ca(2+) uptake and altered gene expression of proapoptotic and antiapoptotic molecules (e.g., Fas ligand, Noxa, and Mcl-1). Nuclear accumulation of NFAT was severely reduced in ORAI1-deficient T cells, and expression of ORAI1 and a constitutively active mutant of NFAT recovered cell death. These results indicate NFAT-mediated cell death pathway as one of the major downstream targets of ORAI1-induced Ca(2+) entry. By expressing various mutants of ORAI1 in wild-type and Orai1(-/-) T cells to generate different levels of intracellular Ca(2+), we have shown that activation-induced cell death is directly proportional to the intracellular Ca(2+) concentration levels. Consistent with the in vitro results, Orai1(-/-) mice showed strong resistance to T cell depletion induced by injection of anti-CD3 Ab. Furthermore, ORAI1-deficient T cells showed enhanced survival after adoptive transfer into immunocompromised hosts. Thus, our results demonstrate a crucial role of the ORAI1-NFAT pathway in T cell death and highlight the important role of ORAI1 as a major route of Ca(2+) entry during activated T cell death.
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Apoptosis/inmunología , Linfocitos T CD4-Positivos/inmunología , Canales de Calcio/inmunología , Señalización del Calcio/inmunología , Animales , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/metabolismo , Canales de Calcio/metabolismo , Separación Celular , Supervivencia Celular , Citometría de Flujo , Humanos , Immunoblotting , Ratones , Ratones Noqueados , Mitocondrias/metabolismo , Factores de Transcripción NFATC/inmunología , Factores de Transcripción NFATC/metabolismo , Proteína ORAI1 , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción GenéticaRESUMEN
TGF-ß1 upregulates microRNA-192 (miR-192) in cultured glomerular mesangial cells and in glomeruli from diabetic mice. miR-192 not only increases collagen expression by targeting the E-box repressors Zeb1/2 but also modulates other renal miRNAs, suggesting that it may be a therapeutic target for diabetic nephropathy. We evaluated the efficacy of a locked nucleic acid (LNA)-modified inhibitor of miR-192, designated LNA-anti-miR-192, in mouse models of diabetic nephropathy. LNA-anti-miR-192 significantly reduced levels of miR-192, but not miR-194, in kidneys of both normal and streptozotocin-induced diabetic mice. In the kidneys of diabetic mice, inhibition of miR-192 significantly increased Zeb1/2 and decreased gene expression of collagen, TGF-ß, and fibronectin; immunostaining confirmed the downregulation of these mediators of renal fibrosis. Furthermore, LNA-anti-miR-192 attenuated proteinuria in these diabetic mice. In summary, the specific reduction of renal miR-192 decreases renal fibrosis and improves proteinuria, lending support for the possibility of an anti-miRNA-based translational approach to the treatment of diabetic nephropathy.
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Diabetes Mellitus Experimental/complicaciones , Nefropatías Diabéticas/patología , Nefropatías Diabéticas/prevención & control , Riñón/patología , MicroARNs/antagonistas & inhibidores , MicroARNs/efectos de los fármacos , Oligonucleótidos/farmacología , Albuminuria/metabolismo , Albuminuria/prevención & control , Animales , Colágeno/metabolismo , Diabetes Mellitus Experimental/inducido químicamente , Diabetes Mellitus Tipo 1/inducido químicamente , Diabetes Mellitus Tipo 1/complicaciones , Nefropatías Diabéticas/metabolismo , Modelos Animales de Enfermedad , Fibronectinas/metabolismo , Fibrosis , Proteínas de Homeodominio/metabolismo , Riñón/efectos de los fármacos , Riñón/metabolismo , Factores de Transcripción de Tipo Kruppel/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Proteinuria/metabolismo , Proteinuria/prevención & control , Proteínas Represoras/metabolismo , Estreptozocina/efectos adversos , Factor de Crecimiento Transformador beta/metabolismo , Caja Homeótica 2 de Unión a E-Box con Dedos de Zinc , Homeobox 1 de Unión a la E-Box con Dedos de ZincRESUMEN
Evidence showing a relationship between the mouse gut microbiome and properties such as phenotype and reaction to therapeutic agents and other treatments has increased significantly over the past 20 to 30 y. Recent concerns regarding the reproducibility of animal experiments have underscored the importance of understanding this relationship and how differences in husbandry practices can affect the gut microbiome. The current study focuses on effects of different barrier practices in 2 barrier facilities at the same institution on the fecal microbiome of breeding C57Bl/6J mice. Ten female and 10 male C57Bl/6J mice were obtained in one shipment from Jackson Laboratories and were housed under different barrier conditions upon arrival. Fecal samples were collected on arrival and periodically thereafter and were sent to TransnetYX for microbiome analysis. Mice used for