RESUMEN
During activity, coordinated vasodilation of microcirculatory networks with upstream supply vessels increases blood flow to skeletal and cardiac muscles and reduces peripheral resistance. Endothelial dysfunction in humans attenuates activity-dependent vasodilation, resulting in exercise-induced hypertension in otherwise normotensive individuals. Underpinning activity-dependent hyperemia is an ascending vasodilation in which the endothelial gap junction protein, connexin (Cx)40, plays an essential role. Because exercise-induced hypertension is proposed as a forerunner to clinical hypertension, we hypothesized that endothelial disruption of Cx40 function in mice may create an animal model of this condition. To this end, we created mice in which a mutant Cx40T152A was expressed alongside wildtype Cx40 selectively in the endothelium. Expression of the Cx40T152A transgene in Xenopus oocytes and mouse coronary endothelial cells in vitro impaired both electric and chemical conductance and acted as a dominant-negative against wildtype Cx40, Cx43, and Cx45, but not Cx37. Endothelial expression of Cx40T152A in Cx40T152ATg mice attenuated ascending vasodilation, without effect on radial coupling through myoendothelial gap junctions. Using radiotelemetry, Cx40T152ATg mice showed an activity-dependent increase in blood pressure, which was significantly greater than in wildtype mice, but significantly less than in chronically hypertensive, Cx40knockout mice. The increase in heart rate with activity was also greater than in wildtype or Cx40knockout mice. We conclude that the endothelial Cx40T152A mutation attenuates activity-dependent vasodilation, producing a model of exercise-induced hypertension. These data highlight the importance of endothelial coupling through Cx40 in regulating blood pressure during activity.
Asunto(s)
Conexinas/deficiencia , Endotelio Vascular/metabolismo , Hipertensión/etiología , Hipertensión/fisiopatología , Condicionamiento Físico Animal/efectos adversos , Animales , Presión Sanguínea/fisiología , Conexinas/genética , Conexinas/metabolismo , Modelos Animales de Enfermedad , Endotelio Vascular/patología , Uniones Comunicantes/fisiología , Frecuencia Cardíaca/fisiología , Técnicas In Vitro , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Mutación/genética , Vasodilatación/fisiología , Proteína alfa-5 de Unión ComunicanteRESUMEN
UNLABELLED: Human respiratory syncytial virus (RSV) is a major cause of morbidity and severe lower respiratory tract disease in the elderly and very young, with some infants developing bronchiolitis, recurrent wheezing, and asthma following infection. Previous studies in humans and animal models have shown that vaccination with formalin-inactivated RSV (FI-RSV) leads to prominent airway eosinophilic inflammation following RSV challenge; however, the roles of pulmonary eosinophilia in the antiviral response and in disease pathogenesis are inadequately understood. In vivo studies in mice with eotaxin and/or interleukin 5 (IL-5) deficiency showed that FI-RSV vaccination did not lead to enhanced pulmonary disease, where following challenge there were reduced pulmonary eosinophilia, inflammation, Th2-type cytokine responses, and altered chemokine (TARC and CCL17) responses. In contrast to wild-type mice, RSV was recovered at high titers from the lungs of eotaxin- and/or IL-5-deficient mice. Adoptive transfer of eosinophils to FI-RSV-immunized eotaxin- and IL-5-deficient (double-deficient) mice challenged with RSV was associated with potent viral clearance that was mediated at least partly through nitric oxide. These studies show that pulmonary eosinophilia has dual outcomes: one linked to RSV-induced airway inflammation and pulmonary pathology and one with innate features that contribute to a reduction in the viral load. IMPORTANCE: This study is critical to understanding the mechanisms attributable to RSV vaccine-enhanced disease. This study addresses the hypothesis that IL-5 and eotaxin are critical in pulmonary eosinophil response related to FI-RSV vaccine-enhanced disease. The findings suggest that in addition to mediating tissue pathology, eosinophils within a Th2 environment also have antiviral activity.
Asunto(s)
Eosinófilos/inmunología , Pulmón/inmunología , Pulmón/patología , Infecciones por Virus Sincitial Respiratorio/inmunología , Infecciones por Virus Sincitial Respiratorio/patología , Vacunas contra Virus Sincitial Respiratorio/inmunología , Virus Sincitial Respiratorio Humano/inmunología , Animales , Femenino , Pulmón/virología , Ratones Endogámicos BALB C , Ratones Noqueados , Ratones Transgénicos , Vacunas de Productos Inactivados/inmunología , Carga ViralRESUMEN
Mutant forms of connexin40 (Cx40) exist in the human population and predispose carriers to atrial fibrillation. Since endothelial expression of Cx40 is important for electrical and chemical communication within the arterial wall, carriers of mutant Cx40 proteins may be predisposed to peripheral arterial dysfunction and dysregulation of blood pressure. We have therefore studied mice expressing either a chemically dysfunctional mutant, Cx40T202S, or wild-type Cx40, with native Cx40, specifically in the endothelium. Blood pressure was measured by telemetry under normal conditions and during cardiovascular stress induced by locomotor activity, phenylephrine or nitric oxide blockade (N(É·)-nitro-L-arginine methyl ester hydroxide, L-NAME). Blood pressure of Cx40T202STg mice was significantly elevated at night when compared with wild-type or Cx40Tg mice, without change in mean heart rate, pulse pressure or locomotor activity. Analysis over 24 h showed that blood pressure of Cx40T202STg mice was significantly elevated at rest and additionally during locomotor activity. In contrast, neither plasma renin concentration nor pressor responses to phenylephrine or L-NAME were altered, the latter indicating that nitric oxide bioavailability was normal. In isolated, pressurised mesenteric arteries, hyperpolarisation and vasodilation evoked by SKA-31, the selective modulator of SKCa and IKCa channels, was significantly reduced in Cx40T202STg mice, due to attenuation of the SKCa component. Acetylcholine-induced ascending vasodilation in vivo was also significantly attenuated in cremaster muscle arterioles of Cx40T202STg mice, compared to wild-type and Cx40Tg mice. We conclude that endothelial expression of the chemically dysfunctional Cx40T202S reduces peripheral vasodilator capacity mediated by SKCa-dependent hyperpolarisation and also increases blood pressure.
