RESUMEN
BACKGROUND: Otosclerosis is a common cause of adult-onset progressive hearing loss, affecting 0.3%-0.4% of the population. It results from dysregulation of bone homeostasis in the otic capsule, most commonly leading to fixation of the stapes bone, impairing sound conduction through the middle ear. Otosclerosis has a well-known genetic predisposition including familial cases with apparent autosomal dominant mode of inheritance. While linkage analysis and genome-wide association studies suggested an association with several genomic loci and with genes encoding structural proteins involved in bone formation or metabolism, the molecular genetic pathophysiology of human otosclerosis is yet mostly unknown. METHODS: Whole-exome sequencing, linkage analysis, generation of CRISPR mutant mice, hearing tests and micro-CT. RESULTS: Through genetic studies of kindred with seven individuals affected by apparent autosomal dominant otosclerosis, we identified a disease-causing variant in SMARCA4, encoding a key component of the PBAF chromatin remodelling complex. We generated CRISPR-Cas9 transgenic mice carrying the human mutation in the mouse SMARCA4 orthologue. Mutant Smarca4+/E1548K mice exhibited marked hearing impairment demonstrated through acoustic startle response and auditory brainstem response tests. Isolated ossicles of the auditory bullae of mutant mice exhibited a highly irregular structure of the incus bone, and their in situ micro-CT studies demonstrated the anomalous structure of the incus bone, causing disruption in the ossicular chain. CONCLUSION: We demonstrate that otosclerosis can be caused by a variant in SMARCA4, with a similar phenotype of hearing impairment and abnormal bone formation in the auditory bullae in transgenic mice carrying the human mutation in the mouse SMARCA4 orthologue.
Asunto(s)
Pérdida Auditiva , Otosclerosis , Adulto , Humanos , Ratones , Animales , Otosclerosis/genética , Otosclerosis/cirugía , Vesícula/complicaciones , Estudio de Asociación del Genoma Completo , Reflejo de Sobresalto , Fenotipo , Ratones Transgénicos , Mutación , ADN Helicasas/genética , Proteínas Nucleares/genética , Factores de Transcripción/genéticaRESUMEN
The disadvantage of current biodegradable metals such as Mg and Fe is the release of hydrogen gas in vivo that can cause gas embolism and the production of voluminous iron oxide that can cause inflammation, respectively. Such considerations have turned focus towards Zn as an alternative. This is based on the fact that Zn plays a crucial role in many physiological processes, as well as potentially being biocompatible and capable of with biodegradation. As such, the purpose of the present study was to evaluate the in vivo performance of pure Zinc and Zn-2%Fe implants. The use of iron as an alloying element was aimed at accelerating the corrosion rate of pure zinc by a micro-galvanic effect so as to maintain the post-implantation biodegradation characteristics of the implant. In vivo assessment was carried out using cylindrical disks implanted in the back midline of 16 male Wistar rats for up to 24 weeks. Post-implantation evaluation included monitoring the well-being of rats, weekly examination of hematological parameters: serum Zn levels, red and white blood cell counts and hemoglobin levels, X-ray radiography, histological analysis and corrosion rate assessment. The results obtained in terms of well-being, hematological tests and histological analysis of the rats indicate that the in vivo behavior of pure Zn and Zn-2%Fe implants was adequate and in line with the results obtained by the control group containing inert Ti-6Al-4V alloy implants. The corrosion rate of Zn-2%Fe alloy in in vivo conditions was relatively increased compared to pure Zn due to micro-galvanic corrosion.
