RESUMEN
BACKGROUND: Mucopolysaccharidosis type I (MPS-I) is a lysosomal storage disorder caused by a deficiency of the enzyme α-l-iduronidase, leading to accumulation of undegraded dermatan and heparan sulfates in the cells and secondary multiorgan dysfunction. In humans, depending upon the nature of the underlying mutation(s) in the IDUA gene, the condition presents with a spectrum of clinical severity. OBJECTIVES: To characterize the clinical and biochemical phenotypes, and the genotype of a family of Golden Retriever dogs. ANIMALS: Two affected siblings and 11 related dogs. METHODS: Family study. Urine metabolic screening and leucocyte lysosomal enzyme activity assays were performed for biochemical characterization. Whole genome sequencing was used to identify the causal mutation. RESULTS: The clinical signs shown by the proband resemble the human attenuated form of the disease, with a dysmorphic appearance, musculoskeletal, ocular and cardiac defects, and survival to adulthood. Urinary metabolic studies identified high levels of dermatan sulfate, heparan sulfate, and heparin. Lysosomal enzyme activities demonstrated deficiency in α-l-iduronidase activity in leucocytes. Genome sequencing revealed a novel homozygous deletion of 287 bp resulting in full deletion of exon 10 of the IDUA gene (NC_006585.3(NM_001313883.1):c.1400-76_1521+89del). Treatment with pentosan polyphosphate improved the clinical signs until euthanasia at 4.5 years. CONCLUSION AND CLINICAL IMPORTANCE: Analysis of the genotype/phenotype correlation in this dog family suggests that dogs with MPS-I could have a less severe phenotype than humans, even in the presence of severe mutations. Treatment with pentosan polyphosphate should be considered in dogs with MPS-I.
Asunto(s)
Enfermedades de los Perros , Exones , Mucopolisacaridosis I , Animales , Enfermedades de los Perros/tratamiento farmacológico , Enfermedades de los Perros/genética , Perros , Exones/genética , Homocigoto , Iduronidasa/genética , Mucopolisacaridosis I/genética , Mucopolisacaridosis I/veterinaria , Mutación , Eliminación de SecuenciaRESUMEN
Mucopolysaccharidosis (MPS) is a metabolic storage disorder caused by the deficiency of any lysosomal enzyme required for the breakdown of glycosaminoglycans. A 15-month-old Boston Terrier presented with clinical signs consistent with lysosomal storage disease including corneal opacities, multifocal central nervous system disease and progressively worsening clinical course. Diagnosis was confirmed at necropsy based on histopathologic evaluation of multiple organs demonstrating accumulation of mucopolysaccharides. Whole genome sequencing was used to uncover a frame-shift insertion affecting the alpha-L-iduronidase (IDUA) gene (c.19_20insCGGCCCCC), a mutation confirmed in another Boston Terrier presented 2 years later with a similar clinical picture. Both dogs were homozygous for the IDUA mutation and shared coat colors not recognized as normal for the breed by the American Kennel Club. In contrast, the mutation was not detected in 120 unrelated Boston Terriers as well as 202 dogs from other breeds. Recent inbreeding to select for recessive and unusual coat colors may have concentrated this relatively rare allele in the breed. The identification of the variant enables ante-mortem diagnosis of similar cases and selective breeding to avoid the spread of this disease in the breed. Boston Terriers carrying this variant represent a promising model for MPS I with neurological abnormalities in humans.
Asunto(s)
Perros/genética , Mucopolisacaridosis I/genética , Mucopolisacaridosis I/veterinaria , Mutación/genética , Secuenciación Completa del Genoma , Animales , Secuencia de Bases , Femenino , Mucopolisacaridosis I/diagnóstico por imagen , Mucopolisacaridosis I/patologíaRESUMEN
OBJECTIVE: This study examined the effect of mucopolysaccharidosis (MPS) type 1 on diffusion tensor imaging (DTI) metrics in the canine brain. We hypothesized 1) white matter regions in the MPS brain would show decreased fractional anisotropy (FA) and increased radial diffusivity (RD) compared to the same regions in normal brain, 2) compared to FA, RD would more closely correlate with myelin density and fiber coherence, and 3) DTI and histological data from the normal brain could be used to accurately predict degree of myelination in the MPS brain using DTI metrics. METHODS: We performed DTI imaging on one normal canine brain and two MPS brains on a 7T MR scanner and generated FA and RD maps. Brains were sectioned and stained with a gold chloride stain for myelin to obtain myelin optical density and fiber coherence values. The three brains were compared using the DTI and histology metrics. RESULTS: Most measured regions in one MPS brain and all measured regions in the other MPS brain exhibited decreased FA, increased RD, and decreased myelin density in white matter. FA and RD significantly correlated with myelin density in the normal brain but failed to reach significance in either MPS brain. A predictive model using FA but not RD was able to accurately predict degree of myelination in one MPS brain. CONCLUSION: Dysmyelination in the MPS brain results in decreased FA and increased RD. However, in the small sample, FA and RD were values not significantly correlated with myelination in either MPS brain.