Asunto(s)
Conexinas/metabolismo , Endotelio Vascular/fisiopatología , Hipertensión/fisiopatología , Canales de Potasio Calcio-Activados/metabolismo , Vasodilatación/fisiología , Animales , Presión Sanguínea , Conexinas/genética , Masculino , Potenciales de la Membrana , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Técnicas de Placa-Clamp , Proteína alfa-5 de Unión ComunicanteRESUMEN
Cardiovascular disease is characterised by reduced nitric oxide bioavailability resulting from oxidative stress. Our previous studies have shown that nitric oxide deficit per se increases the contribution of T-type calcium channels to vascular tone through increased superoxide from NADPH oxidase (Nox). The aim of the present study was therefore to identify the Nox isoform responsible for modulating T-type channel function, as T-type channels are implicated in several pathophysiological conditions involving oxidative stress. We evaluated T-channel function in skeletal muscle arterioles in vivo, using a novel T-channel blocker, TTA-A2 (3 µmol/L), which demonstrated no cross reactivity with L-type channels. Wild-type and Nox2 knockout (Nox2ko) mice were treated with the nitric oxide synthase inhibitor L-NAME (40 mg/kg/day) for 2 weeks. L-NAME treatment significantly increased systolic blood pressure and the contribution of T-type calcium channels to arteriolar tone in wild-type mice, and this was not prevented by Nox2 deletion. In Nox2ko mice, pharmacological inhibition of Nox1 (10 µmol/L ML171), Nox4 (10 µmol/L VAS2870) and Nox4-derived hydrogen peroxide (500 U/mL catalase) significantly reduced the effect of chronic nitric oxide inhibition on T-type channel function. In contrast, in wild-type mice, ML171 and VAS2870, but not catalase, reduced the contribution of T-type channels to vascular tone, suggesting a role for Nox1 and non-selective actions of VAS2870. We conclude that Nox1, but not Nox2 or Nox4, is responsible for the upregulation of T-type calcium channels elicited by chronic nitric oxide deficit. These data point to an important role for this isoform in increasing T-type channel function during oxidative stress.
Asunto(s)
Arteriolas/metabolismo , Canales de Calcio Tipo T/metabolismo , NADH NADPH Oxidorreductasas/metabolismo , Óxido Nítrico/deficiencia , Animales , Arteriolas/efectos de los fármacos , Arteriolas/fisiología , Bloqueadores de los Canales de Calcio/farmacología , Isoenzimas/antagonistas & inhibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Glicoproteínas de Membrana/antagonistas & inhibidores , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Músculo Esquelético/irrigación sanguínea , NADH NADPH Oxidorreductasas/antagonistas & inhibidores , NADH NADPH Oxidorreductasas/genética , NADPH Oxidasa 1 , NADPH Oxidasa 2 , NADPH Oxidasa 4 , NADPH Oxidasas/antagonistas & inhibidores , NADPH Oxidasas/genética , NADPH Oxidasas/metabolismo , Óxido Nítrico/metabolismo , Regulación hacia Arriba , VasoconstricciónRESUMEN
Cytotoxic lymphocytes (CTL) have been reported to show a range of motility patterns from rapid long-range tracking to complete arrest, but how and whether these kinematics affect their ability to kill target cells is not known. Many in vitro killing assays utilize cell lines and tumour-derived cells as targets, which may be of limited relevance to the kinetics of CTL-mediated killing of somatic cells. Here, live-cell microscopy is used to examine the interactions of CTL and primary murine skin cells presenting antigens. We developed a qualitative and quantitative killing assay using extended-duration fluorescence time-lapse microscopy coupled with large-volume objective software-based data analysis to obtain population data of cell-to-cell interactions, motility and apoptosis. In vivo and ex vivo activated antigen-specific cytotoxic lymphocytes were added to primary keratinocyte targets in culture with fluorometric detection of caspase-3 activation in targets as an objective determinant of apoptosis. We found that activated CTL achieved contact-dependent apoptosis of non-tumour targets after a period of prolonged attachment - on average 21 hours - which was determined by target cell type, amount of antigen, and activation status of CTL. Activation of CTL even without engagement of the T cell receptor was sufficient to mobilise cells significantly above baseline, while the addition of cognate antigen further enhanced their motility. Highly activated CTL showed markedly increased vector displacement, and velocity, and lead to increased antigen-specific target cell death. These data show that the inherent kinematics of CTL correlate directly with their ability to kill non-tumour cells presenting cognate antigen.