Asunto(s)
Implantes Absorbibles , Hierro , Zinc , Implantes Absorbibles/efectos adversos , Aleaciones/química , Animales , Materiales Biocompatibles/efectos adversos , Materiales Biocompatibles/química , Corrosión , Hierro/química , Masculino , Ensayo de Materiales , Ratas , Ratas Wistar , Titanio/química , Zinc/sangre , Zinc/químicaRESUMEN
The efforts to develop structural materials for biodegradable metal implants have lately shifted their focus from Magnesium and Iron base alloys towards Zinc. This was mainly due to the accelerated corrosion rate of Mg that is accompanied by hydrogen gas evolution, formation of voluminous iron oxide products with reduced degradation rate in the case of Iron implants and the crucial role of Zn in many physiological processes. However the mechanical properties and degradation capabilities of pure zinc in physiological environment are limited and do not comply with the requirements of biodegradable implants. The present study aims at evaluating the effect of 4%Fe on the in-vitro and in-vivo behavior of pure Zinc. This was carried out in order to address the inherent disadvantages of pure zinc in terms of mechanical properties and biodegradability. The results obtained clearly indicate that the biocompatibility and mechanical properties of the new material system was in accord with the prospective requirements of biodegradable implants. However the corrosion degradation of the new alloy in in-vivo conditions was quite similar to that of pure zinc in spite of the significant micro-galvanic effect created by Delta phase Zn11Fe.
Asunto(s)
Implantes Absorbibles , Aleaciones , Hierro , Zinc , Animales , Materiales Biocompatibles , Corrosión , Masculino , Ratas WistarRESUMEN
MAPKs and inhibitory-kappaB kinase (IKK) were suggested to link various conditions thought to develop in adipose tissue in obesity (oxidative, endoplasmic reticulum stress, inflammation) with insulin resistance. Yet whether in obesity these kinases are affected in a fat-depot-differential manner is unknown. We assessed the expression and phosphorylation of these kinases in paired omental and abdominal-sc fat biopsies from 48 severely obese women (body mass index > 32 kg/m(2)). Protein and mRNAs of p38MAPK, ERK, c-Jun kinase-1, and IKKbeta were increased 1.5-2.5-fold in omental vs. sc fat. The phosphorylated (activated) forms of these kinases were also increased to similar magnitudes as the total expression. However, phosphorylation of insulin receptor substrate-1 on Ser312 (equivalent of murine Ser307) was not increased in omental, compared with sc, fat. Consistently, fat tissue fragments stimulated with insulin demonstrated that tyrosine phosphorylation and signal transduction to Akt/protein kinase B in omental fat was not inferior to that observable in sc fat. Comparison with lean women (body mass index 23.2 +/- 2.9 kg/m(2)) revealed similar ERK2 and IKKbeta expression and phosphorylation in both fat depots. However, as compared with lean controls, obese women exhibited 480 and 270% higher amount of the phosphorylated forms of p38MAPK and c-Jun kinase, respectively, in omental, but not sc, fat, and this expression level correlated with clinical parameters of glycemia and insulin sensitivity. Increased expression of stress-activated kinases and IKK and their phosphorylated forms in omental fat occurs in obesity, potentially contributing to differential roles of omental and sc fat in the pathophysiology of obesity.
Asunto(s)
Quinasa I-kappa B/metabolismo , Insulina/farmacología , Grasa Intraabdominal/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Obesidad/metabolismo , Epiplón/metabolismo , Grasa Subcutánea/metabolismo , Adulto , Estudios de Casos y Controles , Femenino , Humanos , Técnicas In Vitro , Proteínas Sustrato del Receptor de Insulina , Grasa Intraabdominal/enzimología , Grasa Intraabdominal/patología , Persona de Mediana Edad , Proteína Quinasa 8 Activada por Mitógenos/metabolismo , Obesidad/enzimología , Obesidad/patología , Epiplón/enzimología , Epiplón/patología , Proteína Oncogénica v-akt/metabolismo , Fosfoproteínas/metabolismo , Fosforilación , Transducción de Señal/efectos de los fármacos , Grasa Subcutánea/enzimología , Grasa Subcutánea/patología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