collection of feces were housed as breeding pairs, with a total of 5 breeding pairs per barrier. An additional fecal sample was collected from these mice at 8 wk after arrival. One F1 female and one F1 male from each breeding cage were housed as brother-sister breeding pairs and a fecal sample was collected from them at 8 wk of age. Brother-sister breeding colonies were continued through F3, with fecal samples for microbiome analysis were collected from each generation at 8 wk of age. Breeding colonies in the 2 barriers showed differences in relative abundance, α -diversity, and ß -diversity. Our data indicate that differences in barrier husbandry practices, including the use of autoclaved cages, the degree of restricted access, feed treatment practices, and water provision practices, can affect fecal microbiome divergence in both the parental and filial generations of different breeding colonies. To our knowledge, this is the first study to examine the effect of barrier husbandry practices on the microbiome of breeding colonies through the F3 generation.
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Microbioma Gastrointestinal , Microbiota , Ratones , Animales , Masculino , Femenino , Reproducibilidad de los Resultados , Ratones Endogámicos C57BL , HecesRESUMEN
Protein farnesyltransferase (FTase) and protein geranylgeranyltransferase-I (GGTase-I) add 15- or 20-carbon lipids, respectively, to proteins that terminate with a CaaX motif. These posttranslational modifications of proteins with lipids promote protein interactions with membrane surfaces in cells, but the in vivo importance of the CaaX prenyltransferases and the protein lipidation reactions they catalyze remain incompletely defined. One study concluded that a deficiency of FTase was inconsequential in adult mice and led to little or no tissue pathology. To assess the physiologic importance of the CaaX prenyltransferases, we used conditional knockout alleles and an albumin-Cre transgene to produce mice lacking FTase, GGTase-I, or both enzymes in hepatocytes. The hepatocyte-specific FTase knockout mice survived but exhibited hepatocellular disease and elevated transaminases. Mice lacking GGTase-I not only had elevated transaminases but also had dilated bile cannaliculi, hyperbilirubinemia, hepatosplenomegaly, and reduced survival. Of note, GGTase-I-deficient hepatocytes had a rounded shape and markedly reduced numbers of actin stress fibers. Hepatocyte-specific FTase/GGTase-I double-knockout mice closely resembled mice lacking GGTase-I alone, but the disease was slightly more severe. Our studies refute the notion that FTase is dispensable and demonstrate that GGTase-I is crucial for the vitality of hepatocytes.
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Transferasas Alquil y Aril/deficiencia , Dimetilaliltranstransferasa/deficiencia , Farnesiltransferasa/deficiencia , Hepatocitos/enzimología , Hepatopatías/fisiopatología , Prenilación de Proteína/efectos de los fármacos , Animales , Hígado/patología , Hígado/fisiopatología , Hepatopatías/patología , Ratones , Ratones NoqueadosRESUMEN
Tissue factor pathway inhibitor-2 (TFPI-2) inhibits factor XIa, plasma kallikrein, and factor VIIa/tissue factor; accordingly, it has been proposed for use as an anticoagulant. Full-length TFPI-2 or its isolated first Kunitz domain (KD1) also inhibits plasmin; therefore, it has been proposed for use as an antifibrinolytic agent. However, the anticoagulant properties of TFPI-2 or KD1 would diminish its antifibrinolytic function. In this study, structure-based investigations and analysis of the serine protease profiles revealed that coagulation enzymes prefer a hydrophobic residue at the P2' position in their substrates/inhibitors, whereas plasmin prefers a positively charged arginine residue at the corresponding position in its substrates/inhibitors. Based upon this observation, we changed the P2' residue Leu-17 in KD1 to Arg (KD1-L17R) and compared its inhibitory properties with wild-type KD1 (KD1-WT). Both WT and KD1-L17R were expressed in Escherichia coli, folded, and purified to homogeneity. N-terminal sequences and mass spectra confirmed proper expression of KD1-WT and KD1-L17R. Compared with KD1-WT, the KD1-L17R did not inhibit factor XIa, plasma kallikrein, or factor VIIa/tissue factor. Furthermore, KD1-L17R inhibited plasmin with â¼6-fold increased affinity and effectively prevented plasma clot fibrinolysis induced by tissue plasminogen activator. Similarly, in a mouse liver laceration bleeding model, KD1-L17R was â¼8-fold more effective than KD1-WT in preventing blood loss. Importantly, in this bleeding model, KD1-L17R was equally or more effective than aprotinin or tranexamic acid, which have been used as antifibrinolytic agents to prevent blood loss during major surgery/trauma. Furthermore, as compared with aprotinin, renal toxicity was not observed with KD1-L17R.