Asunto(s)
Comunicación Celular , Queratinocitos/fisiología , Activación de Linfocitos , Linfocitos T Citotóxicos/inmunología , Animales , Fenómenos Biomecánicos , Células Cultivadas , Queratinocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Linfocitos T Citotóxicos/fisiologíaRESUMEN
Genetically modified mice have played an important part in elucidating gene function in vivo. However, conclusions from transgenic studies may be compromised by complications arising from the site of transgene integration into the genome and, in inducible systems, the non-innocuous nature of inducer molecules. The aim of the present study was to use the vascular system to validate a technique based on the bacterial lac operon system, in which transgene expression can be repressed and de-repressed by an innocuous lactose analogue, IPTG. We have modified an endothelium specific promoter (TIE2) with synthetic LacO sequences and made transgenic mouse lines with this modified promoter driving expression of mutant forms of connexin40 and an independently translated reporter, EGFP. We show that tissue specificity of this modified promoter is retained in the vasculature of transgenic mice in spite of the presence of LacO sequences, and that transgene expression is uniform throughout the endothelium of a range of adult systemic and cerebral arteries and arterioles. Moreover, transgene expression can be consistently down-regulated by crossing the transgenic mice with mice expressing an inhibitor protein LacI(R), and in one transgenic line, transgene expression could be de-repressed rapidly by the innocuous inducer, IPTG. We conclude that the modified bacterial lac operon system can be used successfully to validate transgenic phenotypes through a simple breeding schedule with mice homozygous for the LacI(R) protein.
Asunto(s)
Células Endoteliales/metabolismo , Expresión Génica , Represoras Lac/fisiología , Animales , Conexinas/biosíntesis , Conexinas/genética , Endotelio Vascular/citología , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/genética , Homocigoto , Ratones Endogámicos C57BL , Ratones Transgénicos , Especificidad de Órganos , Regiones Promotoras Genéticas , Receptor TIE-2/genética , Activación Transcripcional , Transgenes , Proteína alfa-5 de Unión ComunicanteRESUMEN
Heparanase is a ß-d-endoglucuronidase that cleaves heparan sulphate, a key component of the ECM and basement membrane. The remodelling of the ECM by heparanase has been proposed to regulate both normal physiological and pathological processes, including wound healing, inflammation, tumour angiogenesis and cell migration. Heparanase is also known to exhibit non-enzymatic functions by regulating cell adhesion, cell signalling and differentiation. In this study, constitutive heparanase-deficient (Hpse(-/-) ) mice were generated on a C57BL/6 background using the Cre/loxP recombination system, with a complete lack of heparanase mRNA, protein and activity. Although heparanase has been implicated in embryogenesis and development, Hpse(-/-) mice are anatomically normal and fertile. Interestingly, consistent with the suggested function of heparanase in cell migration, the trafficking of dendritic cells from the skin to the draining lymph nodes was markedly reduced in Hpse(-/-) mice. Furthermore, the ability of Hpse(-/-) mice to generate an allergic inflammatory response in the airways, a process that requires dendritic cell migration, was also impaired. These findings establish an important role for heparanase in immunity and identify the enzyme as a potential target for regulation of an immune response.
Asunto(s)
Movimiento Celular/inmunología , Células Dendríticas/inmunología , Glucuronidasa/inmunología , Neumonía/inmunología , Animales , Western Blotting , Movimiento Celular/genética , Células Cultivadas , Células Dendríticas/metabolismo , Femenino , Citometría de Flujo , Expresión Génica/genética , Expresión Génica/inmunología , Glucuronidasa/deficiencia , Glucuronidasa/genética , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Fluorescente , Neumonía/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Piel/inmunología , Piel/metabolismoRESUMEN
Infectious clone technologies allow the rational design of live attenuated viral vaccines with the possibility of vaccine-driven coexpression of immunomodulatory molecules for additional vaccine safety and efficacy. The latter could lead to novel strategies for vaccine protection against infectious diseases where traditional approaches have failed. Here we show for the flavivirus Murray Valley encephalitis virus (MVEV) that incorporation of the internal ribosome entry site (IRES) of Encephalomyocarditis virus between the capsid and prM genes strongly attenuated virulence and that the resulting bicistronic virus was both genetically stable and potently immunogenic. Furthermore, the novel bicistronic genome organization facilitated the generation of a recombinant virus carrying an beta interferon (IFN-ß) gene. Given the importance of IFNs in limiting virus dissemination and in efficient induction of memory B and T cell antiviral immunity, we hypothesized that coexpression of the cytokine with the live vaccine might further increase virulence attenuation without loss of immunogenicity. We found that bicistronic mouse IFN-ß coexpressing MVEV yielded high virus and IFN titers in cultured cells that do not respond to the coexpressed IFN. However, in IFN response-sufficient cell cultures and mice, the virus produced a self-limiting infection. Nevertheless, the attenuated virus triggered robust innate and adaptive immune responses evidenced by the induced expression of Mx proteins (used as a sensitive biomarker for measuring the type I IFN response) and the generation of neutralizing antibodies, respectively. IMPORTANCE The family Flaviviridae includes a number of important human pathogens, such as Dengue virus, Yellow fever virus, Japanese encephalitis virus, West Nile virus, and Hepatitis C virus. Flaviviruses infect large numbers of individuals on all continents. For example, as many as 100 million people are infected annually with Dengue virus, and 150 million people suffer a chronic infection with Hepatitis C virus. However, protective vaccines against dengue and hepatitis C are still missing, and improved vaccines against other flaviviral diseases are needed. The present study investigated the effects of a redesigned flaviviral genome and the coexpression of an antiviral protein (interferon) on virus replication, pathogenicity, and immunogenicity. Our findings may aid in the rational design of a new class of well-tolerated and safe vaccines.
