Dupuytren's disease susceptibility gene, EPDR1, is involved in myofibroblast contractility.

BACKGROUND: Dupuytren's Disease is a common disorder of the connective tissue characterized by progressive and irreversible fibroblastic proliferation affecting the palmar fascia. Progressive flexion deformity appears over several months or years and although usually painless, it can result in a serious handicap causing loss of manual dexterity. There is no cure for the disease and the etiology is largely unknown. A genome-wide association study of Dupuytren's Disease identified nine susceptibility loci with the strongest genetic signal located in an intron of EPDR1, the gene encoding the Ependymin Related 1 protein. OBJECTIVE: Here, we investigate the role of EPDR1 in Dupuytren's Disease. METHODS: We research the role of EPDR1 by assessing gene expression in patient tissue and by gene silencing in fibroblast-populated collagen lattice (FPCL) assay, which is used as an in vitro model of Dupuytren's contractures. RESULTS: The three alternative transcripts produced by the EPDR1 gene are all detected in affected Dupuytren's tissue and control unaffected palmar fascia tissue. Dupuytren's tissue also contracts more in the FPCL paradigm. Dicer-substrate RNA-mediated knockdown of EPDR1 results in moderate late stage attenuation of contraction rate in FPCL, implying a role in matrix contraction. CONCLUSION: Our results suggest functional involvement of EPDR1 in the etiology of Dupuytren's Disease.

Monitoring collagen synthesis in fibroblasts using fluorescently labeled tRNA pairs.

There is a critical need for techniques that directly monitor protein synthesis within cells isolated from normal and diseased tissue. Fibrotic disease, for which there is no drug treatment, is characterized by the overexpression of collagens. Here, we use a bioinformatics approach to identify a pair of glycine and proline isoacceptor tRNAs as being specific for the decoding of collagen mRNAs, leading to development of a FRET-based approach, dicodon monitoring of protein synthesis (DiCoMPS), that directly monitors the synthesis of collagen. DiCoMPS aimed at detecting collagen synthesis will be helpful in identifying novel anti-fibrotic compounds in cells derived from patients with fibrosis of any etiology, and, suitably adapted, should be widely applicable in monitoring the synthesis of other proteins in cells.

A blinded placebo-controlled randomized trial on the use of astaxanthin as an adjunct to splinting in the treatment of carpal tunnel syndrome.

BACKGROUND: Nutritional supplementation is a potential adjunct in the conservative management of carpal tunnel syndrome (CTS). This study investigated whether astaxanthin (a beta-carotenoid) increased the effectiveness of splinting in managing CTS. METHODS: This is a triple-blinded randomized controlled trial where 63 patients with electrodiagnostically confirmed CTS were randomly allocated into either the experimental group (n = 32) (astaxanthin-4-mg capsules + splinting) or the control group (n = 31) (placebo + splinting). Medications were taken for 9 weeks followed by a 3-week washout. The primary outcome measure was the Symptom Severity Scale (SSS). Secondary outcome measures in the study included physical impairments, disability, and health status measures. Electrodiagnostic testing was performed before entry into the study and again at 12 weeks. All other outcomes were measured at baseline, 6, and 12 weeks. RESULTS: There was a reduction in symptoms as measured by the SSS over the course of treatment in both groups (p = 0.002), but no differences between the groups (p = 0.18). The Disability of Arm, Shoulder and Hand questionnaire and the Short Form 36-item Health Survey showed no effects over time or between treatment groups. The baseline difference between the groups in the level of total cholesterol and low-density lipoproteins remained constant over the course of the study. Impairment measures demonstrated no significant changes in grip, dexterity, or sensation. CONCLUSION: At present, the role for astaxanthin as an adjunct in conservative management of CTS has not been established.

Protein biomarker analysis of primary Peyronie's disease cells.

INTRODUCTION: The molecular pathogenesis of Peyronie's Disease (PD) remains unclear more than 250 years after its initial description. Because of this, no test is currently available to accurately predict PD progression among those affected. AIM: To investigate the expression of wound healing and fibrosis-associated proteins in primary cell cultures of PD fibroblasts to determine whether altered protein expression patterns can be used as predictors of clinical course and natural history. METHODS: Primary cell cultures derived from normal Tunica albuginea tissue and PD plaque tissue were examined by immuno-cytochemistry. Protein expression profiles were analyzed by Surface-Enhanced Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (SELDI-TOF-MS) and Western immunoblotting. MAIN OUTCOME MEASURES: Expression of wound healing and fibrosis-associated proteins and protein expression patterns were assessed. RESULTS: Statistically significant increases in smooth muscle alpha-actin, beta-catenin, and Heat shock proteins (Hsp47) were identified in cells derived from PD relative to cells derived from normal Tunica albuginea tissue. Changes in TGFbeta-1 receptor and Fibronectin were also observed. In addition, altered expression of additional as yet unidentified proteins at 4.7, 8.9, 10.8, 16.8, and 76.8 kDa were detected by complementary SELDI-TOF-MS approaches. CONCLUSIONS: Primary cells derived from PD plaques display up-regulated expression of several proteins that are established components of fibrosis and wound healing. In addition, changes in other, as yet unidentified proteins were measured. It will be of interest to conduct further studies to see whether these dysregulated protein peaks represent potential biological markers of disease progression.