Asunto(s)
Enfermedades de los Perros/patología , Mucopolisacaridosis I/patología , Mucopolisacaridosis I/veterinaria , Fibras Nerviosas Mielínicas/patología , Animales , Encéfalo/patología , Imagen de Difusión Tensora , Perros , Sustancia Gris/patología , Procesamiento de Imagen Asistido por Computador , Fibras Nerviosas/patología , Valor Predictivo de las Pruebas , Tomografía de Coherencia Óptica , Sustancia Blanca/patologíaRESUMEN
Mucopolysaccharidosis I (MPS I) is a lysosomal storage disease characterized by deficient α-L-iduronidase activity, leading to the accumulation of poorly degraded glycosaminoglycans (GAGs). Children with MPS I exhibit high incidence of spine disease, including accelerated disc degeneration and vertebral dysplasia, which in turn lead to spinal cord compression and kyphoscoliosis. In this study we investigated the efficacy of neonatal enzyme replacement therapy (ERT), alone or in combination with oral simvastatin (ERT + SIM) for attenuating cervical spine disease progression in MPS I, using a canine model. Four groups were studied: normal controls; MPS I untreated; MPS I ERT-treated; and MPS I ERT + SIM-treated. Animals were euthanized at age 1 year. Intervertebral disc condition and spinal cord compression were evaluated from magnetic resonance imaging (MRI) images and plain radiographs, vertebral bone condition and odontoid hypoplasia were evaluated using micro-computed tomography (µCT), and epiphyseal cartilage to bone conversion was evaluated histologically. Untreated MPS I animals exhibited more advanced disc degeneration and more severe spinal cord compression than normal animals. Both treatment groups resulted in partial preservation of disc condition and cord compression, with ERT + SIM not significantly better than ERT alone. Untreated MPS I animals had significantly lower vertebral trabecular bone volume and mineral density, whereas ERT treatment resulted in partial preservation of these properties. ERT + SIM treatment demonstrated similar, but not greater, efficacy. Both treatment groups partially normalized endochondral ossification in the vertebral epiphyses (as indicated by absence of persistent growth plate cartilage), and odontoid process size and morphology. These results indicate that ERT begun from a very early age attenuates the severity of cervical spine disease in MPS I, particularly for the vertebral bone and odontoid process, and that additional treatment with simvastatin does not provide a significant additional benefit over ERT alone.
Asunto(s)
Enfermedades de los Perros , Terapia de Reemplazo Enzimático , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Iduronidasa/uso terapéutico , Degeneración del Disco Intervertebral , Mucopolisacaridosis I , Simvastatina/farmacología , Animales , Modelos Animales de Enfermedad , Enfermedades de los Perros/tratamiento farmacológico , Enfermedades de los Perros/metabolismo , Enfermedades de los Perros/patología , Perros , Femenino , Degeneración del Disco Intervertebral/tratamiento farmacológico , Degeneración del Disco Intervertebral/metabolismo , Degeneración del Disco Intervertebral/patología , Degeneración del Disco Intervertebral/veterinaria , Masculino , Mucopolisacaridosis I/tratamiento farmacológico , Mucopolisacaridosis I/metabolismo , Mucopolisacaridosis I/patología , Mucopolisacaridosis I/veterinariaRESUMEN
Intrathecal (IT) recombinant human α-l-iduronidase (rhIDU) has been shown to reduce mean brain glycosaminoglycans (GAGs) to normal levels in mucopolysaccharidosis I (MPS I) dogs. In this study, we examined storage in neuroanatomical regions of the MPS I dog brain, including frontal lobe, cerebellum, basal ganglia, thalamus, hippocampal formation, and brainstem, to determine the response of these functional regions to treatment with IT rhIDU. GAG storage in untreated MPS I dogs was significantly different from normal dogs in all examined sections. GAG levels in normal dogs varied by region: frontal lobe (mean: 2.36 ± 0.54 µg/mg protein), cerebellum (2.67 ± 0.33), basal ganglia and thalamus (3.51 ± 0.60), hippocampus (3.30 ± 0.40), and brainstem (3.73 ± 1.10). Following IT treatment, there was a reduction in GAG storage in each region in all treatment groups, except for the brainstem. Percent reduction in GAG levels from untreated to treated MPS I dogs in the deeper regions of the brain was 30% for basal ganglia and thalamus and 30% for hippocampus, and storage reduction was greater in superficial regions, with 61% reduction in the frontal lobe and 54% in the cerebellum compared with untreated MPS I dogs. Secondary lipid storage in neurons was also reduced in frontal lobe, but not in the other brain regions examined. Response to therapy appeared to be greater in more superficial regions of the brain, particularly in the frontal lobe cortex.