Asunto(s)
Clonación Molecular/métodos , Virus de la Encefalitis del Valle Murray/genética , Virus de la Encefalomiocarditis/genética , Inmunidad Celular/inmunología , Ribosomas/genética , Vacunas Sintéticas/genética , Vacunas Virales/biosíntesis , Animales , Anticuerpos Neutralizantes/inmunología , Chlorocebus aethiops , Cartilla de ADN/genética , Virus de la Encefalitis del Valle Murray/patogenicidad , Ingeniería Genética/métodos , Inmunohistoquímica , Interferón beta/metabolismo , Estimación de Kaplan-Meier , Ratones , Ratones Endogámicos C57BL , Proteínas de Resistencia a Mixovirus/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Vacunas Sintéticas/virología , Células Vero , Vacunas Virales/genéticaRESUMEN
UNLABELLED: Non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis are components of the metabolic syndrome. Serum leptin levels are elevated in obesity, but the role of leptin in the pathophysiology of the liver involvement is still unclear. To identify the effects and mechanisms by which leptin influences the pathogenesis of NAFLD, we performed epididymal white adipose tissue (eWAT) transplantation from congenic wild-type mice into the subcutaneous dorsal area of Lep(ob/ob) recipient mice and compared the results with those of the Lep(ob/ob) sham-operated mice. The mice were followed for 102-216 days. During killing, the transplanted mice had significantly lost body weight and exhibited significantly higher leptin levels, improved glucose tolerance, and lower liver injury scores than the sham-operated mice. Liver microarray analysis showed that novel pathways related to GA-binding protein (GABP) transcription factor targets, pheromone binding, and olfactory signaling were differentially expressed in the transplanted mice. Our data also replicate pathways known to be involved in NAFLD, such as those involved in the regulation of microRNAs, lipid, glucose, and glutathione metabolism, peroxisome proliferator-activated receptor signaling, cellular regulation, carboxylic acid processes, iron, heme, and tetrapyrrole binding, immunity and inflammation, insulin signaling, cytochrome P450 function, and cancer. CONCLUSION: wild-type eWAT transplantation into Lep(ob/ob) mice led to improvements in metabolism, body weight, and liver injury, possibly attributed to the production of leptin by the transplanted eWAT. These improvements were accompanied by the differential expression of novel pathways. The causal relationship between GABP downregulation and NAFLD improvement remains to be determined.
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Hígado Graso/genética , Hígado Graso/metabolismo , Transducción de Señal , Tejido Adiposo Blanco/metabolismo , Tejido Adiposo Blanco/trasplante , Animales , Ácidos Grasos/metabolismo , Hígado Graso/inmunología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Prueba de Tolerancia a la Glucosa , Hormonas/sangre , Mediadores de Inflamación/inmunología , Mediadores de Inflamación/metabolismo , Insulina/metabolismo , Hígado/metabolismo , Hígado/patología , Masculino , Ratones , Ratones Congénicos , Ratones Transgénicos , Enfermedad del Hígado Graso no Alcohólico , Esteroides/metabolismoRESUMEN
OBJECTIVE: To determine whether impairment of endothelial connexin40 (Cx40), an effect that can occur in hypertension and aging, contributes to the arterial dysfunction and stiffening in these conditions. APPROACH AND RESULTS: A new transgenic mouse strain, expressing a mutant Cx40, (Cx40T202S), specifically in the vascular endothelium, has been developed and characterized. This mutation produces nonfunctional hemichannels, whereas gap junctions containing the mutant are electrically, but not chemically, patent. Mesenteric resistance arteries from Cx40T202S mice showed increased sensitivity of the myogenic response to intraluminal pressure in vitro, compared with wild-type mice, whereas transgenic mice overexpressing native Cx40 (Cx40Tg) showed reduced sensitivity. In control and Cx40Tg mice, the sensitivity to pressure of myogenic constriction was modulated by both NO and endothelium-derived hyperpolarization; however, the endothelium-derived hyperpolarization component was absent in Cx40T202S arteries. Analysis of passive mechanical properties revealed that arterial stiffness was enhanced in vessels from Cx40T202S mice, but not in wild-type or Cx40Tg mice. CONCLUSIONS: Introduction of a mutant form of Cx40 in the endogenous endothelial Cx40 population prevents endothelium-derived hyperpolarization activation during myogenic constriction, enhancing sensitivity to intraluminal pressure and increasing arterial stiffness. We conclude that genetic polymorphisms in endothelial Cx40 can contribute to the pathogenesis of arterial disease.