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Sustitución de Aminoácidos , Antifibrinolíticos , Fibrinólisis/efectos de los fármacos , Glicoproteínas , Hemorragia/tratamiento farmacológico , Pliegue de Proteína , Animales , Antifibrinolíticos/química , Antifibrinolíticos/farmacología , Aprotinina/química , Aprotinina/farmacología , Factores de Coagulación Sanguínea/química , Modelos Animales de Enfermedad , Escherichia coli , Glicoproteínas/química , Glicoproteínas/genética , Glicoproteínas/farmacología , Humanos , Ratones , Mutación Missense , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacología , Relación Estructura-Actividad , Ácido Tranexámico/química , Ácido Tranexámico/farmacologíaRESUMEN
Neurotensin receptor-1 (NTR-1) is overexpressed in colon cancers and colon cancer cell lines. Signaling through this receptor stimulates proliferation of colonocyte-derived cell lines and promotes inflammation and mucosal healing in animal models of colitis. Given the causal role of this signaling pathway in mediating colitis and the importance of inflammation in cancer development, we tested the effects of NTR-1 in mouse models of inflammation-associated and sporadic colon cancer using NTR-1-deficient (Ntsr1(-) (/-)) and wild-type (Ntsr1(+/+)) mice. In mice treated with azoxymethane (AOM) to model sporadic cancer, NTR-1 had a significant effect on tumor development with Ntsr1(+/+) mice developing over twofold more tumors than Ntsr1(-) (/-) mice (p = 0.04). There was no effect of NTR-1 on the number of aberrant crypt foci or tumor size, suggesting that NT/NTR-1 signaling promotes the conversion of precancerous cells to adenomas. Interestingly, NTR-1 status did not affect tumor development in an inflammation-associated cancer model where mice were treated with AOM followed by two cycles of 5% dextran sulfate sodium (DSS). In addition, colonic molecular and histopathologic analyses were performed shortly after a single cycle of DSS. NTR-1 status did not affect colonic myeloperoxidase activity or histopathologic scores for damage and inflammation. However, Ntsr1(-) (/-) mice were more resistant to DSS-induced mortality (p = 0.01) and had over twofold higher colonic expression levels of Il6 and Cxcl2 (p < 0.04), cytokines known to promote tumor development. These results represent the first direct demonstration that targeted disruption of the Ntsr1 gene reduces susceptibility to colon tumorigenesis.
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Colitis/genética , Neoplasias del Colon/genética , Modelos Animales de Enfermedad , Receptores de Neurotensina/genética , Animales , Azoximetano , Quimiocina CXCL2/genética , Quimiocina CXCL2/metabolismo , Colitis/inducido químicamente , Colitis/metabolismo , Colon/efectos de los fármacos , Colon/metabolismo , Colon/patología , Neoplasias del Colon/inducido químicamente , Neoplasias del Colon/metabolismo , Sulfato de Dextran , Femenino , Expresión Génica/efectos de los fármacos , Humanos , Inmunohistoquímica , Interleucina-6/genética , Interleucina-6/metabolismo , Estimación de Kaplan-Meier , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Pirazoles/farmacología , Quinolinas/farmacología , Receptores de Neurotensina/antagonistas & inhibidores , Receptores de Neurotensina/deficiencia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
OBJECTIVES: Many autoimmune diseases are characterised by a female predominance. This may be caused by sex hormones, sex chromosomes or both. This report uses a transgenic mouse model to investigate how sex chromosome complement, not confounded by differences in gonadal type, might contribute to lupus pathogenesis. METHODS: Transgenic NZM2328 mice were created by deletion of the Sry gene from the Y chromosome, thereby separating genetic from gonadal sex. Survival, renal histopathology and markers of immune activation were compared in mice carrying the XX versus the XY(-) sex chromosome complement, with each genotype being ovary bearing. RESULTS: Mice with XX sex chromosome complement compared with XY(-) exhibited poorer survival rates and increased kidney pathology. Splenic T lymphocytes from XX mice demonstrated upregulated X-linked CD40 ligand expression and higher levels of activation markers ex vivo. Increased MMP, TGF and IL-13 production was found, while IL-2 was lower in XX mice. An accumulation of splenic follicular B cells and peritoneal marginal zone B cells was observed, coupled with upregulated costimulatory marker expression on B cells in XX mice. CONCLUSION: These data show that the XX sex chromosome complement, compared with XY(-), is associated with accelerated spontaneous lupus.