Asunto(s)
Encéfalo/metabolismo , Enfermedades de los Perros/metabolismo , Glicosaminoglicanos/metabolismo , Iduronidasa/administración & dosificación , Mucopolisacaridosis I/veterinaria , Animales , Modelos Animales de Enfermedad , Enfermedades de los Perros/tratamiento farmacológico , Enfermedades de los Perros/enzimología , Perros , Femenino , Histocitoquímica/veterinaria , Inyecciones Espinales/veterinaria , Masculino , Mucopolisacaridosis I/tratamiento farmacológico , Mucopolisacaridosis I/enzimología , Mucopolisacaridosis I/metabolismo , Proteínas Recombinantes/administración & dosificación , Distribución TisularRESUMEN
Mucopolysaccharidosis-I (MPS-I) is an inherited deficiency of α-L-iduronidase (IdU) that causes lysosomal accumulation of glycosaminoglycans (GAG) in a variety of parenchymal cell types and connective tissues. The fundamental link between genetic mutation and tissue GAG accumulation is clear, but relatively little attention has been given to the morphology or pathogenesis of associated lesions, particularly those affecting the vascular system. The terminal parietal branches of the abdominal aorta were examined from a colony of dogs homozygous (MPS-I affected) or heterozygous (unaffected carrier) for an IdU mutation that eliminated all enzyme activity, and in affected animals treated with human recombinant IdU. High-resolution computed tomography showed that vascular wall thickenings occurred in affected animals near branch points, and associated with low endothelial shear stress. Histologically these asymmetric 'plaques' entailed extensive intimal thickening with disruption of the internal elastic lamina, occluding more than 50% of the vascular lumen in some cases. Immunohistochemistry was used to show that areas of sclerosis contained foamy (GAG laden) macrophages, fibroblasts and smooth muscle cells, with loss of overlying endothelial basement membrane and claudin-5 expression. Lesions contained scattered cells expressing nuclear factor-κß (p65), increased fibronectin and transforming growth factor ß-1 signaling (with nuclear Smad3 accumulation) in comparison to unaffected vessels. Intimal lesion development and morphology was improved by intravenous recombinant enzyme treatment, particularly with immune tolerance to this exogenous protein. The progressive sclerotic vasculopathy of MPS-I shares some morphological and molecular similarities to atherosclerosis, including formation in areas of low shear stress near branch points, and can be reduced or inhibited by intravenous administration of recombinant IdU.
Asunto(s)
Arterias/patología , Mucopolisacaridosis I/veterinaria , Enfermedades Vasculares/veterinaria , Animales , Perros , Femenino , Humanos , Iduronidasa/administración & dosificación , Iduronidasa/genética , Iduronidasa/uso terapéutico , Inmunohistoquímica , Masculino , Mucopolisacaridosis I/genética , Mucopolisacaridosis I/patología , Mucopolisacaridosis I/terapia , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/genética , Proteínas Recombinantes/uso terapéutico , Tomografía Computarizada por Rayos X , Enfermedades Vasculares/genética , Enfermedades Vasculares/patología , Enfermedades Vasculares/terapiaRESUMEN
PURPOSE: Mucopolysaccharidosis I (MPS I) is an inherited metabolic disorder resulting from deficiency of α-L-iduronidase and lysosomal accumulation of glycosaminoglycans (GAG) in multiple tissues. Accumulation of GAG in corneal stromal cells causes corneal opacity and reduced vision. The purpose of this study was to determine the extent of ocular GAG accumulation and investigate the effectiveness of intravenous enzyme replacement therapy (ERT) on corneal GAG accumulation in dogs. METHODS: Ocular tissues were obtained from 58 dogs with mucopolysaccharidosis I and four unaffected controls. Affected dogs received either low-dose ERT, high-dose ERT, or no treatment; some low-dose dogs also received intrathecal treatments. Histologic severity of corneal stromal GAG accumulation was scored. RESULTS: Accumulation of GAG was found in corneal stromal cells and scleral fibroblasts but not in corneal epithelium, endothelium, ciliary epithelium, choroid, retina, retinal pigment epithelium, or optic nerve. Corneal GAG accumulation increased in severity with increasing age. Although low-dose ERT did not significantly reduce corneal stromal GAG accumulation in comparison with untreated animals, high-dose ERT did result in significantly less GAG accumulation compared with the untreated dogs (adjusted P = 0.0143) or the low-dose ERT group (adjusted P = 0.0031). Intrathecal treatments did not significantly affect GAG accumulation. Dogs that began ERT shortly after birth also had significantly less (P < 0.0001) GAG accumulation in the corneal stroma than dogs with a later onset of treatment. CONCLUSIONS: These data suggest that high-dose, intravenous ERT is effective at preventing and/or clearing corneal stromal GAG accumulation, particularly if initiated early after birth.
