Isoform-specific expression of the Coxsackie and adenovirus receptor (CAR) in neuromuscular junction and cardiac intercalated discs

BACKGROUND: The Coxsackie and adenovirus receptor (CAR) has a restricted expression pattern in the adult. In skeletal muscle, although CAR is expressed in immature fibers, its transcript levels are barely detectable in mature muscle. This is in contrast to the robust expression observed in the heart. However, both heart and skeletal muscle are susceptible to infection with the Coxsackie B virus which utilizes primarily CAR for cellular internalization. The specific point of viral entry in skeletal and heart muscle remains unknown. RESULTS: Using antibodies directed against the extracellular and the cytoplasmic domains of CAR, we show CAR in normal human and mouse skeletal muscle to be a novel component of the neuromuscular junction. In cardiac muscle, CAR immunoreactivity is observed at the level of intercalated discs. We demonstrate a single isoform of CAR to be expressed exclusively at the human neuromuscular junction whereas both predominant CAR isoforms are expressed at the intercalated discs of non-diseased human heart. CONCLUSION: The localization of CAR to these important junctional complexes suggests that CAR may play both a structural and a regulatory role in skeletal and cardiac muscle, and that these complexes may serve as a point of entry for Coxsackie B virus

Ultrasound increases plasmid-mediated gene transfer to dystrophic muscles without collateral damage

Studies have shown that ultrasound, used either alone or in combination with microbubble contrast agents, can increase cell membrane permeability to plasmid DNA. Because ultrasound is a non-painful and well-established tool in clinical medicine, its potential to enhance DNA uptake into the muscles of patients with muscular dystrophy is conceptually attractive. Therefore, we evaluated the ability of ultrasound pulses (1 MHz; 1.5 W/cm2) to increase exogenous (LacZ) gene expression in normal wild-type and dystrophic Dmd(mdx/mdx) mice after plasmid DNA injection into muscle. We also ascertained whether co-injection of lipid-encapsulated perfluoropropane microbubbles (Definity) or pretreatment with hyaluronidase could further increase the level of gene transfer to ultrasound-treated muscles. The use of ultrasound did not increase transfection efficiency in normal mice. In contrast, dystrophic mice demonstrated an increase in the number of transfected fibers (threefold) as well as the amount of LacZ protein (22-fold) after ultrasound exposure, provided that Definity was also co-injected with the DNA. Pretreatment of muscles with hyaluronidase before ultrasound exposure was not effective in augmenting the level of gene transfer. Under the optimal conditions for dystrophic muscle transfection (ultrasound + Definity), there was no associated increase in muscle damage. Hence ultrasound may provide a safe and effective method for enhancing gene transfer to dystrophic muscles, thereby increasing the prospects for therapeutic application of naked DNA in muscular dystrophy patients.Peer reviewed: YesNRC publication: N

Pathogenesis of myonecrosis induced by crude venom and a myotoxin of Bothrops asper.

The pathogenesis of skeletal muscle necrosis induced by crude Bothrops asper venom and isolated myotoxic phospholipase was studied using light and electron microscopy. White mice were injected intramuscularly with a dose of 2.5 micrograms/g and tissue samples were taken at 30 min and 1, 3, 6, 12, 24, and 48 hr. Toxin-injected muscle showed localized wedge-shaped lesions ("delta lesions") by 30 min, which included disrupted plasma membranes. At 1 and 3 hr the predominant type of necrotic cell contained clumped myofibrils in which individual myofilaments were indistinguishable. At later time periods there was a relaxation and redistribution of myofilaments resulting in a more homogeneous and hyaline appearance of necrotic cells. Some mitochondria were swollen and had flocculent densities, and most of them were disrupted, having only one membrane and vesiculated cristae. The basal lamina was intact at all time intervals. Phagocytosis of muscle cell debris started at 3 hr and was prominent by 24-48 hr. In crude venom-injected muscle many cells showed pathologic features identical to those observed after myotoxin injection. Crude venom also induced hemorrhage which was evident 30 min after injection, reaching its highest level by 12 hr. At 3, 6, and 12 hr some cells were undergoing different pathologic changes which appeared to be due to ischemia. Although these cells were irreversibly damaged, as indicated by ruptured plasma membrane, their myofibrillar structure was better preserved than that of toxin-affected cells. The Z line was absent, but A, I, H, and M bands were intact. As a result of Z line loss, sarcomeres were disoriented. It is proposed that the myotoxin induces myonecrosis by first altering the integrity of the plasma membrane, thereby increasing the permeability to calcium, other ions, and molecules which leads to death of the cell. Crude venom affects muscle cells in two ways: by direct action of myotoxin (s) and by ischemia due to hemorrhage.UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP

Colonial refractions: the 'Gypsy camp' as a spatio-racial political technology

Camps for civilians first appeared in the colonies. Largely drawing on the literature on colonialism and race, this article conceptualizes the 'Gypsy camp' in Western European cities as a spatio-racial political technology. We first discuss the shift, starting with decolonization, from colonial to metropolitan technologies of the governance of social heterogeneity. We then relate this broad historical framing to the ideas and ideologies that since the 1960s have been underpinning the planning and governance of the ‘Gypsy camp' in both the UK and Italy. We document the 1970s emergence of a new and distinctive type of camp that was predicated upon a racially connoted tension between policies criminalizing sedentarization and ideologies of cultural protection. Given that the imposition of the ‘Gypsy camp' was essentially uncontested, we argue that the conditions of possibility for it to emerge and become institutionalized were both a spatio-racial similarity with typically colonial technologies of governance, and the fact that it was largely perceived as a self-evident necessity for the governance and control of one specific population. We conclude by calling for more analyses on this and other forms of urban confinement in both the Global North and South, in order to account for the increasingly disquieting mushrooming of confining and controlling governance devices, practices and ideologies

