RESUMO
Powerful masticatory muscles are found in most primates, including chimpanzees and gorillas, and were part of a prominent adaptation of Australopithecus and Paranthropus, extinct genera of the family Hominidae. In contrast, masticatory muscles are considerably smaller in both modern and fossil members of Homo. The evolving hominid masticatory apparatus--traceable to a Late Miocene, chimpanzee-like morphology--shifted towards a pattern of gracilization nearly simultaneously with accelerated encephalization in early Homo. Here, we show that the gene encoding the predominant myosin heavy chain (MYH) expressed in these muscles was inactivated by a frameshifting mutation after the lineages leading to humans and chimpanzees diverged. Loss of this protein isoform is associated with marked size reductions in individual muscle fibres and entire masticatory muscles. Using the coding sequence for the myosin rod domains as a molecular clock, we estimate that this mutation appeared approximately 2.4 million years ago, predating the appearance of modern human body size and emigration of Homo from Africa. This represents the first proteomic distinction between humans and chimpanzees that can be correlated with a traceable anatomic imprint in the fossil record.
Assuntos
Evolução Molecular , Fósseis , Mutação da Fase de Leitura/genética , Hominidae/anatomia & histologia , Hominidae/genética , Cadeias Pesadas de Miosina/genética , Miosinas/genética , Filogenia , Sequência de Aminoácidos , Animais , Sequência de Bases , Biologia Computacional , Cães , Éxons/genética , História Antiga , Humanos , Macaca/anatomia & histologia , Macaca/genética , Músculos da Mastigação/anatomia & histologia , Dados de Sequência Molecular , Cadeias Pesadas de Miosina/química , Miosinas/química , Pan troglodytes/anatomia & histologia , Pan troglodytes/genética , Pongo pygmaeus/anatomia & histologia , Pongo pygmaeus/genética , Crânio/anatomia & histologia , Fatores de TempoRESUMO
The essential product of the Duchenne muscular dystrophy (DMD) gene is dystrophin1, a rod-like protein2 that protects striated myocytes from contraction-induced injury3,4. Dystrophin-related protein (or utrophin) retains most of the structural and protein binding elements of dystrophin5. Importantly, normal thymic expression in DMD patients6 should protect utrophin by central immunologic tolerance. We designed a codon-optimized, synthetic transgene encoding a miniaturized utrophin (µUtro), deliverable by adeno-associated virus (AAV) vectors. Here, we show that µUtro is a highly functional, non-immunogenic substitute for dystrophin, preventing the most deleterious histological and physiological aspects of muscular dystrophy in small and large animal models. Following systemic administration of an AAV-µUtro to neonatal dystrophin-deficient mdx mice, histological and biochemical markers of myonecrosis and regeneration are completely suppressed throughout growth to adult weight. In the dystrophin-deficient golden retriever model, µUtro non-toxically prevented myonecrosis, even in the most powerful muscles. In a stringent test of immunogenicity, focal expression of µUtro in the deletional-null German shorthaired pointer model produced no evidence of cell-mediated immunity, in contrast to the robust T cell response against similarly constructed µDystrophin (µDystro). These findings support a model in which utrophin-derived therapies might be used to treat clinical dystrophin deficiency, with a favorable immunologic profile and preserved function in the face of extreme miniaturization.
Assuntos
Terapia Genética , Distrofias Musculares/terapia , Distrofia Muscular Animal/terapia , Distrofia Muscular de Duchenne/terapia , Utrofina/genética , Animais , Dependovirus/genética , Modelos Animais de Doenças , Cães , Distrofina/genética , Humanos , Camundongos , Camundongos Endogâmicos mdx , Contração Muscular/genética , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Distrofias Musculares/genética , Distrofias Musculares/patologia , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/patologia , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/patologia , Transgenes/genética , Utrofina/uso terapêuticoRESUMO
BACKGROUND: The muscular dystrophies exemplify a class of systemic disorders for which widespread protein replacement in situ is essential for treatment of the underlying genetic disorder. Somatic gene therapy will require efficient, scale-independent transport of DNA-containing macromolecular complexes too large to cross the continuous endothelia under physiological conditions. Previous studies in large-animal models have revealed a trade-off between the efficiency of gene transfer and the inherent safety of the required surgical and pharmacological interventions to achieve this. METHODS AND RESULTS: Rats and dogs underwent limb or hemibody isolation via atraumatic tourniquet placement or myocardial isolation via heterotopic transplantation. Recombinant adenovirus (10(13) particles per kilogram) or recombinant adeno-associated virus (10(14) genome copies/kg) encoding the lacZ transgene was delivered through pressurized venous infusion without pharmacological mediators. Muscle exhibited almost 100% myofiber transduction in rats and dogs by X-galactosidase staining and significantly higher beta-galactosidase levels compared with nonpressurized delivery. No significant difference was seen in beta-galactosidase levels between 100- or 400-mm Hg groups. The <50-mm Hg group yielded inhomogeneous and significantly lower transgene expression. CONCLUSIONS: Uniform scale- and vector-independent skeletal and cardiac myofiber transduction is facilitated by pressurized venous infusion in anatomic domains isolated from the central circulation without pharmacological interference with cardiovascular homeostasis. We provide the first demonstration of uniform gene transfer to muscle fibers of an entire extremity in the dog, providing a firm foundation for further translational studies of efficacy in canine models for human diseases.