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Conexinas/fisiología , Endotelio Vascular/metabolismo , Polimorfismo Genético , Rigidez Vascular , Animales , Presión Sanguínea , Peso Corporal , Conexinas/análisis , Conexinas/genética , Conductividad Eléctrica , Uniones Comunicantes/fisiología , Frecuencia Cardíaca , Masculino , Arterias Mesentéricas/fisiología , Ratones , Ratones Transgénicos , Proteína alfa-5 de Unión Comunicante , Proteína alfa-4 de Unión ComunicanteRESUMEN
Regulation of blood flow in microcirculatory networks depends on spread of local vasodilatation to encompass upstream arteries; a process mediated by endothelial conduction of hyperpolarization. Given that endothelial coupling is reduced in hypertension, we used hypertensive Cx40ko mice, in which endothelial coupling is attenuated, to investigate the contribution of the renin-angiotensin system and reduced endothelial cell coupling to conducted vasodilatation of cremaster arterioles in vivo. When the endothelium was disrupted by light dye treatment, conducted vasodilatation, following ionophoresis of acetylcholine, was abolished beyond the site of endothelial damage. In the absence of Cx40, sparse immunohistochemical staining was found for Cx37 in the endothelium, and endothelial, myoendothelial and smooth muscle gap junctions were identified by electron microscopy. Hyperpolarization decayed more rapidly in arterioles from Cx40ko than wild-type mice. This was accompanied by a shift in the threshold potential defining the linear relationship between voltage and diameter, increased T-type calcium channel expression and increased contribution of T-type (3 µmol l(-1) NNC 55-0396), relative to L-type (1 µmol l(-1) nifedipine), channels to vascular tone. The change in electromechanical coupling was reversed by inhibition of the renin-angiotensin system (candesartan, 1.0 mg kg(-1) day(-1) for 2 weeks) or by acute treatment with the superoxide scavenger tempol (1 mmol l(-1)). Candesartan and tempol treatments also significantly improved conducted vasodilatation. We conclude that conducted vasodilatation in Cx40ko mice requires the endothelium, and attenuation results from both a reduction in endothelial coupling and an angiotensin II-induced increase in oxidative stress. We suggest that during cardiovascular disease, the ability of microvascular networks to maintain tissue integrity may be compromised due to oxidative stress-induced changes in electromechanical coupling.
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Endotelio Vascular/fisiopatología , Hipertensión/fisiopatología , Estrés Oxidativo , Angiotensina II/fisiología , Bloqueadores del Receptor Tipo 1 de Angiotensina II/farmacología , Animales , Arteriolas/fisiología , Bencimidazoles/farmacología , Compuestos de Bifenilo , Canales de Calcio Tipo L/fisiología , Canales de Calcio Tipo T/fisiología , Conexinas/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microcirculación , Modelos Cardiovasculares , Renina/sangre , Tetrazoles/farmacología , Vasodilatación , Proteína alfa-5 de Unión ComunicanteRESUMEN
BACKGROUND: Immunological mechanisms involved in the survival and development of human filarial species in the vertebrate host are poorly known due to the lack of suitable experimental models. In order to understand the role of cytokines in the survival and development of filarial larvae in the vertebrate host, we infected different strains of BALB/c mice deficient in a number of cytokine or cytokine receptor genes with Loa loa. The survival and development of larvae were monitored. METHODS: BALB/c mice genetically deficient in IL-4R, IFN-γ, IFN-γ/IL-5, IL-5, and IL-4R/IL-5 cytokine or cytokine receptor genes were infected with a human strain of L. loa and necropsies were performed at different time intervals up to 70 days post infection to monitor the survival and development of L. loa larvae. The larvae were teased out of the skin, muscles, peritoneal and pleural cavities, heart and lung tissues. The length and width of the recovered larvae were measured to assess their growth. RESULTS: In mice deficient for IL-4R, IFN-γ, IFN-γ/IL-5, IL-5 and IL-4R/IL-5, the larvae survived up to 5, 20, 40, 50 and 70 days respectively. Worms recovered 70 days post infection in IL-4R/IL-5 DKO mice were young adults and measured 10.12 mm in length and 0.1 mm in width. Overall, 47% of larvae were recovered from subcutaneous tissues, 40% from muscles, 6% from the peritoneal cavity and 4% from the pleural cavity, lungs and heart. CONCLUSION: L. loa exhibits a differential survival and development in different strains of cytokine or cytokine receptor gene knockout mice with IL-4R and IL-5 playing critical roles in the host resistance to L. loa infection. The knock out BALB/c mouse therefore represents a useful tool to explore the key effectors of adaptive immunity involved in the killing of the L. loa parasite in a mammal host.