CONTEXT: Macrophage infiltration into adipose tissue has been demonstrated to accompany obesity, with a potential preferential infiltration into intraabdominal vs. sc fat. OBJECTIVE: Our objective was to determine whether this occurs across different populations with a range of body mass indexes and to assess the relationship with regional adiposity and comorbidity of obesity. SETTING AND PATIENTS: In two independent cohorts, we used paired omental (OM) and sc fat biopsies from lean controls or predominantly sc or intraabdominally obese persons with minimal comorbidity (n = 60, cohort 1), or from severely obese women with a significant rate of comorbidity (n = 29, cohort 2). RESULTS: Elevated macrophage infiltration into OM vs. sc fat was observable in lean subjects and exaggerated by obesity, particularly if predominantly intraabdominal. This was paralleled by increased monocyte chemoattractant protein-1 (MCP1) and colony-stimulating factor-1 (CSF1) mRNA levels. Level of CSF1 and MCP1 mRNA correlated with the number of OM macrophages (r = 0.521, P < 0.0001 and r = 0.258, P < 0.051, respectively). In severely obese women (mean body mass index = 43.0 +/- 1.1 kg/m(2)), higher protein expression of both MCP1 and CSF1 was detected in OM vs. sc fat. Number of OM macrophages, but not of sc macrophages, correlated with waist circumference (r = 0.636, P = 0.001 vs. r = 0.170, P = 0.427) and with the number of metabolic syndrome parameters (r = 0.385, P = 0.065 vs. r = -0.158, P = 0.472, respectively). Preferential macrophage infiltration into OM fat was mainly observed in a subgroup in whom obesity was associated with impaired glucose homeostasis. CONCLUSIONS: Preferential macrophage infiltration into OM fat is a general phenomenon exaggerated by central obesity, potentially linking central adiposity with increased risk of diabetes and coronary heart disease.
Asunto(s)
Macrófagos/citología , Obesidad/inmunología , Obesidad/mortalidad , Epiplón/citología , Grasa Subcutánea Abdominal/citología , Adulto , Anciano , Biopsia , Índice de Masa Corporal , Movimiento Celular/inmunología , Quimiocina CCL2/genética , Estudios de Cohortes , Comorbilidad , Femenino , Humanos , Factor Estimulante de Colonias de Macrófagos/genética , Macrófagos/inmunología , Masculino , Persona de Mediana Edad , Obesidad/patología , Epiplón/inmunología , ARN Mensajero/metabolismo , Grasa Subcutánea Abdominal/inmunologíaRESUMEN
BACKGROUND: Previous studies have indicated that beta adrenergic receptor stimulation has no effect on the cognitive functioning of the prefrontal cortex (PFC). Blockade of beta-1 and beta-2 receptors in the PFC with the mixed beta-1/beta-2 antagonist, propanolol, had no effect on spatial working memory performance. However, more selective blockade of beta-1 or beta-2 receptors might show efficacy if the two receptors have opposite effects on PFC function. The current study examined the effects of the selective beta-1 antagonist, betaxolol, on working memory in rats and monkeys. METHODS: In rats, betaxolol (.0011-1.11 microg/.5 microL) was infused into the PFC 5 min before delayed alternation testing. Monkeys were systemically injected with betaxolol (.0000011-.11 mg/kg) 2 hours before delayed response testing. RESULTS: Betaxolol produced a dose-related improvement in working memory performance following either direct PFC infusion in rats, or systemic administration in monkeys. However, some aged monkeys developed serious pancreatic problems over the course of this study. CONCLUSIONS: These findings suggest that endogenous activation of the beta-1 adrenergic receptor impairs PFC cognitive function. These results may have therapeutic relevance to post-traumatic stress disorder or other disorders with excessive noradrenergic activity and PFC dysfunction. Pancreatic side effects in aged subjects taking betaxolol warrants further investigation.