Asunto(s)
Enfermedades de la Córnea/veterinaria , Enfermedades de los Perros/tratamiento farmacológico , Terapia de Reemplazo Enzimático/veterinaria , Iduronidasa/uso terapéutico , Mucopolisacaridosis I/veterinaria , Envejecimiento/fisiología , Animales , Enfermedades de la Córnea/tratamiento farmacológico , Enfermedades de la Córnea/enzimología , Sustancia Propia/metabolismo , Enfermedades de los Perros/enzimología , Perros , Fibroblastos/metabolismo , Glicosaminoglicanos/metabolismo , Glicosaminoglicanos/ultraestructura , Iduronidasa/administración & dosificación , Inyecciones Intravenosas , Inyecciones Espinales , Mucopolisacaridosis I/tratamiento farmacológico , Mucopolisacaridosis I/enzimología , Estudios Retrospectivos , Esclerótica/metabolismoRESUMEN
Mucopolysaccharidosis (MPS) types I and VII are inborn errors of metabolism caused by mutation of enzymes involved in glycosaminoglycan catabolism, which leads to intralysosomal accumulation of glycosaminoglycans. In children, severe forms of MPS I and VII are characterized by somatic and neurologic manifestations, including a poorly understood hearing loss. The purpose of this study is to describe the age-related histopathologic changes of the ear in spontaneous canine models of MPS I and VII. Pathologic changes in the ear were assessed in MPS I and VII dogs ranging from 1.6 to 9.3 months of age. Paraffin-embedded sections of the whole ear and Epon-embedded semithin sections of the cochlea were examined. The following lesions were blindly scored in the middle and inner ear: inflammation, cells vacuolization, thickening of osseous and membranous structures, perivascular vacuolated macrophages infiltration, and bone resorption. All dogs had lysosomal storage within cells of tympanic membrane, ossicles, tympanic bone and mucosa, cochlear bone, spiral ligament, limbus, and stria vascularis. The MPS I dogs mainly had progressive cochlear lesions. The MPS VII dogs had severe and early middle ear lesions, including chronic otitis media and bone resorption. The MPS I dog only partially recapitulates the pathology seen in humans; specifically, the dog model lacks inflammatory middle ear disease. In contrast, the MPS VII dog has severe inflammatory middle ear disease similar to that reported in the human. In conclusion, the canine MPS VII model appears to be a good model to study MPS VII-related deafness.
Asunto(s)
Enfermedades de los Perros/patología , Enfermedades del Oído/veterinaria , Mucopolisacaridosis I/veterinaria , Mucopolisacaridosis VII/veterinaria , Animales , Enfermedades de los Perros/etiología , Perros , Enfermedades del Oído/etiología , Enfermedades del Oído/patología , Oído Interno/patología , Oído Medio/patología , Humanos , Masculino , Mucopolisacaridosis I/complicaciones , Mucopolisacaridosis I/patología , Mucopolisacaridosis VII/complicaciones , Mucopolisacaridosis VII/patologíaRESUMEN
Renal failure was diagnosed in an 11-mo-old male domestic shorthair cat from a colony with mucopolysaccharidosis type I lysosomal storage disease. Grossly, the kidneys were enlarged and bulged on cut section. Histology revealed tubular necrosis and regeneration with severe interstitial macrophage accumulation. Tubular epithelial cells and interstitial macrophages were distended by abundant, large cytoplasmic vacuoles. Electron microscopy demonstrated severe tubular epithelial vacuolar degeneration with lysosomes distended by granular debris and mineral precipitates. Interstitial macrophages contained similarly distended lysosomes. Although the initial cause of the tubular injury was not identified, the presence of macrophages laden with storage product most likely exacerbated the disease. The macrophage infiltrate may have caused tubular ischemia by compressing peritubular capillaries and separating tubules from their blood supply. Because the kidney is not normally affected in MPS I, this case is an unusual presentation of a well-characterized disease. Furthermore, this report documents the diagnostic workflow used to investigate a single case of feline acute renal failure in the setting of numerous at-risk laboratory animals.