Danon disease due to a novel splice mutation in the LAMP2 gene

Danon disease is a rare X-linked dominant disorder caused by lysosomal-associated membrane protein 2 (LAMP2) deficiency and is characterized by hypertrophic cardiomyopathy, cardiac conduction abnormalities, skeletal vacuolar myopathy, variable degree of mental retardation, and peripheral pigmentary retinopathy. We describe a novel splice mutation in the LAMP2 gene in a French Canadian family. By identifying this novel mutation we were able to offer genetic screening and counseling to all family members. Presymptomatic diagnosis is important as cardiac surveillance can be life-saving

Differentiation of murine embryonic stem cells in skeletal muscles of mice

Possible myogenic differentiation of SSEA-1- and OCT-4-positive murine embryonic stem cells (ESCs) and embryoid bodies (EBs) was studied in vitro and in vivo. In vitro, ESC- or EB-derived ESCs (EBs/ESCs) showed only traces of Pax 3 and 7 expression by immunocytochemistry and Pax 3 expression by immunoblot. By RT-PCR, myogenic determinant molecules (myf5, myoD, and myogenin) were expressed by EBs/ESCs but not by ESCs. However, in such cultures, very rare contracting myotubes were still present. Suspensions of LacZ-labeled ESCs or EBs were injected into anterior tibialis muscles (ATM) of different cohorts of mice for the study of their survival and possible myogenic differentiation. The different cohorts of mice included isogenic adult 129/Sv, nonisogenic CD1 and mdx, as well as mdx immunosuppressed with 2.5 mg/kg daily injections of tacrolimus. Ten to 90 days postinjections, the injected ATM of nonisogenic mice did not contain cells positive for LacZ, SSEA-1, OCT-4, or embryonic myosin heavy chain. The ATM of intact mdx mice contained very rare examples of muscle fibers positive for dystrophin and/or embryonic myosin heavy chain. In the ATM of the isogenic normal and the immunosuppressed mdx mice, as expected, large teratomas developed containing the usual diverse cell types. In some teratomas of immunosuppressed mdx mice, small pockets of muscle fibers expressed dystrophin and myosin heavy chain. Our studies indicated that in muscles of animals nonisogenic with the used ESCs, only very rare ESCs survived with myogenic differentiation. These studies also indicated that ESCs will not undergo significant, selective, and preferential myogenic differentiation in vitro or in vivo in any of the models studied. It is probable that this strain of murine ESC requires some experimentally induced alteration of its gene expression profile to secure significant myogenicity and suppress tumorogenicity.NRC publication: Ye

Gene transfer into skeletal muscles by isogenic myoblasts

NRC publication: Ye

Successful compensation for dystrophin deficiency by a helper-dependent adenovirus expressing full-length utrophin

Helper-dependent adenovirus vector (AdV)\u2013mediated full-length dystrophin expression leads to significant mitigation of the dystrophic phenotype of the mdx mouse. However, dystrophin, as a neoantigen, elicits antibody formation. As an alternative approach, we evaluated gene transfer of full-length murine utrophin, a functional homologue of dystrophin that is normally present only at the neuromuscular junction. A single injection in the tibialis anterior (TA) muscle of the helper-dependent adenovirus vector encoding utrophin provided very good transduction, with 58% of fibers demonstrating sarcolemmal utrophin expression in the neonates, and 35% utrophin-positive (Utr\u207a) fibers in adults. The presence of utrophin prevented extensive necrosis in the neonates, halted further necrosis in the adults, and led to restoration of sarcolemmal expression of dystrophin-associated proteins up to 1 year after injection. Marked physiological improvement was observed in both neonates and adults. Neither increased humoral responses nor cellular immune responses were evident. However, there was a time-related decline of the initial high utrophin expression. Although viral DNA persisted in animals that were injected in the neonatal stage, viral DNA levels decreased in muscles of adult mice. These results demonstrate that although utrophin gene transfer leads to amelioration of the dystrophic phenotype, the effects are not sustained upon loss of utrophin expression.NRC publication: Ye

Improved Performance of a Fully Gutted Adenovirus Vector Containing Two Full-Length Dystrophin cDNAs Regulated by a Strong Promoter

Dystrophin gene transfer using gutted or helper-dependent adenoviruses (HDAd), which have most of their genes deleted, is a promising approach to treat Duchenne muscular dystrophy. In an attempt to boost the amount of dystrophin produced after gene transfer, we have constructed a fully deleted HDAd (HDCBDys2x) containing two human dystrophin cDNAs controlled by the powerful hybrid cytomegalovirus enhancer/beta-actin promoter. We demonstrated high dystrophin expression after infection of muscle cultures with HDCBDys2x. Similarly, high (mean=583) and moderate (mean=124) numbers of muscle fibers were transduced in anterior tibialis muscle after intramuscular injection of HDCBDys2x in neonate and adult dystrophindeficient (mdx) mice 10 days postinjection. In fact, in the neonatally injected mdx mice, the transferred dystrophin was five times more abundant than in normal human muscle. However, the high early transduction level was transient in both animal groups, and we observed a humoral response against the human dystrophin. In contrast, we demonstrated sustained dystrophin expression in immunodeficient mouse muscles. Dystrophin expression of HDCBDys2x could be further increased in the presence of an E1/E3-deleted (first-generation) adenovirus, thus demonstrating that the latter vector synthesizes trans-acting enhancing factors. We have achieved abundant dystrophin expression with our new, improved HDAd. It is anticipated that high longterm transgene expression will be possible by employing weaker immunogenic transgenes.NRC publication: N