The effects of botulinum toxin type A on muscle blood perfusion and metabolism.

BACKGROUND: Botulinum toxin type A is approved by the U.S. Food and Drug Administration for the treatment of facial rhytides. However, the complete spectrum of action of botulinum toxin A has not yet been completely defined. Little is known about the metabolism of muscle after botulinum toxin A injection. This information may give insight into the additional effects botulinum toxin A may have on muscle. The authors assessed the influence of botulinum toxin A on the metabolism of muscle using dynamic investigative techniques. METHODS: Twenty New Zealand White rabbits were divided into control, paralysis, and sham groups. Masseter muscle paralysis was achieved with botulinum toxin A. Dynamic computed tomographic and positron emission tomographic scans were obtained. Masseter muscle blood flow, blood volume, permeability surface, and mean transit time and glucose uptake were measured. RESULTS: Eighteen animals completed the study. Masseter blood perfusion showed consistent results across all parameters. Blood flow, blood volume, and permeability surface were significantly increased at weeks 4 and 8 on the paralyzed side. Mean transit time at week 4 was decreased on the paralyzed side. Positron emission tomographic scans showed that injected muscles in the botulinum toxin A group tended to have increased glucose uptake compared with untreated muscles. CONCLUSIONS: Botulinum toxin A injection increases muscle blood perfusion parameters and glucose uptake for a transient period. This increase is similar in duration to the known interval of botulinum toxin A-induced paralysis. These changes have been identified in a dynamic fashion and may represent changes in calcitonin gene-related peptide release.

The effects of masseter muscle paralysis on facial bone growth.

BACKGROUND: Understanding the effects of muscle function on facial bone growth may help us treat children with facial anomalies. Facial bone growth is known to be a result of both genetic and epigenetic influences. One of the main epigenetic factors controlling growth is thought to be muscle action. The purpose of this study was to establish a model of single facial muscle paralysis and to identify the effects masseter muscle paralysis has on mandible and zygoma growth. METHODS: Twenty New Zealand white rabbits were divided into control, paralysis, and sham groups. Masseter muscle paralysis was achieved with botulinum toxin A (BTX). Computed tomographic and single-photon emission computed tomography (SPECT) scans and cephalometric measurements were performed. Masseter weights and mandible and zygoma volumes, shapes, and metabolism were measured. RESULTS: Eighteen animals completed the study. Significant decreases in zygoma and mandible volumes with minimal changes in shape were seen on the paralyzed sides. SPECT showed a decrease in bone production in both zygomas and mandibles on the paralyzed sides. CONCLUSIONS: An animal model has been created in which the effects of single muscle paralysis on bone growth can be studied. Masseter muscle function may be responsible in maintaining mandible and zygoma volume by controlling bone production. Masseter function alone has less influence on mandible and zygoma shape.

Viable group A streptococci in macrophages during acute soft tissue infection.

BACKGROUND: Group A streptococcal severe soft tissue infections, such as necrotizing fasciitis, are rapidly progressive infections associated with high mortality. Group A streptococcus is typically considered an extracellular pathogen, but has been shown to reside intracellularly in host cells. METHODS AND FINDINGS: We characterized in vivo interactions between group A streptococci (GAS) and cells involved in innate immune responses, using human biopsies (n = 70) collected from 17 patients with soft tissue infections. Immunostaining and in situ image analysis revealed high amounts of bacteria in the biopsies, even in those collected after prolonged antibiotic therapy. Viability of the streptococci was assessed by use of a bacterial viability stain, which demonstrated viable bacteria in 74% of the biopsies. GAS were present both extracellularly and intracellularly within phagocytic cells, primarily within macrophages. Intracellular GAS were predominantly noted in biopsies from newly involved tissue characterized by lower inflammation and bacterial load, whereas purely extracellular GAS or a combination of intra- and extracellular GAS dominated in severely inflamed tissue. The latter tissue was also associated with a significantly increased amount of the cysteine protease streptococcal pyrogenic exotoxin SpeB. In vitro studies confirmed that macrophages serve as reservoirs for viable GAS, and infection with a speB-deletion mutant produced significantly lower frequencies of cells with viable GAS following infection as compared to the wild-type bacteria. CONCLUSIONS: This is the first study to demonstrate that GAS survive intracellularly in macrophages during acute invasive infections. This intracellular presence may have evolved as a mechanism to avoid antibiotic eradication, which may explain our finding that high bacterial load is present even in tissue collected after prolonged intravenous antibiotic therapy. This new insight into the pathogenesis of streptococcal soft tissue infections highlights a need for alternative therapeutic strategies.