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Lupus Eritematoso Sistémico/genética , Aberraciones Cromosómicas Sexuales , Trastornos de los Cromosomas Sexuales/genética , Cromosoma X/genética , Cromosoma Y/genética , Animales , Biomarcadores/metabolismo , Antígenos CD28/inmunología , Complejo CD3/inmunología , Ligando de CD40/metabolismo , Duplicación Cromosómica , Femenino , Riñón , Enfermedades Renales , Longevidad , Lupus Eritematoso Sistémico/inmunología , Lupus Eritematoso Sistémico/metabolismo , Masculino , Ratones , Ratones Transgénicos , Bazo/inmunología , Linfocitos T/inmunología , Regulación hacia ArribaRESUMEN
Patients with neurofibromatosis type 1 (NF1) carry approximately a 10% lifetime risk of developing a malignant peripheral nerve sheath tumor (MPNST). Although the molecular mechanisms underlying NF1 to MPNST malignant transformation remain unclear, alterations of both the RAS/RAF/MAPK and PI3K/AKT/mTOR signaling pathways have been implicated. In a series of genetically engineered murine models, we perturbed RAS/RAF/MAPK or/and PTEN/PI3K/AKT pathway, individually or simultaneously, via conditional activation of K-ras oncogene or deletion of Nf1 or Pten tumor suppressor genes. Only K-Ras activation in combination with a single Pten allele deletion led to 100% penetrable development of NF lesions and subsequent progression to MPNST. Importantly, loss or decrease in PTEN expression was found in all murine MPNSTs and a majority of human NF1-associated MPNST lesions, suggesting that PTEN dosage and its controlled signaling pathways are critical for transformation of NFs to MPNST. Using noninvasive in vivo PET-CT imaging, we demonstrated that FDG can be used to identify the malignant transformation in both murine and human MPNSTs. Our data suggest that combined inhibition of RAS/RAF/MAPK and PTEN/PI3K/AKT pathways may be beneficial for patients with MPNST.
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Transformación Celular Neoplásica/genética , Dosificación de Gen , Neurofibroma/genética , Neurofibroma/patología , Fosfohidrolasa PTEN/genética , Animales , Transformación Celular Neoplásica/patología , Fluorodesoxiglucosa F18 , Humanos , Ratones , Ratones Mutantes , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Proteína Oncogénica p21(ras)/metabolismo , Tomografía de Emisión de Positrones , Quinasas raf/metabolismoRESUMEN
Previous studies have shown that the alternative pathway of complement activation plays an important role in protection against infection with Cryptococcus neoformans. Cryptococcus gattii does not activate the alternative pathway as well as C. neoformans in vitro. The role of complement in C. gattii infection in vivo has not been reported. In this study, we used mice deficient in complement components to investigate the role of complement in protection against a C. gattii isolate from an ongoing outbreak in northwestern North America. While factor B-deficient mice showed an enhanced rate of death, complement component C3-deficient mice died even more rapidly, indicating that the alternative pathway was not the only complement pathway contributing to protection against disease. Both C3- and factor B-deficient mice had increased fungal burdens in comparison to wild-type mice. Histopathology revealed an overwhelming fungal burden in the lungs of these complement-deficient mice, which undoubtedly prevented efficient gas exchange, causing death. Following the fate of radiolabeled organisms showed that both factor B- and C3-deficient mice were less effective than wild-type mice in clearing organisms. However, opsonization of C. gattii with complement components was not sufficient to prolong life in mice deficient in complement. Killing of C. gattii by macrophages in vitro was decreased in the presence of serum from factor B- and C3-deficient versus wild-type mice. In conclusion, we have demonstrated that complement activation is crucial for survival in C. gattii infection. Additionally, we have shown that the alternative pathway of complement activation is not the only complement pathway contributing to protection.
