Neuronal Nitric Oxide Synthase-Rescue of Dystrophin/Utrophin Double Knockout Mice does not Require nNOS Localization to the Cell Membrane

Survival of dystrophin/utrophin double-knockout (dko) mice was increased by muscle-specific expression of a neuronal nitric oxide synthase (nNOS) transgene. Dko mice expressing the transgene (nNOS TG+/dko) experienced delayed onset of mortality and increased life-span. The nNOS TG+/dko mice demonstrated a significant decrease in the concentration of CD163+, M2c macrophages that can express arginase and promote fibrosis. The decrease in M2c macrophages was associated with a significant reduction in fibrosis of heart, diaphragm and hindlimb muscles of nNOS TG+/dko mice. The nNOS transgene had no effect on the concentration of cytolytic, CD68+, M1 macrophages. Accordingly, we did not observe any change in the extent of muscle fiber lysis in the nNOS TG+/dko mice. These findings show that nNOS/NO (nitric oxide)-mediated decreases in M2c macrophages lead to a reduction in the muscle fibrosis that is associated with increased mortality in mice lacking dystrophin and utrophin. Interestingly, the dramatic and beneficial effects of the nNOS transgene were not attributable to localization of nNOS protein at the cell membrane. We did not detect any nNOS protein at the sarcolemma in nNOS TG+/dko muscles. This important observation shows that sarcolemmal localization is not necessary for nNOS to have beneficial effects in dystrophic tissue and the presence of nNOS in the cytosol of dystrophic muscle fibers can ameliorate the pathology and most importantly, significantly increase life-span

Arginine Metabolism by Macrophages Promotes Cardiac and Muscle Fibrosis in mdx Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is the most common, lethal disease of childhood. One of 3500 new-born males suffers from this universally-lethal disease. Other than the use of corticosteroids, little is available to affect the relentless progress of the disease, leading many families to use dietary supplements in hopes of reducing the progression or severity of muscle wasting. Arginine is commonly used as a dietary supplement and its use has been reported to have beneficial effects following short-term administration to mdx mice, a genetic model of DMD. However, the long-term effects of arginine supplementation are unknown. This lack of knowledge about the long-term effects of increased arginine metabolism is important because elevated arginine metabolism can increase tissue fibrosis, and increased fibrosis of skeletal muscles and the heart is an important and potentially life-threatening feature of DMD.We use both genetic and nutritional manipulations to test whether changes in arginase metabolism promote fibrosis and increase pathology in mdx mice. Our findings show that fibrotic lesions in mdx muscle are enriched with arginase-2-expressing macrophages and that muscle macrophages stimulated with cytokines that activate the M2 phenotype show elevated arginase activity and expression. We generated a line of arginase-2-null mutant mdx mice and found that the mutation reduced fibrosis in muscles of 18-month-old mdx mice, and reduced kyphosis that is attributable to muscle fibrosis. We also observed that dietary supplementation with arginine for 17-months increased mdx muscle fibrosis. In contrast, arginine-2 mutation did not reduce cardiac fibrosis or affect cardiac function assessed by echocardiography, although 17-months of dietary supplementation with arginine increased cardiac fibrosis. Long-term arginine treatments did not decrease matrix metalloproteinase-2 or -9 or increase the expression of utrophin, which have been reported as beneficial effects of short-term treatments.Our findings demonstrate that arginine metabolism by arginase promotes fibrosis of muscle in muscular dystrophy and contributes to kyphosis. Our findings also show that long-term, dietary supplementation with arginine exacerbates fibrosis of dystrophic heart and muscles. Thus, commonly-practiced dietary supplementation with arginine by DMD patients has potential risk for increasing pathology when performed for long periods, despite reports of benefits acquired with short-term supplementation

Antiepileptic drugs’ tolerability and safety – a systematic review and meta-analysis of adverse effects in dogs

<p>Various anti-epileptic drugs (AEDs) are used for the management of idiopathic epilepsy (IE) in dogs. Their safety profile is an important consideration for regulatory bodies, owners and prescribing clinicians. However, information on their adverse effects still remains limited with most of it derived from non-blinded non-randomized uncontrolled trials and case reports.</p><p><span>This poster won third place, which was presented at the Veterinary Evidence Today conference, Edinburgh November 1-3, 2016. </span></p><br /> <img src="https://www.veterinaryevidence.org/rcvskmod/icons/oa-icon.jpg" alt="Open Access" /

A Deletion in the N-Myc Downstream Regulated Gene 1 (NDRG1) Gene in Greyhounds with Polyneuropathy