Asunto(s)
Animales de Laboratorio , Enfermedades de los Gatos/patología , Mucopolisacaridosis I/veterinaria , Insuficiencia Renal/veterinaria , Animales , Gatos , Resultado Fatal , Técnicas Histológicas/veterinaria , Inmunohistoquímica/veterinaria , Túbulos Renales/ultraestructura , Masculino , Microscopía Electrónica/veterinaria , Mucopolisacaridosis I/complicaciones , Mucopolisacaridosis I/patología , Insuficiencia Renal/etiología , Insuficiencia Renal/patologíaRESUMEN
Enzyme replacement therapy (ERT) with intravenous recombinant human alpha-l-iduronidase (IV rhIDU) is a treatment for patients with mucopolysaccharidosis I (MPS I). Spinal cord compression develops in MPS I patients due in part to dural and leptomeningeal thickening from accumulated glycosaminoglycans (GAG). We tested long-term and every 3-month intrathecal (IT) and weekly IV rhIDU in MPS I dogs age 12-15months (Adult) and MPS I pups age 2-23days (Early) to determine whether spinal cord compression could be reversed, stabilized, or prevented. Five treatment groups of MPS I dogs were evaluated (n=4 per group): IT+IV Adult, IV Adult, IT + IV Early, 0.58mg/kg IV Early and 1.57mg/kg IV Early. IT + IV rhIDU (Adult and Early) led to very high iduronidase levels in cervical, thoracic, and lumber spinal meninges (3600-29,000% of normal), while IV rhIDU alone (Adult and Early) led to levels that were 8.2-176% of normal. GAG storage was significantly reduced from untreated levels in spinal meninges of IT + IV Early (p<.001), IT+IV Adult (p=.001), 0.58mg/kg IV Early (p=.002) and 1.57mg/kg IV Early (p<.001) treatment groups. Treatment of dogs shortly after birth with IT+IV rhIDU (IT + IV Early) led to normal to near-normal GAG levels in the meninges and histologic absence of storage vacuoles. Lysosomal storage was reduced in spinal anterior horn cells in 1.57mg/kg IV Early and IT + IV Early animals. All dogs in IT + IV Adult and IV Adult groups had compression of their spinal cord at 12-15months of age determined by magnetic resonance imaging and was due to protrusion of spinal disks into the canal. Cord compression developed in 3 of 4 dogs in the 0.58mg/kg IV Early group; 2 of 3 dogs in the IT + IV Early group; and 0 of 4 dogs in the 1.57mg/kg IV Early group by 12-18months of age. IT + IV rhIDU was more effective than IV rhIDU alone for treatment of meningeal storage, and it prevented meningeal GAG accumulation when begun early. High-dose IV rhIDU from birth (1.57mg/kg weekly) appeared to prevent cord compression due to protrusion of spinal disks.
Asunto(s)
Terapia de Reemplazo Enzimático/veterinaria , Iduronidasa/uso terapéutico , Mucopolisacaridosis I/tratamiento farmacológico , Mucopolisacaridosis I/veterinaria , Compresión de la Médula Espinal/tratamiento farmacológico , Compresión de la Médula Espinal/veterinaria , Animales , Perros , Humanos , Inyecciones Espinales , Imagen por Resonancia Magnética/veterinaria , Médula Espinal/patología , Compresión de la Médula Espinal/patologíaRESUMEN
Mucopolysaccharidosis I (MPS I) and MPS VII are due to loss-of-function mutations within the genes that encode the lysosomal enzymes alpha-l-iduronidase and beta-glucuronidase, respectively, and result in accumulation of glycosaminoglycans and multisystemic disease. Both disorders are associated with elastin fragmentation and dilatation of the aorta. Here, the pathogenesis and effect of gene therapy on aortic disease in canine models of MPS was evaluated. We found that cathepsin S is upregulated at the mRNA and enzyme activity level, while matrix metalloproteinase 12 (MMP-12) is upregulated at the mRNA level, in aortas from untreated MPS I and MPS VII dogs. Both of these proteases can degrade elastin. In addition, mRNA levels for the interleukin 6-like cytokine oncostatin M were increased in MPS I and MPS VII dog aortas, while mRNA for tumor necrosis factor alpha and toll-like receptor 4 were increased in MPS VII dog aortas. These cytokines could contribute to upregulation of the elastases. Neonatal intravenous injection of a retroviral vector expressing beta-glucuronidase to MPS VII dogs reduced RNA levels of cathepsin S and MMP-12 and aortic dilatation was delayed, albeit dilatation developed at late times after gene therapy. A post-mortem aorta from a patient with MPS VII also exhibited elastin fragmentation. We conclude that aortic dilatation in MPS I and MPS VII dogs is likely due to degradation of elastin by cathepsin S and/or MMP-12. Inhibitors of these enzymes or these cytokine-induced signal transduction pathways might reduce aortic disease in patients with MPS.