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Complemento C3/inmunología , Factor B del Complemento/inmunología , Criptococosis/inmunología , Animales , Activación de Complemento/inmunología , Complemento C3/deficiencia , Factor B del Complemento/deficiencia , Criptococosis/patología , Cryptococcus/inmunología , Ensayo de Inmunoadsorción Enzimática , Humanos , Pulmón/microbiología , Pulmón/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factor de Necrosis Tumoral alfa/inmunología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Replication-competent retrovirus (RCR) vectors are intrinsically incapable of infecting quiescent cells and have been shown to achieve highly efficient and tumor-restricted replicative spread and gene transfer in vivo after direct intratumoral injection in a variety of primary cancer models. However, i.v. delivery of RCR vectors expressing therapeutic genes has never previously been tested, particularly in an immunocompetent tumor model. Therefore, in the present study, we sought to test the therapeutic effect of an RCR vector (ACE-CD) carrying the yeast cytosine deaminase (CD) gene, which converts the nontoxic prodrug 5-fluorocytosine (5FC) into the chemotoxin 5-fluorouracil, after delivery by infusion into the locoregional circulation in a multifocal hepatic metastasis model of colon cancer. After confirmation of suicide gene cytotoxicity in vitro, multifocal hepatic tumors were established in syngeneic mice with murine CT26 colorectal cancer cells expressing firefly luciferase (CT26-Luc), and the ACE-CD vector was infused via intrasplenic injection into the portal circulation. Fourteen days after locoregional infusion, systemic administration of 5FC resulted in significant inhibition of bioluminescent signals in mice whose tumors had been infected with RCR but not in control mice. Notably, there was no detectable RCR vector spread to normal liver or bone marrow by quantitative PCR analysis. Our results thus show that locoregional delivery of a suicide gene by RCR vectors infused into the portal circulation results in progressive transduction of multiple tumor foci in the liver, without evidence of spread to adjacent normal parenchyma or extrahepatic tissues, and can achieve significant tumor growth inhibition.
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Neoplasias Colorrectales/terapia , Genes Transgénicos Suicidas , Terapia Genética/métodos , Retroviridae/fisiología , Animales , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/virología , Citosina Desaminasa/genética , Citosina Desaminasa/metabolismo , Modelos Animales de Enfermedad , Femenino , Flucitosina/farmacocinética , Fluorouracilo/farmacocinética , Fluorouracilo/farmacología , Vectores Genéticos/genética , Humanos , Neoplasias Hepáticas Experimentales/genética , Neoplasias Hepáticas Experimentales/secundario , Neoplasias Hepáticas Experimentales/terapia , Neoplasias Hepáticas Experimentales/virología , Ratones , Ratones Endogámicos BALB C , Retroviridae/genética , Replicación ViralRESUMEN
Polycyclic aromatic hydrocarbons like benzo[a]pyrene (BaP) are ubiquitous environmental contaminants formed during incomplete combustion of organic materials. Our prior work showed that transplacental exposure to BaP depletes ovarian follicles and increases prevalence of epithelial ovarian tumors later in life. We used the MutaMouse transgenic rodent model to address the hypothesis that ovarian mutations play a role in tumorigenesis caused by prenatal exposure to BaP. Pregnant MutaMouse females were treated with 0, 10, 20, or 40 mg/(kg day) BaP orally on gestational days 7-16, covering critical windows of ovarian development. Female offspring were euthanized at 10 weeks of age; some ovaries with oviducts were processed for follicle counting; other ovaries/oviducts and bone marrow were processed for determination of lacZ mutant frequency (MF). Mutant plaques were pooled within dose groups and sequenced to determine the mutation spectrum. BaP exposure caused highly significant dose-related decreases in ovarian follicles and increases in ovarian/oviductal and bone marrow mutant frequencies at all doses. Absence of follicles, cell packets, and epithelial tubular structures were observed with 20 and 40 mg/(kg day) BaP. Depletion of ovarian germ cells was inversely associated with ovarian MF. BaP induced primarily G > T and G > C transversions and deletions in ovaries/oviducts and bone marrow cells and produced a mutation signature highly consistent with that of tobacco smoking in human cancers. Overall, our results show that prenatal BaP exposure significantly depletes ovarian germ cells, causes histopathological abnormalities, and increases the burden of ovarian/oviductal mutations, which may be involved in pathogenesis of epithelial ovarian tumors. Environ. Mol. Mutagen. 60:410-420, 2019. © 2018 Her Majesty the Queen in Right of Canada.
