A translational approach for limb vascular delivery of the micro-dystrophin gene without high volume or high pressure for treatment of Duchenne muscular dystrophy

Background  

Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder with monogenic mutations setting the stage for successful gene therapy treatment. We have completed a study that directly deals with the following key issues that can be directly adapted to a gene therapy clinical trial using rAAV considering the following criteria: 1) A regional vascular delivery approach that will protect the patient from widespread dissemination of virus; 2) an approach to potentially facilitate safe passage of the virus for efficient skeletal muscle transduction; 3) the use of viral doses to accommodate current limitations imposed by vector production methods; 4) and at the same time, achieve a clinically meaningful outcome by transducing multiple muscles in the lower limb to prolong ambulation.     

Effect of denervation on the endogenous phosphorylating activity in the cytosol of rat skeletal muscle

An endogenous phosphorylating activity is demonstrated in the cytosol from soleus muscle of the rat which is markedly stimulated after severing the motor nerve fibers to this muscle. The [γ-³²P]ATP phosphotransferase reaction is heat-labile, dependent upon Mg²⁺ but not Ca²⁺ or cyclic GMP, inhibited by a cyclic AMP dependent protein kinase inhibitor, and directly related to the amount of cytosolic protein which provides the endogenous source of both the protein kinase enzyme, ATP, cyclic AMP and phosphorylatable protein substrate. The time-course of the delayed transitory stimulation of the cytosolic phosphorylating activity of the denervated soleus may involve neurotropic factors.     

Bone morphogenetic protein receptor type II C-terminus interacts with c-Src: implication for a role in pulmonary arterial hypertension

Mutations of bone morphogenetic protein receptor type II (BMPR-II) have been associated with familial and idiopathic pulmonary arterial hypertension (PAH). BMPR-II is a member of the transforming growth factor-beta receptor superfamily. It consists of extracellular, transmembrane, and kinase domains, and a unique C-terminus with mostly unknown function. However, a number of PAH-causing mutations are predicted to truncate the C-terminus, suggesting that this domain plays an important role in the homeostasis of pulmonary vessels. In this study, we sought to elucidate the functional role of this C-terminus by seeking its interacting partners. Using yeast two-hybrid screening, we identified c-Src tyrosine kinase as a binding partner of this C-terminus. In vitro co-immunoprecipitation confirmed their interaction. Mutations truncating the C-terminus disrupted their interaction, while missense mutation within kinase domain reduced their interaction. In addition, BMPR-II and c-Src tyrosine kinase colocalized within intracellular aggregates when overexpressed in HEK293 cells. Moreover, mutations truncating the C-terminus disrupted their colocalization, whereas missense mutation within kinase domain had no effect on their colocalization. Furthermore, BMP ligand stimulation decreased c-Src-activating phosphorylation at Tyrosine 418 in pulmonary smooth muscle cells in both time- and concentration-dependent manners. Mutations that truncated the C-terminus abolished this response. Taken together, these results suggest a model in which proliferative effect of c-Src by vasoactive molecules is balanced by opposing effect of BMP signaling in basal state, and the loss of this balance due to BMPR2 mutations leads to increased c-Src activity and subsequently cell growth.     

A systemic view of higher education and professional psychology: implications of the Combined-Integrated model of doctoral training

This article was developed in response to the proceedings of the Consensus Conference on Combined and Integrated Doctoral Training in Psychology held at James Madison University in Harrisonburg, VA, May 2 to 4, 2003. The authors approach the recommendations of the conference from the perspective of their experiences in higher education administration at the national, regional, and state levels. The authors conclude that the Consensus Conference represents an exemplar of best practice in program planning. They suggest that a major reconceptualization of higher education is under way that emphasizes broad collaboration among various professional groups as a means of providing appropriate mental health and health care services. Consequently, professional psychology will need to reconceptualize its role in the broader context of other professions within the university setting. Recommendations for the education of psychologists and the development of future training programs are provided. Suggestions for implementation of various recommendations flowing from the Consensus Conference are delineated.     

Proapoptotic Bak is sequestered by Mcl-1 and Bcl-xL, but not Bcl-2, until displaced by BH3-only proteins

Commitment of cells to apoptosis is governed largely by the interaction between members of the Bcl-2 protein family. Its three subfamilies have distinct roles: The BH3-only proteins trigger apoptosis by binding via their BH3 domain to prosurvival relatives, while the proapoptotic Bax and Bak have an essential downstream role involving permeabilization of organellar membranes and induction of caspase activation. We have investigated the regulation of Bak and find that, in healthy cells, Bak associates with Mcl-1 and Bcl-x(L) but surprisingly not Bcl-2, Bcl-w, or A1. These interactions require the Bak BH3 domain, which is also necessary for Bak dimerization and killing activity. When cytotoxic signals activate BH3-only proteins that can engage both Mcl-1 and Bcl-x(L) (such as Noxa plus Bad), Bak is displaced and induces cell death. Accordingly, the BH3-only protein Noxa could bind to Mcl-1, displace Bak, and promote Mcl-1 degradation, but Bak-mediated cell death also required neutralization of Bcl-x(L) by other BH3-only proteins. The results indicate that Bak is held in check solely by Mcl-1 and Bcl-x(L) and induces apoptosis only if freed from both. The finding that different prosurvival proteins have selective roles has notable implications for the design of anti-cancer drugs that target the Bcl-2 family.     