Asunto(s)
Aorta/enzimología , Enfermedades de los Perros/enzimología , Mucopolisacaridosis I/enzimología , Mucopolisacaridosis I/veterinaria , Mucopolisacaridosis VII/enzimología , Elastasa Pancreática/metabolismo , Regulación hacia Arriba , Animales , Enfermedades de la Aorta/complicaciones , Catepsinas/metabolismo , Perros , Elastina/metabolismo , Humanos , Masculino , Metaloproteinasa 12 de la Matriz/metabolismo , Mucopolisacaridosis VII/veterinaria , Adulto JovenRESUMEN
BACKGROUND: Somatic cell nuclear transfer in cats offers a useful tool for the generation of valuable research models. However, low birth rates after nuclear transfer hamper exploitation of the full potential of the technology. Poor embryo development after activation of the reconstructed oocytes seems to be responsible, at least in part, for the low efficiency. The objective of this study was to characterize the response of cat oocytes to various stimuli in order to fine-tune existing and possibly develop new activation methods for the generation of cat disease models by somatic cell nuclear transfer. METHODS: First, changes in the intracellular free calcium concentration [Ca2+]i in the oocytes induced by a number of artificial stimuli were characterized. The stimuli included electroporation, ethanol, ionomycin, thimerosal, strontium-chloride and sodium (Na+)-free medium. The potential of the most promising treatments (with or without subsequent incubation in the presence of cycloheximide and cytochalasin B) to stimulate oocyte activation and support development of the resultant parthenogenetic embryos was then evaluated. Finally, the most effective methods were selected to activate oocytes reconstructed during nuclear transfer with fibroblasts from mucopolysaccharidosis I- and alpha-mannosidosis-affected cats. RESULTS: All treatments were able to elicit a [Ca2+]i elevation in the ooplasm with various characteristics. Pronuclear formation and development up to the blastocyst stage was most efficiently triggered by electroporation (60.5 +/- 2.9 and 11.5 +/- 1.7%) and the combined thimerosal/DTT treatment (67.7 +/- 1.8 and 10.6 +/- 1.9%); incubation of the stimulated oocytes with cycloheximide and cytochalasin B had a positive effect on embryo development. When these two methods were used to activate oocytes reconstructed during nuclear transfer, up to 84.9% of the reconstructed oocytes cleaved. When the 2 to 4-cell embryos (a total of 220) were transferred into 19 recipient females, 4 animals became pregnant. All of the fetuses developed from oocytes activated by electroporation followed by cycloheximide and cytochalasin B incubation; no fetal development was detected as a result of thimerosal/DTT activation. Although heartbeats were detected in two of the cloned fetuses, no term development occurred. CONCLUSION: Electroporation proved to be the most effective method for the activation of cat oocytes reconstructed by nuclear transfer. The combined thimerosal/DTT treatment followed by cycloheximide and cytochalasin B incubation triggered development effectively to the blastocyst stage; whether it is a viable option to stimulate term development of cloned cat embryos needs further investigations.
Asunto(s)
Enfermedades de los Gatos/genética , Técnicas de Transferencia Nuclear , Oocitos/fisiología , Animales , Calcio/metabolismo , Gatos , Cicloheximida/farmacología , Citocalasina B/farmacología , Modelos Animales de Enfermedad , Electroporación , Técnicas de Cultivo de Embriones , Desarrollo Embrionario/fisiología , Femenino , Fibroblastos/fisiología , Mucopolisacaridosis I/genética , Mucopolisacaridosis I/veterinaria , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Partenogénesis , Embarazo , Conservadores Farmacéuticos , Inhibidores de la Síntesis de la Proteína/farmacología , Estimulación Química , Timerosal/farmacología , alfa-Manosidosis/genética , alfa-Manosidosis/veterinariaRESUMEN
Issues of cost and genetics can result in inbreeding of canine genetic disease colonies. Beagles often are used to maintain such colonies, providing stock for outcrosses. Factor VII (FVII) deficiency is a hemostatic disorder found at increased frequency in beagles and has been characterized at the DNA level. Deficiency of FVII presents obstacles in colonies founded with beagles. An initial finding of a FVII-deficient pup from a longstanding colony prompted us to evaluate FVII deficiency fully in this colony. Current and archival records and tissues were used to reconstruct the colony pedigree, assess the contribution from beagles, and test samples to document the source and frequency of the mutant FVII allele. As part of this study we developed a PCR-based diagnostic assay that was simpler than what was previously available. Pedigree analysis revealed a founder effect implicating beagles that led to high frequency (55%) of the mutant allele. In addition, affected animals were identified. The complete picture of the clinical effect within the colony remains unclear, but unusual neonatal presentations, including hemoabdomen, have occurred in pups affected with FVII deficiency. Use of a PCR-based diagnostic assay to screen all potential beagle breeding stock will prevent similar occurrences of FVII deficiency in future canine research colonies.
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Modelos Animales de Enfermedad , Deficiencia del Factor VII/veterinaria , Mucopolisacaridosis I/veterinaria , Animales , Secuencia de Bases , Cartilla de ADN , Perros , Deficiencia del Factor VII/complicaciones , Femenino , Masculino , Mucopolisacaridosis I/complicaciones , Linaje , Reacción en Cadena de la PolimerasaRESUMEN
PURPOSE: To determine the versatility of retroviral vector-mediated rat beta-glucuronidase cDNA expression in the normal retinal pigment epithelium (RPE) of eyes of various species and in RPE of eyes with three types of mucopolysaccharidosis (MPS types I, VI, and VII) and to evaluate the effect of multiple transductions and long-term stable expression in the RPE. METHODS: A retroviral construct containing a rat beta-glucuronidase cDNA (NTK-BGEO) was used to infect RPE cells at subconfluence. The transduced cells were selected in G418, an antibiotic toxic to normal mammalian cells. Beta-glucuronidase activity was measured in transduced cells and media, using a fluorogenic substrate. Glycosaminoglycan profiles were examined by metabolically labeling RPE with Na2(35)SO4. RESULTS: Transduced RPE cells, regardless of species or disease status, expressed rat beta-glucuronidase. The expressed enzyme restored normal levels of glycosaminoglycans in the RPE cells of homozygous MPS VII-affected dogs by metabolizing stored glycosaminoglycans. The expressed enzyme failed to metabolize stored glycosaminoglycans of MPS I and MPS VI, indicating that overexpression could not bypass the exoglycosidase restriction. Multiple transductions increased beta-glucuronidase activity several times in the cell layer and in the media. The expression was stable in vitro for at least 12 weeks. CONCLUSIONS: A retroviral vector can mediate transfer of beta-glucuronidase in various species of normal and MPS-affected RPE. The expression is stable in vitro. The metabolism of stored glycosaminoglycans in MPS needs replacement of only the deficient enzyme to reverse the storage.