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Interleucina-5/genética , Loa/crecimiento & desarrollo , Loiasis/parasitología , Receptores de Interleucina-4/genética , Inmunidad Adaptativa , Animales , Dípteros/parasitología , Técnicas de Inactivación de Genes , Humanos , Interferón gamma/genética , Interleucina-4/genética , Larva , Loa/genética , Loa/fisiología , Loiasis/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones NoqueadosRESUMEN
Malignant hyperthermia susceptibility (MHS) is primarily conferred by mutations within ryanodine receptor type 1 (RYR1). Here we address how the MHS mutation T4826I within the S4-S5 linker influences excitation-contraction coupling and resting myoplasmic Ca(2+) concentration ([Ca(2+)](rest)) in flexor digitorum brevis (FDB) and vastus lateralis prepared from heterozygous (Het) and homozygous (Hom) T4826I-RYR1 knock-in mice (Yuen, B. T., Boncompagni, S., Feng, W., Yang, T., Lopez, J. R., Matthaei, K. I., Goth, S. R., Protasi, F., Franzini-Armstrong, C., Allen, P. D., and Pessah, I. N. (2011) FASEB J. doi:22131268). FDB responses to electrical stimuli and acute halothane (0.1%, v/v) exposure showed a rank order of Hom â« Het â« WT. Release of Ca(2+) from the sarcoplasmic reticulum and Ca(2+) entry contributed to halothane-triggered increases in [Ca(2+)](rest) in Hom FDBs and elicited pronounced Ca(2+) oscillations in â¼30% of FDBs tested. Genotype contributed significantly elevated [Ca(2+)](rest) (Hom > Het > WT) measured in vivo using ion-selective microelectrodes. Het and Hom oxygen consumption rates measured in intact myotubes using the Seahorse Bioscience (Billerica, MA) flux analyzer and mitochondrial content measured with MitoTracker were lower than WT, whereas total cellular calpain activity was higher than WT. Muscle membranes did not differ in RYR1 expression nor in Ser(2844) phosphorylation among the genotypes. Single channel analysis showed highly divergent gating behavior with Hom and WT favoring open and closed states, respectively, whereas Het exhibited heterogeneous gating behaviors. [(3)H]Ryanodine binding analysis revealed a gene dose influence on binding density and regulation by Ca(2+), Mg(2+), and temperature. Pronounced abnormalities inherent in T4826I-RYR1 channels confer MHS and promote basal disturbances of excitation-contraction coupling, [Ca(2+)](rest), and oxygen consumption rates. Considering that both Het and Hom T4826I-RYR1 mice are viable, the remarkable isolated single channel dysfunction mediated through this mutation in S4-S5 cytoplasmic linker must be highly regulated in vivo.
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Dosificación de Gen , Heterocigoto , Homocigoto , Hipertermia Maligna/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Mutación Missense , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Sustitución de Aminoácidos , Animales , Calcio/metabolismo , Hipertermia Maligna/genética , Ratones , Ratones Transgénicos , Estructura Terciaria de Proteína , Canal Liberador de Calcio Receptor de Rianodina/genética , Retículo Sarcoplasmático/genética , Retículo Sarcoplasmático/metabolismoRESUMEN
Mutation T4825I in the type 1 ryanodine receptor (RYR1(T4825I/+)) confers human malignant hyperthermia susceptibility (MHS). We report a knock-in mouse line that expresses the isogenetic mutation T4826I. Heterozygous RYR1(T4826I/+) (Het) or homozygous RYR1(T4826I/T4826I) (Hom) mice are fully viable under typical rearing conditions but exhibit genotype- and sex-dependent susceptibility to environmental conditions that trigger MH. Hom mice maintain higher core temperatures than WT in the home cage, have chronically elevated myoplasmic[Ca(2+)](rest), and present muscle damage in soleus with a strong sex bias. Mice subjected to heat stress in an enclosed 37°C chamber fail to trigger MH regardless of genotype, whereas heat stress at 41°C invariably triggers fulminant MH in Hom, but not Het, mice within 20 min. WT and Het female mice fail to maintain euthermic body temperature when placed atop a bed whose surface is 37°C during halothane anesthesia (1.75%) and have no hyperthermic response, whereas 100% Hom mice of either sex and 17% of the Het males develop fulminant MH. WT mice placed on a 41°C bed maintain body temperature while being administered halothane, and 40% of the Het females and 100% of the Het males develop fulminant MH within 40 min. Myopathic alterations in soleus were apparent by 12 mo, including abnormally distributed and enlarged mitochondria, deeply infolded sarcolemma, and frequent Z-line streaming regions, which were more severe in males. These data demonstrate that an MHS mutation within the S4-S5 cytoplasmic linker of RYR1 confers genotype- and sex-dependent susceptibility to pharmacological and environmental stressors that trigger fulminant MH and promote myopathy.