Asunto(s)
Antagonistas de Receptores Adrenérgicos beta 1 , Antagonistas Adrenérgicos beta/farmacología , Betaxolol/farmacología , Memoria a Corto Plazo/efectos de los fármacos , Desempeño Psicomotor/efectos de los fármacos , Antagonistas Adrenérgicos beta/toxicidad , Animales , Atención/efectos de los fármacos , Betaxolol/toxicidad , Cognición/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Hipnóticos y Sedantes , Macaca mulatta , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Microinyecciones , Enfermedades Pancreáticas/inducido químicamente , Corteza Prefrontal , Ratas , Técnicas EstereotáxicasRESUMEN
A juvenile (1 year old ) female rhesus macaque (Macaca mulatta) developed a chronic active skin condition characterized by pruritus, erythema, alopecia, scaling, exfoliation, and lichenification. Lesions were limited to the ventrum, specifically rostral mandible and neck, axilla and inguinal regions, distal extremities, and interdigital regions. Differential diagnoses included infection, dietary deficiency, metabolic abnormality, endocrinopathy, and immunological injury. Diagnostic tests included complete hemogram, serum chemistry, skin scrapes for ectoparasite detection, hair plucks for dermatophyte culture, and a serum-based hypersensitivity panel. All results were within normal limits. Dermal biopsies revealed lesions consistent with active allergic dermatitis, and a diagnosis of atopic dermatitis was made. Oral cyclosporine (5 mg/kg daily) rapidly eliminated clinical evidence of dermatitis. Histologically, lesions resolved after 12 months of treatment. Atopic dermatitis is an inflammatory skin condition for which there are neither pathognomonic clinical or diagnostic features nor a single successful therapy. Basic criteria such as pruritus, lichenification, a chronic course, and history of allergies strongly support the diagnosis. One successful therapeutic agent is a macrolide calcineurin inhibitor, cyclosporine. It represents a safer class of immunomodulatory drugs than corticosteroids and provides targeted alteration of lymphocyte function. To our knowledge this case represents the first reported successful treatment of atopic dermatitis in a nonhuman primate utilizing cyclosporine.
Asunto(s)
Ciclosporina/uso terapéutico , Dermatitis Atópica/tratamiento farmacológico , Fármacos Dermatológicos/uso terapéutico , Inmunosupresores/uso terapéutico , Macaca mulatta , Enfermedades de los Monos/tratamiento farmacológico , Administración Oral , Animales , Biopsia/veterinaria , Dermatitis Atópica/patología , Femenino , Enfermedades de los Monos/patología , Resultado del TratamientoRESUMEN
Low bone density and large muscle mass predispose rabbits to femoral fractures. However, there are few reports describing treatment and prognosis. Two New Zealand White rabbits presented with unilateral left rear limb abduction and lateral rotation of the distal left rear limb 2 and 17 days after experimental surgery to create a "stair step" in the patellar groove of the left medial femoral chondyle. This procedure was performed after approval by the Institutional Animal Care and Use Committee. Radiography revealed a spiral oblique mid-shaft fracture of the left femur in both rabbits. Open fracture reduction was undertaken. Because of the presence of screws and Kirschner-wires in the medial femoral condyle, a lateral approach to surgical correction was chosen. Intramedullary fixation was used to reduce and stabilize the fractures. A 0.062" Kirschner wire was selected for the intramedullary device, because it was sufficiently flexible to allow easy passage into the femoral canal while being sufficiently stiff to promote reduction of the fracture. In addition, the ends of the fracture were secured with a 0.032" Kirschner cerclage wire to provide additional control of rotation and angulation. Then we assessed the range of motion of the knee joint to determine fracture stability and ensure that the hardware did not impinge on soft-tissue elements. After closure and application of sterile dressing, the hind legs were hobbled proximal to the hock by using elastic veterinary wrap in a figure-eight pattern to maintain limb alignment and prevent formation of pressure ulcers. Intraoperative fluoroscopic evaluation and postoperative radiographs confirmed fracture reduction. Bruising and seroma formation occurred at the surgical site, and transient anorexia developed. Rabbits were treated with fluids, analgesics, antibiotics, and fitted with Elizabethan collars. They were housed in isolation to limit excessive environmental stimulation, which could alarm them and provoke "thumping" of the rear legs. Muscular weakness and atrophy developed in the affected legs, but the fractures remained immobilized. Radiographs obtained 21 days after surgery confirmed marked callus formation and integrity of the implanted hardware. Four weeks after surgical fixation, both rabbits showed increased muscle development in the repaired leg and were ambulating normally. The long-term prognosis was excellent. These cases demonstrate that repair of femoral fractures in rabbits can be achieved by using basic orthopedic techniques and diligent post-operative management.