Asunto(s)
ADN Complementario/metabolismo , Técnicas de Transferencia de Gen , Vectores Genéticos , Glucuronidasa/metabolismo , Epitelio Pigmentado Ocular/enzimología , Retroviridae/genética , Animales , Northern Blotting , Gatos , Bovinos , Células Cultivadas , Perros , Regulación Enzimológica de la Expresión Génica , Glucuronidasa/genética , Glicosaminoglicanos/metabolismo , Caballos , Macaca mulatta , Mucopolisacaridosis I/enzimología , Mucopolisacaridosis I/veterinaria , Mucopolisacaridosis VI/enzimología , Mucopolisacaridosis VI/veterinaria , Mucopolisacaridosis VII/enzimología , Mucopolisacaridosis VII/veterinaria , Epitelio Pigmentado Ocular/citología , Epitelio Pigmentado Ocular/virología , ARN/aislamiento & purificación , RatasRESUMEN
Nodular regenerative hyperplasia (NRH) is an uncommon hepatic lesion often associated with noncirrhotic portal hypertension (PHT). We have noted that NRH and PHT are frequent occurrences in a colony of dogs with the genetic storage disease, mucopolysaccharidosis I (MPS-I). This observation provides the opportunity to study the histology and pathogenesis of NRH and noncirrhotic PHT in a new animal model. Thirteen of 32 dogs (41%) with MPS-I developed multiple portocaval shunts between 4 and 48 months of age that were grossly visible at necropsy. Seven of the 13 developed marked ascites, whereas all those without shunts and littermates (n = 24) heterozygous for the mutated alpha-L-iduronidase allele (carriers unaffected by the storage disease) did not. The large and medium-sized portal veins were widely patent without thrombosis or vascular malformations. Hepatic parenchymal fibrosis was absent or mild and did not correlate with shunt formation. All 32 livers had varying degrees of diffuse periportal hepatocellular hyperplasia with multifocal atrophy and compression of centrolobular cords (NRH) most prominent in dogs with shunts. Many small portal veins were reduced in diameter or absent, especially in animals with shunts. Noncirrhotic PHT and NRH appear to be related to the obliteration of small portal veins in these dogs, but the pathogenesis of this vascular change remains unknown.
Asunto(s)
Modelos Animales de Enfermedad , Enfermedades de los Perros/patología , Hipertensión Portal/veterinaria , Hepatopatías/veterinaria , Mucopolisacaridosis I/veterinaria , Animales , Atrofia , Perros , Femenino , Hiperplasia , Hígado/patologíaRESUMEN
A seven-month-old, female domestic shorthair cat was presented to the Veterinary Teaching Hospital, University of Zurich, with abnormal facial features, retarded growth and progressive hindlimb paresis. On physical examination the cat had a flat, broad face with hypertelorism, frontal bossing, small ears and thickened upper and lower eyelids. The corneas of both eyes were clear and the pupils were dilated. The skin was generally thickened, most prominently on the dorsal aspect of the neck. Radiography of the entire skeleton revealed a severely deformed spinal column, bilateral hip luxation with hip dysplasia, an abnormally shaped skull and generalised decreased bone opacity. The clinical features and radiographic changes were suggestive of mucopolysaccharidosis. The toluidine blue spot test on a urine sample, however, was negative for glycosaminoglycans. Further biochemical investigations revealed a deficiency of the enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-phosphotransferase, EC 2.7.8.17) in peripheral leukocytes and an elevation of many lysosomal enzymes in the serum of the cat which is diagnostic for mucolipidosis type II. Histology and electron microscopy of different tissues are briefly summarised. The findings of this cat, the first reported case of mucolipidosis type II are compared with other similar storage diseases described in the cat.