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Predisposición Genética a la Enfermedad/genética , Hipertermia Maligna/genética , Músculo Esquelético/metabolismo , Mutación , Canal Liberador de Calcio Receptor de Rianodina/genética , Sustitución de Aminoácidos , Anestésicos por Inhalación/administración & dosificación , Animales , Temperatura Corporal/efectos de los fármacos , Temperatura Corporal/genética , Temperatura Corporal/fisiología , Femenino , Expresión Génica , Genotipo , Halotano/administración & dosificación , Calor , Humanos , Masculino , Potenciales de la Membrana , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Electrónica , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/fisiología , Músculo Esquelético/patología , Canal Liberador de Calcio Receptor de Rianodina/fisiología , Sarcolema/metabolismo , Sarcolema/ultraestructura , Factores SexualesRESUMEN
Glutathione transferase kappa (GSTK1-1) is a highly conserved, mitochondrial enzyme potentially involved in redox reactions. GSTK1-1-deficient mice were generated to further study the enzyme's biological role. Reduced and total glutathione levels in liver and kidney were unchanged by GSTK1-1 deficiency and NADPH quinone oxidoreductase 1 expression was not elevated indicating that there is no general underlying oxidative stress in Gstk1(-/-) mice. Electron microscopy of liver and kidney showed no changes in mitochondrial morphology with GSTK1-1 deficiency. The death of a number of Gstk1(-/-) males with urinary tract problems prompted close examination of the kidneys. Electron microscopy revealed glomerular basement membrane changes at 3 months, accompanied by detectable microalbuminuria in male mice (albumin:creatinine ratio of 2.66±0.83 vs 1.13±0.20 mg/mmol for Gstk1(-/-) and wild-type (WT), respectively, P=0.001). This was followed by significant foot process effacement (40-55% vs 10% for Gstk1(-/-) and WT, respectively) at 6 months of age in all Gstk1(-/-) mice examined. Kidney tubules were ultrastructurally normal. Compared with human disease, the Gstk1(-/-) kidneys show changes seen in glomerulopathies causing nephrotic syndrome. Gstk1(-/-) mice may offer insights into the early development of glomerular nephropathies.
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Glomerulonefritis/etiología , Glomerulonefritis/patología , Glutatión Transferasa/deficiencia , Albuminuria/etiología , Animales , Análisis Químico de la Sangre , Western Blotting , Ensayo de Inmunoadsorción Enzimática , Inmunohistoquímica , Riñón/ultraestructura , Hígado/ultraestructura , Masculino , Ratones , Ratones Noqueados , Microscopía Electrónica , Estrés Oxidativo/fisiología , UrinálisisRESUMEN
The gelsolin related actin binding protein, Flii, is able to regulate wound healing; mice with decreased Flii expression show improved wound healing whereas mice with elevated Flii expression exhibit impaired wound healing. In both mice and humans Flii expression increases with age and amelioration of FLII activity represents a possible therapeutic strategy for improved wound healing in humans. Despite analysis of Flii function in a variety of organisms we know little of the molecular mechanisms underlying Flii action. Two new murine alleles of Flii have been produced to drive constitutive or tissue-specific expression of Flii. Each strain is able to rescue the embryonic lethality associated with a Flii null allele and to impair wound healing. These strains provide valuable resources for ongoing investigation of Flii function in a variety of biological processes.
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Proteínas del Citoesqueleto/genética , Perfilación de la Expresión Génica , Piel/metabolismo , Cicatrización de Heridas/genética , Animales , Western Blotting , Encéfalo/metabolismo , Proteínas Portadoras , Proteínas del Citoesqueleto/metabolismo , Femenino , Genotipo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Ratones Transgénicos , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Músculos/metabolismo , Miocardio/metabolismo , Proteínas/genética , Proteínas/metabolismo , ARN no Traducido , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Piel/fisiopatología , Especificidad de la Especie , Bazo/metabolismo , Factores de Tiempo , Transactivadores , Cicatrización de Heridas/fisiologíaRESUMEN
Human respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infection in infants. In human infants, plasmacytoid dendritic cells (pDC) are recruited to the nasal compartment during infection and initiate host defense through the secretion of type I IFN, IL-12, and IL-6. However, RSV-infected pDC are refractory to TLR7-mediated activation. In this study, we used the rodent-specific pathogen, pneumonia virus of mice (PVM), to determine the contribution of pDC and TLR7 signaling to the development of the innate inflammatory and early adaptive immune response. In wild-type, but not TLR7- or MyD88-deficient mice, PVM inoculation led to a marked infiltration of pDC and increased expression of type I, II, and III IFNs. The delayed induction of IFNs in the absence of TLR7 or MyD88 was associated with a diminished innate inflammatory response and augmented virus recovery from lung tissue. In the absence of TLR7, PVM-specific CD8(+) T cell cytokine production was abrogated. The adoptive transfer of TLR7-sufficient, but not TLR7-deficient pDC to TLR7 gene-deleted mice recapitulated the antiviral responses observed in wild-type mice and promoted virus clearance. In summary, TLR7-mediated signaling by pDC is required for appropriate innate responses to acute pneumovirus infection. It is conceivable that as-yet-unidentified defects in the TLR7 signaling pathway may be associated with elevated levels of RSV-associated morbidity and mortality among otherwise healthy human infants.