Asunto(s)
Fracturas del Fémur/veterinaria , Fémur/lesiones , Curación de Fractura/fisiología , Cuidados Posoperatorios/veterinaria , Animales , Modelos Animales de Enfermedad , Fracturas del Fémur/terapia , Fémur/diagnóstico por imagen , Fijación Interna de Fracturas/veterinaria , Curación de Fractura/efectos de los fármacos , Suspensión Trasera/fisiología , Articulación de la Rodilla/fisiología , Articulación de la Rodilla/cirugía , Masculino , Cuidados Posoperatorios/métodos , Conejos , Radiografía , Rango del Movimiento Articular/fisiología , Organismos Libres de Patógenos EspecíficosAsunto(s)
Anorexia/veterinaria , Estenosis Esofágica/veterinaria , Enfermedades de los Roedores/patología , Sialorrea/veterinaria , Animales , Anorexia/etiología , Estenosis Esofágica/complicaciones , Masculino , Ratas , Ratas Sprague-Dawley , Enfermedades de los Roedores/etiología , Sialorrea/etiologíaRESUMEN
Colony-stimulating factor-1 (CSF1) is one of two cytokines required for normal osteoclastogenesis. There are two major isoforms of CSF1, the cell-surface or membrane-bound isoform (mCSF1) and soluble CSF1 (sCSF1). Whether these isoforms serve nonredundant functions in bone is unclear. To explore this question, we generated transgenic mice expressing human sCSF1, human mCSF1, or both (s/mCSF1) in osteoblasts using the 2.3-kb rat alphaI-collagen promoter. Bone density determined by peripheral quantitative computed tomography was significantly reduced in mCSF1, sCSF1, and s/mCSF1 transgenic mice compared with wild-type animals. When analyzed by sex, sCSF1, and s/mCSF1, female animals but not mCSF1 female mice were found to have greater bone loss than their male littermates (-20 vs. -9.2%; P<0.05 for sCSF1 and -21.6 vs. -11.2% for s/mCSF1; P<0.01). By breeding CSF1 isoform-selective transgenic mice to an op/op background, mice were generated in which a single CSF1 isoform was the only source of the cytokine (sCSF1op/op and mCSF1op/op). Unlike osteoblast-targeted overexpression of mCSF1, selective transgenic expression of sCSF1 did not completely correct the op/op phenotype in 5-mo-old animals. Interestingly, compared with sham-ovariectomized mice of the same genotype, ovariectomy in sCSF1op/op mice led to a greater loss of spinal bone mineral density (22.1%) than was seen in either mCSF1op/op mice (12.9%) or in wild-type animals (10.9%). Our findings support the conclusion that sCSF1 and mCSF1 serve nonredundant functions in bone and that sCSF1 may play a role in mediating estrogen-deficiency bone loss.
Asunto(s)
Resorción Ósea/genética , Factor Estimulante de Colonias de Macrófagos/genética , Osteoblastos/metabolismo , Ovariectomía , Animales , Animales Recién Nacidos , Densidad Ósea/efectos de los fármacos , Densidad Ósea/genética , Resorción Ósea/metabolismo , Células Cultivadas , Estradiol/farmacología , Femenino , Marcación de Gen/métodos , Factor Estimulante de Colonias de Macrófagos/sangre , Factor Estimulante de Colonias de Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Especificidad de Órganos/genética , Osteopetrosis/genética , Osteopetrosis/metabolismo , Ovariectomía/veterinaria , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transfección/métodos , Regulación hacia Arriba/fisiologíaRESUMEN
Intra-abdominal (IA) fat functionally differs from subcutaneous (SC) adipose tissue, likely contributing to its stronger association with obesity-induced morbidity and to differential response to medications. Drug-induced partial lipodystrophy, like in response to antiretroviral agents, is an extreme manifestation of the different response of different fat depots, with loss of SC but not IA. Investigating depot-specific adipocyte differences is limited by the low accessibility to IA fat and by the heterogenous cell population comprising adipose tissue. Here, we aimed at utilizing immortalized preadipocyte cell lines from IA (epididymal) or SC (inguinal) fat to investigate whether they differentially respond to the HIV protease inhibitor nelfinavir. Preadipocytes were readily amenable to adipogenesis, as evidenced by lipid accumulation, expression of adipose-specific genes, measurable lipolysis, and insulin responsiveness. Leptin secretion was higher by the SC line, consistent with known differences between IA and SC fat. As previously reported, nelfinavir inhibited adipogenesis downstream of C/EBPbeta, but similarly in both cell lines. In contrast, nelfinavir's capacity to diminish insulin signaling, decrease leptin secretion, enhance basal lipolysis, and decrease expression of the lipid droplet-associated protein perilipin occurred more robustly and/or at lower nelfinavir concentrations in the SC line. This was despite similar intracellular concentrations of nelfinavir (23.8 +/- 5.6 and 33.6 +/- 12.2 microg/mg protein for inguinal and epididymal adipocytes, respectively, P = 0.46). The cell lines recapitulated depot-differential effects of nelfinavir observed in differentiated primary preadipocytes and with whole tissue explants. Thus, we report the use of fat depot-specific adipocyte cell lines for unraveling depot-differential responses to a drug causing partial lipodystrophy.