Asunto(s)
Enfermedades de los Gatos/diagnóstico , Mucolipidosis/veterinaria , Animales , Axones/ultraestructura , Huesos/anomalías , Huesos/diagnóstico por imagen , Huesos/ultraestructura , Cartílago/ultraestructura , Enfermedades de los Gatos/metabolismo , Enfermedades de los Gatos/patología , Gatos , Tejido Conectivo/ultraestructura , Femenino , Leucocitos/enzimología , Leucocitos/ultraestructura , Microscopía Electrónica/veterinaria , Mucolipidosis/diagnóstico , Mucolipidosis/patología , Mucopolisacaridosis I/diagnóstico , Mucopolisacaridosis I/metabolismo , Mucopolisacaridosis I/veterinaria , Mucopolisacaridosis VI/diagnóstico , Mucopolisacaridosis VI/metabolismo , Mucopolisacaridosis VI/veterinaria , Mucopolisacaridosis VII/diagnóstico , Mucopolisacaridosis VII/metabolismo , Mucopolisacaridosis VII/veterinaria , Radiografía , Piel/ultraestructuraRESUMEN
Livers from normal cats and dogs, cats with mucopolysaccharidoses (MPS) I and VI, and dogs with MPS VII were analyzed biochemically and morphometrically to determine the lysosomal storage of glycosaminoglycans (GAG) in these animal models of human genetic disease. Analyses were performed on liver samples from seven normal cats ranging in age from 13 weeks to 15 months; six MPS I-affected cats ranging in age from 10 weeks to 26 months; four MPS VI-affected cats ranging in age from 9 months to 32 months; four normal dogs ranging in age from 1 month to 47 months; and three MPS VII-affected dogs, 5 days, 11 days, and 14 months of age. All of the animals were from the breeding colony at the University of Pennsylvania School of Veterinary Medicine and were maintained in accordance with national standards for the care and use of laboratory animals. Each GAG subclass was quantitated, and total GAG concentration was determined. Liver from cats with MPS I had the highest total GAG concentration (5.7 times that of the control), followed by liver from dogs with MPS VII (1.8 times) and cats with MPS VI (1.5 times). These data were very closely correlated (R2 = 0.982) with the results of the morphometric analyses of hepatocyte and Kupffer cell vacuolation associated with lysosomal storage and support the validity of both methods. This is particularly important for the quantification of total and individual GAG concentrations in tissue preparations. The values obtained should prove useful in future assessments of therapeutic regimes, such as enzyme replacement, bone marrow transplantation, and gene therapy, for these genetic diseases.
Asunto(s)
Enfermedades de los Gatos/patología , Enfermedades de los Perros/patología , Glicosaminoglicanos/metabolismo , Hígado/patología , Mucopolisacaridosis/veterinaria , Animales , Enfermedades de los Gatos/metabolismo , Gatos , Densitometría , Enfermedades de los Perros/metabolismo , Perros , Electroforesis en Acetato de Celulosa , Glicosaminoglicanos/análisis , Macrófagos del Hígado/patología , Macrófagos del Hígado/ultraestructura , Hígado/química , Hígado/metabolismo , Hígado/ultraestructura , Microscopía Electrónica , Mucopolisacaridosis/metabolismo , Mucopolisacaridosis/patología , Mucopolisacaridosis I/metabolismo , Mucopolisacaridosis I/patología , Mucopolisacaridosis I/veterinaria , Mucopolisacaridosis VI/metabolismo , Mucopolisacaridosis VI/patología , Mucopolisacaridosis VI/veterinaria , Mucopolisacaridosis VII/metabolismo , Mucopolisacaridosis VII/patología , Mucopolisacaridosis VII/veterinaria , Vacuolas/ultraestructuraRESUMEN
Two long-haired Siamese cats are reported with clinical manifestations of human mucopolysaccharidosis VI (Maroteaux-Lamy disease): facial dysmorphia, dysostosis multiplex, paralysis. Urine of the two affected animals contained a high concentration of glycosaminoglycans, as detected by the dimethylmethylene blue test. Qualitative analysis, performed by thin-layer chromatography of the cetylpyridinium chloride-precipitable material, showed dermatan sulphate. Excessive incorporation of [35S]sulphate in the intracellular mucopolysaccharide of cultured fibroblasts and deficiency of arylsulphatase B in such cells indicate that these cats are affected by Maroteaux-Lamy disease. They should thus be considered the first European case of feline mucopolysaccharidosis VI.
Asunto(s)
Mucopolisacaridosis I/veterinaria , Animales , Huesos/anomalías , Gatos , Condro-4-Sulfatasa/deficiencia , Condro-4-Sulfatasa/genética , Modelos Animales de Enfermedad , Femenino , Glicosaminoglicanos/orina , Humanos , Mucopolisacaridosis I/patología , Mucopolisacaridosis I/orinaRESUMEN
Five dogs with mucopolysaccharidosis I, 3 of which had been treated with bone marrow transplantation (BMT), were evaluated for 20 months with electrocardiography, thoracic radiography, and M-mode and 2-dimensional echocardiography. Treated and untreated (control) dogs had widened P waves on ECG. Thoracic radiographs remained normal for all dogs throughout the study. Thickening of the mitral valve was observed on echocardiograms of dogs in both groups, but the untreated dogs appeared to have thicker valves. Concentrations of glycosaminoglycans in the mitral valves and myocardium were higher in control dogs than in treated dogs. Markedly large aortic root diameters were observed on echocardiograms in both untreated dogs, but aortic root diameters remained normal in treated dogs. Echocardiography, but not electrocardiography, was useful in monitoring heart enlargement in each dog. Dogs treated with BMT generally had less severe cardiac changes and slower disease progression than control dogs.