Assuntos
Dependovirus/genética , Músculo Esquelético/fisiologia , Animais , Cães , Técnicas de Transferência de Genes , Vetores Genéticos , Coração , Transplante de Coração/fisiologia , Masculino , Ratos , Ratos Sprague-Dawley , beta-Galactosidase/genéticaRESUMO
OBJECTIVE: With the increased availability of lower extremity percutaneous transluminal angioplasty (PTA), the conventional, non-interventional management of claudication may be evolving. This study evaluated changes in the use and short-term outcomes of PTA among patients with claudication and other manifestations of peripheral arterial disease (PAD). METHODS: A retrospective cohort study was conducted using the linked Washington State hospital discharge database (CHARS). Cases included all patients undergoing inpatient lower extremity PTA from 1997 to 2004. Patients with claudication were compared with those having PTA for other lower extremity diagnoses. The main outcome measures were readmission, reintervention (angiography, angioplasty/stent, surgical revascularization, or amputation), and death =30 days. RESULTS: A total of 1718 patients (mean age 69.7 +/- 11.2, 52.4% male) underwent PTA for claudication (51.9%), rest pain (12.1%), ulceration (23.2%), or not otherwise specified (12.9%). Yearly PTA use nearly doubled between 1997 and 2004, from 182 to 360, with a more dramatic increase in PTA among patients with claudication. Patients undergoing PTA for claudication were younger (67.9 +/- 10.3 vs 71.7 +/- 11.7 years, P < .01), more likely male (58.2% vs 46.2%, P < .01), and had a lower comorbidity index (0.7 vs 1.1, P < .01) compared with all others. A total of 65.3% were Medicare eligible. Among 555 patients aged <65 years, the indication for PTA was claudication more often when they had private insurance compared with uninsured or Medicaid beneficiaries (70.3% vs 49.1%, P < .01). Patients with claudication had shorter hospitalizations (2.4 +/- 2.3 vs 5.2 +/- 5.8 days, P < .01), lower rates of in-hospital death (0.8% vs 3.3%, P < .01), 30-day mortality (1.2% vs 4.7%, P < .01), and 30-day readmission (10% vs 23.1%, P < .05). Reintervention was required in 28.1% of readmitted patients with claudication, but none underwent amputation =30 days. CONCLUSION: The use of PTA for claudication dramatically increased during the 8-year study period. Claudication was more often the diagnosis for PTA in patients who were younger, healthier, and privately insured. PTA for claudication had a higher-than-expected morbidity, 30-day readmission, and rate of reintervention. Future studies should focus on the factors motivating the use of PTA, its associated outcomes, and global impact on patients and the health care system.
Assuntos
Angioplastia com Balão/estatística & dados numéricos , Claudicação Intermitente/terapia , Úlcera da Perna/terapia , Extremidade Inferior/irrigação sanguínea , Doenças Vasculares Periféricas/complicações , Idoso , Idoso de 80 Anos ou mais , Amputação Cirúrgica/estatística & dados numéricos , Angioplastia com Balão/efeitos adversos , Estudos de Coortes , Feminino , Seguimentos , Mortalidade Hospitalar , Humanos , Claudicação Intermitente/etiologia , Claudicação Intermitente/mortalidade , Úlcera da Perna/etiologia , Úlcera da Perna/mortalidade , Tempo de Internação/estatística & dados numéricos , Masculino , Sistemas Computadorizados de Registros Médicos , Pessoa de Meia-Idade , Readmissão do Paciente/estatística & dados numéricos , Doenças Vasculares Periféricas/mortalidade , Doenças Vasculares Periféricas/terapia , Vigilância da População , Reoperação/estatística & dados numéricos , Reprodutibilidade dos Testes , Estudos Retrospectivos , Fatores de Tempo , Resultado do Tratamento , Washington/epidemiologiaRESUMO
BACKGROUND: Previously, we used cardiopulmonary bypass with incomplete cardiac isolation and antegrade administration of vector for global cardiac gene delivery. Here we present a translatable cardiac surgical procedure that allows for complete surgical isolation of the heart in situ with retrograde (through the coronary venous circulation) administration of both vector and endothelial permeabilizing agents to increase myocyte transduction efficiency. METHODS: In 6 adult dogs the heart was completely isolated with tourniquets placed around both vena cavae and cannulas and all pulmonary veins. On cardiopulmonary bypass, the aorta and pulmonary artery were crossclamped, and the heart was isolated. Crystalloid cardioplegia at 4 degrees C containing 10(13) particles of adenovirus encoding LacZ and 15 microg of vascular endothelial growth factor was infused retrograde into the coronary sinus and recirculated for a total of 30 minutes. The dogs were then weaned from cardiopulmonary bypass and allowed to recover. With a catheter, 3 control dogs underwent retrograde infusion of the same cocktail without cardiac isolation or cardiopulmonary bypass. RESULTS: Beta-galactosidase activities in the cardiopulmonary bypass group were several orders of magnitude higher in both the right and left ventricles when compared with those in the control group (P < .05). X-gal staining from the cardiopulmonary bypass group showed unequivocal evidence of myocyte gene expression globally in a significant proportion of cardiac myocytes. No myocyte gene expression was observed in the control group. CONCLUSION: A novel cardiac surgical technique has been developed. This approach with cardiac isolation and retrograde delivery of vector through the coronary sinus results in efficient myocyte transduction in an adult large animal in vivo.