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Benzo(a)pireno/toxicidad , Exposición Materna , Intercambio Materno-Fetal/fisiología , Mutágenos/toxicidad , Efectos Tardíos de la Exposición Prenatal/patología , Animales , Contaminantes Ambientales/toxicidad , Femenino , Operón Lac/genética , Ratones , Mutación/efectos de los fármacos , Mutación/genética , Folículo Ovárico/citología , Folículo Ovárico/patología , EmbarazoRESUMEN
Astronauts traveling in deep space are exposed to high-charge and energy (HZE) particles from galactic cosmic rays. We have previously determined that irradiation of adult female mice with iron HZE particles induces DNA double-strand breaks, oxidative damage and apoptosis in ovarian follicles, causing premature ovarian failure. These effects occur at lower doses than with conventional photon irradiation. Ovarian failure with resultant loss of negative feedback and elevated levels of gonadotropin hormones is thought to play a role in the pathophysiology of ovarian cancer. Therefore, we hypothesized that charged-iron-particle irradiation induces ovarian tumorigenesis in mice. In this study, three-month-old female mice were exposed to 0 cGy (sham) or 50 cGy iron ions and aged to 18 months. The 50 cGy irradiated mice had increased weight gain with age and lack of estrous cycling, consistent with ovarian failure. A total of 47% and 7% of mice irradiated with 50 cGy had unilateral and bilateral ovarian tumors, respectively, whereas 14% of mice in the 0 cGy group had unilateral tumors. The tumors contained multiple tubular structures, which were lined with cells positive for the epithelial marker cytokeratin, and had few proliferating cells. In some tumors, packets of cells between the tubular structures were immunopositive for the granulosa cell marker FOXL2. Based on these findings, tumors were diagnosed as tubular adenomas or mixed tubular adenoma/granulosa cell tumors. In conclusion, charged-iron-particle-radiation induces ovarian tumors in mice, raising concerns about ovarian tumors as late sequelae of deep space travel in female astronauts.
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Radiación Cósmica/efectos adversos , Medio Ambiente Extraterrestre , Hierro/efectos adversos , Neoplasias Glandulares y Epiteliales/etiología , Neoplasias Inducidas por Radiación/etiología , Neoplasias Ováricas/etiología , Animales , Astronautas , Peso Corporal/efectos de la radiación , Carcinoma Epitelial de Ovario , Daño del ADN , Relación Dosis-Respuesta en la Radiación , Células Epiteliales/patología , Células Epiteliales/efectos de la radiación , Ciclo Estral/efectos de la radiación , Femenino , Ratones , Ratones Endogámicos C57BL , Neoplasias Glandulares y Epiteliales/genética , Neoplasias Glandulares y Epiteliales/patología , Neoplasias Glandulares y Epiteliales/fisiopatología , Neoplasias Inducidas por Radiación/genética , Neoplasias Inducidas por Radiación/patología , Neoplasias Inducidas por Radiación/fisiopatología , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Neoplasias Ováricas/fisiopatología , Ovario/patología , Ovario/efectos de la radiaciónRESUMEN
Protein inhibitor of activated STAT Y (PIASy) is the shortest member of the PIAS family and has been reported to modulate the transcriptional activities of STAT1, lymphoid enhancer factor 1 (LEF-1), and the androgen receptor. PIAS proteins have also been identified as E3 ligases for the small ubiquitin-like modifier (SUMO) proteins. PIASy in particular has been reported to mediate SUMO-2/3 modification of LEF-1, sequestering it into nuclear bodies, and SUMO-1 ligation to c-Myb, modulating its transcriptional activation properties. We have cloned murine Piasy and a splice variant which omits exon 6, containing the nuclear retention PINIT motif. Cell culture studies indicate that both the full length and the splice variant are localized in the nucleus but differentially enhance SUMO ligation. To further understand the functions of PIASy, we have generated PIASy-deficient mice. Surprisingly, Piasy(-/-) mice appear phenotypically normal. Activation of STAT1 is not significantly perturbed in Piasy(-/-) cells, and sumoylation patterns for SUMO-1 or SUMO-3 modification are similar when comparing tissues and embryonic fibroblasts from wild-type and knockout mice. Our study demonstrates that at steady state, PIASy is either dispensable or compensated for by other PIAS family members or by other mechanisms when deleted.