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Células Dendríticas/inmunología , Glicoproteínas de Membrana/inmunología , Glicoproteínas de Membrana/metabolismo , Virus de la Neumonía Murina/inmunología , Factor 88 de Diferenciación Mieloide/metabolismo , Infecciones por Pneumovirus/inmunología , Receptor Toll-Like 7/inmunología , Receptor Toll-Like 7/metabolismo , Inmunidad Adaptativa , Traslado Adoptivo , Animales , Interferones/genética , Interferones/inmunología , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Factor 88 de Diferenciación Mieloide/genética , Transducción de Señal , Receptor Toll-Like 7/genéticaAsunto(s)
Adenosina Desaminasa/genética , Adenosina Desaminasa/metabolismo , Desarrollo Embrionario/genética , Mutación/genética , Virus SSPE/genética , Panencefalitis Esclerosante Subaguda/genética , Replicación Viral/genética , Animales , Antígenos CD/metabolismo , Línea Celular , Cartilla de ADN/genética , Técnica del Anticuerpo Fluorescente , Técnicas de Inactivación de Genes , Proteínas Fluorescentes Verdes , Ratones , Ratones Endogámicos C57BL , Isoformas de Proteínas/genética , Proteínas de Unión al ARN , Receptores de Superficie Celular/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Miembro 1 de la Familia de Moléculas Señalizadoras de la Activación LinfocitariaRESUMEN
Interleukin-13 (IL-13) has been linked to the pathogenesis of inflammatory diseases of the gastrointestinal tract. It is postulated that IL-13 drives inflammatory lesions through the modulation of both hematopoietic and nonhematopoietic cell function in the intestine. To delineate the relevant contribution of elevated levels of intestinal IL-13 to intestinal structure and function, we generated an intestinal IL-13 transgenic mouse (iIL-13Tg). We show that constitutive overexpression of IL-13 in the small bowel induces modification of intestinal epithelial architecture (villus blunting, goblet cell hyperplasia, and increased epithelial proliferation) and epithelial function (altered basolateral â apical Cl(-) ion conductance). Pharmacological analyses in vitro and in vivo determined that elevated Cl(-) conductance is mediated by altered cystic fibrosis transmembrane conductance regulator expression and activity. Generation of iIL-13Tg/Il13rα1(-/-), iIL-13Tg/Il13rα2(-/-), and iIL-13Tg/Stat6(-/-) mice revealed that IL-13-mediated dysregulation of epithelial architecture and Cl(-) conductance is dependent on IL-13Rα1 and STAT-6. These observations demonstrate a central role for the IL-13/IL-13Rα1 pathway in the regulation of intestinal epithelial cell Cl(-) secretion via up-regulation of cystic fibrosis transmembrane conductance regulator, suggesting an important role for this pathway in secretory diarrhea.
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Cloruros/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Subunidad alfa1 del Receptor de Interleucina-13/metabolismo , Interleucina-13/metabolismo , Enfermedades Intestinales/metabolismo , Mucosa Intestinal/metabolismo , Animales , Células CACO-2 , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Quistes/genética , Quistes/metabolismo , Quistes/patología , Diarrea/genética , Diarrea/metabolismo , Diarrea/patología , Fibrosis , Humanos , Interleucina-13/genética , Subunidad alfa1 del Receptor de Interleucina-13/genética , Enfermedades Intestinales/genética , Enfermedades Intestinales/patología , Mucosa Intestinal/patología , Transporte Iónico/genética , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Factor de Transcripción STAT6/genética , Factor de Transcripción STAT6/metabolismoRESUMEN
Using Toll-like receptor (TLR) and MyD88 gene knock-out (GKO) mice the effect of TLRs and MyD88 on virus replication, interferon (IFN)-ß production, natural killer (NK) cell and CD8T cell responses were assessed following ectromelia virus (ECTV) and recombinant vaccinia virus (rVV) infection. The capacity for rVVs encoding cytokines to restore immune function in MyD88(-/-) mice was clearly demonstrated. Results showed that TLR2(-/-), TLR4(-/-)and TLR7(-/-) mice survived ECTV infection whereas MyD88(-/-) and TLR9(-/-)mice, in contrast, were highly susceptible. Next, following infection with rVV, MyD88(-/-) mice elicited reduced serum IFN-ß, NK cell and CD8T cell responses compared with wild-type mice, whereas TLR9(-/-) mice showed elevated CD8T cell responses. When MyD88(-/-)mice were infected with rVV co-expressing IFN-ß these mice were able to restore IFN-ß levels and CD8T cell responses but not NK cell activation. Interestingly, even though rVV co-expressing interleukin (IL)-2 enhanced NK cell activation in MyD88(-/-) mice, this was not associated with an antiviral effect, as observed in normal mice. Surprisingly, co-infection with rVV IL-2/rVV IL-12, but not rVV IL-2/rVV IFN-ß, restored the attenuated phenotype of rVV IL-2 in MyD88(-/-) mice indicating that the IL-2/IL-12 combination promotes antiviral responses. Our results clearly show that the CD8T cell defect observed in MyD88(-/-) mice to vaccinia virus infection can be restored by rVV-encoding IFN-ß demonstrating the critical role of this cytokine in T cell mediated immunity and illustrates that the model can provide an effective platform for the elucidation of cytokine immunobiology.