Asunto(s)
Adipocitos Blancos/efectos de los fármacos , Tejido Adiposo/citología , Distribución de la Grasa Corporal , Línea Celular , Síndrome de Lipodistrofia Asociada a VIH/etiología , Nelfinavir/farmacología , Adipocitos Blancos/fisiología , Adipogénesis/efectos de los fármacos , Adipogénesis/genética , Tejido Adiposo/efectos de los fármacos , Animales , Terapia Antirretroviral Altamente Activa/efectos adversos , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Línea Celular/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Infecciones por VIH/complicaciones , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/patología , Síndrome de Lipodistrofia Asociada a VIH/patología , Ratones , Nelfinavir/efectos adversos , Especificidad de Órganos/efectos de los fármacos , Ratas , Ratas Sprague-DawleyRESUMEN
The complete genetic absence of colony stimulating factor 1 (CSF1) in CSF1-deficient Csf1(op)/Csf1(op) mice leads to reproductive defects in males and females. Although the cell-surface or membrane-bound isoform of CSF1 (mCSF1) is biologically active in bone, little is known about its role in reproduction. Transgenic mice expressing mCSF1 under the control of the 2.4-kb rat collagen type I alpha promoter were developed [Tg(Col1a1-mCSF1)1Gqy] and bred onto a Csf1(op)/Csf1(op) background [Csf1(op)/Csf1(op); Tg(Col1a1-mCSF1)1Gqy] to examine the effects of the mCSF1 isoform in bone in vivo. Surprisingly, when interbred, these mice were fertile. The Csf1(op)/Csf1(op); Tg(Col1a1-mCSF1)1Gqy transgenic male mice have normal libido, sperm number and percent of motile sperm. In Csf1(op)/Csf1(op); Tg(Col1a1-mCSF1)1Gqy females, puberty and estrus cycles are at expected age and duration. Further, females are able to carry pregnancies to term and nurse their offspring. Crosses of Csf1(op)/Csf1(op); Tg(Col1a1-mCSF1)1Gqy males or females with their control littermates showed no significant differences in either number or viability of offspring. However, crossing Csf1(op)/Csf1(op); Tg(Col1a1-mCSF1)1Gqy males with Csf1(op); Tg(Col1a1-mCSF1)1Gqy females resulted in a decline in both the number and viability of offspring, suggesting that a subtle reproductive defect might persist in the transgenic animals that was only manifest when the animals were interbred. Although the gravid murine uterus expresses extremely high levels of CSF1 that are thought to be important for reproduction, uterine tissue levels of CSF1 remained low and unchanged during pregnancy in Csf1(op)/Csf1(op); Tg(Col1a1-mCSF1)1Gqy mice. Low levels of CSF1 protein were detected in serum and in lung and uterine tissue in Csf1(op)/Csf1(op); Tg(Col1a1-mCSF1)1Gqy mouse, which likely result from the known proteolytic shedding of mCSF1 from the cell surface. These data are consistent with the conclusion that mCSF1, when shed from the cell surface, can support reproduction and that high uterine tissue levels of CSF1 may not be required for mouse reproduction.