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Proteínas Portadoras/genética , Proteínas Portadoras/fisiología , Péptidos y Proteínas de Señalización Intracelular , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/química , Núcleo Celular/metabolismo , Células Cultivadas , Proteínas de Unión al ADN/metabolismo , Desarrollo Embrionario y Fetal/genética , Desarrollo Embrionario y Fetal/fisiología , Exones , Femenino , Marcación de Gen , Masculino , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Virus de la Leucemia Murina de Moloney/genética , Virus de la Leucemia Murina de Moloney/patogenicidad , Virus de la Leucemia Murina de Moloney/fisiología , Fenotipo , Proteínas Inhibidoras de STAT Activados , Factor de Transcripción STAT1 , Proteína SUMO-1/metabolismo , Homología de Secuencia de Aminoácido , Transactivadores/metabolismo , Ubiquitinas/metabolismo , Replicación Viral/fisiologíaRESUMEN
The entry of infectious agents in rodent colonies occurs despite robust sentinel monitoring programs, strict quarantine measures, and stringent biosecurity practices. In light of several outbreaks with Aspiculuris tetraptera in our facilities, we investigated the presence of anthelmintic resistance and the use of exhaust air dust (EAD) PCR for early detection of A. tetraptera infection. To determine anthelmintic resistance, C57BL/6, DBA/2, and NCr nude mice were experimentally inoculated with embryonated A. tetraptera ova harvested from enzootically infected mice, followed by treatment with 150 ppm fenbendazole in feed, 150 ppm fenbendazole plus 5 ppm piperazine in feed, or 2.1 mg/mL piperazine in water for 4 or 8 wk. Regardless of the mouse strain or treatment, no A. tetraptera were recovered at necropsy, indicating the lack of resistance in the worms to anthelmintic treatment. In addition, 10 of 12 DBA/2 positive-control mice cleared the A. tetraptera infection without treatment. To evaluate the feasibility of EAD PCR for A. tetraptera, 69 cages of breeder mice enzootically infected with A. tetraptera were housed on a Tecniplast IVC rack as a field study. On day 0, 56% to 58% of the cages on this rack tested positive for A. tetraptera by PCR and fecal centrifugation flotation (FCF). PCR from EAD swabs became positive for A. tetraptera DNA within 1 wk of placing the above cages on the rack. When these mice were treated with 150 ppm fenbendazole in feed, EAD PCR reverted to pinworm-negative after 1 mo of treatment and remained negative for an additional 8 wk. The ability of EAD PCR to detect few A. tetraptera positive mice was investigated by housing only 6 infected mice on another IVC rack as a field study. The EAD PCR from this rack was positive for A. tetraptera DNA within 1 wk of placing the positive mice on it. These findings demonstrate that fenbendazole is still an effective anthelmintic and that EAD PCR is a rapid, noninvasive assay that may be a useful diagnostic tool for antemortem detection of A. tetraptera infection, in conjunction with fecal PCR and FCF.