Enhanced Pseudomonas aeruginosa biofilm development mediated by human neutrophils

Cystic fibrosis (CF) lung disease features persistent neutrophil accumulation to the airways from the time of infancy. CF children are frequently exposed to Pseudomonas aeruginosa, and by adulthood, 80% of CF patients are chronically infected. The formation of biofilms is a particularly important phenotypic characteristic of P. aeruginosa that allows for bacterial survival despite aggressive antibiotic therapy and an exuberant immune response. Here, we show that the presence of neutrophils enhances initial P. aeruginosa biofilm development over a period of 72 h through the formation of polymers comprised of actin and DNA. F-actin was found to be a site of attachment for P. aeruginosa. These actin and DNA polymers are present in CF sputum, and disruption of the polymers dispersed the associated P. aeruginosa cells and reduced biofilm development. These findings demonstrate a potential maladaptation of the primary innate response. When the host fails to eradicate the infection, cellular components from necrotic neutrophils can serve as a biological matrix to facilitate P. aeruginosa biofilm formation.     

Immune privilege in the anterior chamber of the eye

Since the 19th century, it has been recognized that the anterior chamber of the eye permits the prolonged, and sometimes permanent, survival of foreign tissue and tumor grafts. It was initially believed that the absence of patent lymphatics draining the interior of the eye prevented antigens from reaching regional lymphoid tissues. However, sequestration of intraocular antigens alone cannot account for ocular immune privilege, and a clear picture is now emerging that a constellation of anatomical, physiological, and dynamic immunoregulatory factors contribute to ocular immune privilege. Ocular fluids contain a potpourri of immunosuppressive and immunoregulatory factors that suppress T-cell proliferation and secretion of proinflammatory cytokines. The interior of the eye is decorated with Fas ligand (CD95L), which induces apoptosis of infiltrating inflammatory cells. Antigens introduced into the eye induce a unique immune deviation in which TH1 responses, namely delayed-type hypersensitivity (DTH), are actively suppressed. Thus, ocular immune privilege is sustained by factors that suppress immune cell proliferation and purge immune cells that enter the eye and by a dynamic immunoregulatory process that suppresses antigen-specific DTH. Suppression of certain inflammatory responses is an important adaptation for preventing immune-mediated injury to ocular tissues that have little or no capacity to regenerate. Thus, immune privilege is a crucial adaptation for preserving vision.     

Conversion of nonfusing mumps virus infections to fusing infections by selective proteolysis of the HN glycoprotein

Mumps virus strains differ in their ability to induce cell fusion following an infection: strains with activeneuraminidase (NANase) fail to cause cell fusion, while strains with less active NANases cause cell fusion. When chymotrypsin is added to infected cells, cell fusion is amplified in a concentration-dependent manner for all mumps virus strains. Virions produced in such infections do not express HN glycoprotein-associated activities. Chymotrypsin treatment of purified mumps virus in vitro results in sequential cleavage into two glycopolypeptides, HNc1 (32K) and HNc2′ (41K), with concomitant loss of hemagglutinating and NANase activities, and infectivity. Further incubation with chymotrypsin causes complete degradation of HNc1 and digestion of HNc2′ to HNc2 (13K–19K). Both HNc2′ and HNc2 contain the [³H]palmitic acid label found in the HN polypeptide, which suggests that these fragments are associated with the viral membrane. Analyses of infected cells and released virions indicate that chymotrypsin acts similarly on HN exposed at the cell surface. Exogenous NAnase does not abolish the protease-augmented cell fusion though it does reduce cell fusion of untreated fusing strain infections. These results confirm that mumps virus HN glycoprotein is critically linked to cell fusion cytopathology and show that cyrptic cell fusion activity in nonfusing strain infections can be unmasked by the proteolytic removal of the HN glycoprotein.     

Retroesophageal right subclavian artery with persistent ductus arteriosus

We report an adult male cadaver with persistence of the ductus arteriosus and a right retroesophageal artery. The retroesophageal artery was first appreciated as a retropharyngeal mass during the dissection of this area. Both the left and right vertebral arteries originated in normal fashion. The thoracic duct of our specimen drained into the junction of the left internal jugular and left subclavian veins. We believe this to be the first report of simultaneous right retroesophageal subclavian artery and patent ductus arteriosus. The anatomy and embryology of these two anomalies in the same specimen are discussed.