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Monitoreo Epidemiológico/veterinaria , Oxiuriasis/veterinaria , Oxyuroidea/aislamiento & purificación , Animales , Antihelmínticos/farmacología , Brotes de Enfermedades , Polvo/análisis , Heces/parasitología , Femenino , Fenbendazol/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Desnudos , Oxiuriasis/epidemiología , Oxiuriasis/parasitología , Oxyuroidea/clasificación , Oxyuroidea/efectos de los fármacos , Oxyuroidea/crecimiento & desarrollo , Reacción en Cadena de la PolimerasaRESUMEN
Because surface disinfectants are an important means of pathogen control within laboratory animal facilities, these products must have an appropriate spectrum of antimicrobial activity. However, many other factors must also be considered, including effects on human health, environmental safety, and animal behavior. Aqueous solutions of sodium hypochlorite often are considered to be the 'gold standard' for surface disinfection, but these products can be corrosive, caustic, and aversive in odor. This study was designed to identify disinfectants that are as effective as hypochlorite solutions but more acceptable for use in a laboratory animal setting. An antiviral disinfectant-efficacy assay was developed by using viral vectors that expressed green fluorescence protein as surrogates for wild-type viruses of concern in laboratory animals. Efficacy testing revealed that most of the products were highly effective when used against viral vectors in suspension. However, when the disinfectants were challenged by buffering virus in protein or drying virus on nonporous surfaces, the hypochlorite and peroxymonosulfate products performed the best. Review of safety data sheets for the agents indicated that a peroxide-based product was considerably safer than the other products tested and that the pH of most products was not conducive to disposal down a drain. Behavioral testing of Swiss Webster, C57Bl/6, and BALB/c mice showed that the hypochlorite- and peroxide-based products were clearly aversive, given that the mice consistently avoided these products. All of these factors must be considered when choosing the appropriate disinfectant.
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Desinfectantes/química , Animales , Animales de Laboratorio , Antivirales/química , Antivirales/farmacología , Conducta Animal , Desinfectantes/farmacología , Desinfectantes/toxicidad , Desinfección , Vivienda para Animales , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Peróxidos/farmacología , Peróxidos/toxicidad , Hipoclorito de Sodio/farmacología , Hipoclorito de Sodio/toxicidadRESUMEN
In this study, we fed a standard NIH-31 diet fortified with vitamin E to C57BL/6 mice and strains of mice with a C57BL/6 background that had spontaneously developed ulcerative dermatitis (UD). In addition to the therapeutic response to increased levels of vitamin E, we also defined the occurrence of UD within our facility in terms of age, sex, coat color, and lesion location on the body. Mice with spontaneous UD were fed a vitamin E-fortified diet (3000 IU/kg) for a period of 8 weeks and entered the study without regard to vendor source, age, sex, coat color, or the site or number of UD lesions. We found that lesions occurred most commonly on the dorsal cervical and scapular regions and spared the ventral abdomen and thorax. No sex or coat color predilection was noted for the development of UD, however males were older than females at the time of lesion development. Of 71 mice, 32 (45%) had complete lesion re-epithelialization with hair regrowth. Complete lesion repair was not influenced by sex, age, or coat color. The average time to complete lesion repair ranged from 2 to 5 weeks, and there was no correlation with sex or coat color. The positive response to vitamin E suggests that protection from oxidative injury may play a role in the resolution of UD lesions and offers veterinarians and investigators a new treatment option with ease of compliance.
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Dermatitis Atópica/veterinaria , Dieta , Alimentos Fortificados , Enfermedades de los Roedores/tratamiento farmacológico , Úlcera Cutánea/veterinaria , Medicina Veterinaria/métodos , Vitamina E/administración & dosificación , Animales , Dermatitis Atópica/tratamiento farmacológico , Dermatitis Atópica/patología , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Enfermedades de los Roedores/patología , Úlcera Cutánea/tratamiento farmacológico , Úlcera Cutánea/patología , Resultado del TratamientoRESUMEN
Hematologic and serum biochemical values are of great importance in assessing animal health. Normal reference ranges for vervet monkeys (Chlorocebus aethiops sabaeus) have seldom been reported, making it difficult for clinicians to interpret blood values. The purpose of this study was to determine what effects age and sex have on hematologic and serum biochemical values of vervet monkeys and to establish clinically relevant reference ranges for all life stages of each gender. Blood samples were collected from 140 healthy vervet monkeys of Caribbean origin consisting of 60 females and 80 males. Male and female data were displayed separately within six life-stage categories (yearlings, juveniles, adolescents, young adults, adults, and aged). The effects of sex and age on these values were examined statistically. Significant age-related factors included red blood cell count, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, alkaline phosphatase, albumin, total protein, globulin, direct bilirubin, blood urea nitrogen, creatinine, glucose, calcium, phosphorus, potassium, albumin/globulin ratio, blood urea nitrogen/creatinine ratio, and sodium/potassium ratio values. Significant sex-related values included red blood cell count, hemoglobin, hematocrit, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, neutrophil count, total bilirubin, direct bilirubin, creatinine, glucose, calcium, phosphorus, total carbon dioxide, chloride, potassium, and sodium/potassium ratio values.