The polyneuropathy of juvenile Greyhound show dogs shows clinical similarities to the genetically heterogeneous Charcot-Marie-Tooth (CMT) disease in humans. The pedigrees containing affected dogs suggest monogenic autosomal recessive inheritance and all affected dogs trace back to a single male. Here, we studied the neuropathology of this disease and identified a candidate causative mutation. Peripheral nerve biopsies from affected dogs were examined using semi-thin histology, nerve fibre teasing and electron microscopy. A severe chronic progressive mixed polyneuropathy was observed. Seven affected and 17 related control dogs were genotyped on the 50k canine SNP chip. This allowed us to localize the causative mutation to a 19.5 Mb interval on chromosome 13 by homozygosity mapping. The NDRG1 gene is located within this interval and NDRG1 mutations have been shown to cause hereditary motor and sensory neuropathy-Lom in humans (CMT4D). Therefore, we considered NDRG1 a positional and functional candidate gene and performed mutation analysis in affected and control Greyhounds. A 10 bp deletion in canine NDRG1 exon 15 (c.1080_1089delTCGCCTGGAC) was perfectly associated with the polyneuropathy phenotype of Greyhound show dogs. The deletion causes a frame shift (p.Arg361SerfsX60) which alters several amino acids before a stop codon is encountered. A reduced level of NDRG1 transcript could be detected by RT-PCR. Western blot analysis demonstrated an absence of NDRG1 protein in peripheral nerve biopsy of an affected Greyhound. We thus have identified a candidate causative mutation for polyneuropathy in Greyhounds and identified the first genetically characterized canine CMT model which offers an opportunity to gain further insights into the pathobiology and therapy of human NDRG1 associated CMT disease. Selection against this mutation can now be used to eliminate polyneuropathy from Greyhound show dogs

IRES-Mediated Translation of Utrophin A Is Enhanced by Glucocorticoid Treatment in Skeletal Muscle Cells

Glucocorticoids are currently the only drug treatment recognized to benefit Duchenne muscular dystrophy (DMD) patients. The nature of the mechanisms underlying the beneficial effects remains incompletely understood but may involve an increase in the expression of utrophin. Here, we show that treatment of myotubes with 6α−methylprednisolone-21 sodium succinate (PDN) results in enhanced expression of utrophin A without concomitant increases in mRNA levels thereby suggesting that translational regulation contributes to the increase. In agreement with this, we show that PDN treatment of cells transfected with monocistronic reporter constructs harbouring the utrophin A 5′UTR, causes an increase in reporter protein expression while leaving levels of reporter mRNAs unchanged. Using bicistronic reporter assays, we further demonstrate that PDN enhances activity of an Internal Ribosome Entry Site (IRES) located within the utrophin A 5′UTR. Analysis of polysomes demonstrate that PDN causes an overall reduction in polysome-associated mRNAs indicating that global translation rates are depressed under these conditions. Importantly, PDN causes an increase in the polysome association of endogenous utrophin A mRNAs and reporter mRNAs harbouring the utrophin A 5′UTR. Additional experiments identified a distinct region within the utrophin A 5′UTR that contains the inducible IRES activity. Together, these studies demonstrate that a translational regulatory mechanism involving increased IRES activation mediates, at least partially, the enhanced expression of utrophin A in muscle cells treated with glucocorticoids. Targeting the utrophin A IRES may thus offer an important and novel therapeutic avenue for developing drugs appropriate for DMD patients

Asthmatics Exhibit Altered Oxylipin Profiles Compared to Healthy Individuals after Subway Air Exposure

Asthma is a chronic inflammatory lung disease that causes significant morbidity and mortality worldwide. Air pollutants such as particulate matter (PM) and oxidants are important factors in causing exacerbations in asthmatics, and the source and composition of pollutants greatly affects pathological implications.This randomized crossover study investigated responses of the respiratory system to Stockholm subway air in asthmatics and healthy individuals. Eicosanoids and other oxylipins were quantified in the distal lung to provide a measure of shifts in lipid mediators in association with exposure to subway air relative to ambient air.Sixty-four oxylipins representing the cyclooxygenase (COX), lipoxygenase (LOX) and cytochrome P450 (CYP) metabolic pathways were screened using liquid chromatography-tandem mass spectrometry (LC-MS/MS) of bronchoalveolar lavage (BAL)-fluid. Validations through immunocytochemistry staining of BAL-cells were performed for 15-LOX-1, COX-1, COX-2 and peroxisome proliferator-activated receptor gamma (PPARγ). Multivariate statistics were employed to interrogate acquired oxylipin and immunocytochemistry data in combination with patient clinical information.Asthmatics and healthy individuals exhibited divergent oxylipin profiles following exposure to ambient and subway air. Significant changes were observed in 8 metabolites of linoleic- and α-linolenic acid synthesized via the 15-LOX pathway, and of the COX product prostaglandin E(2) (PGE(2)). Oxylipin levels were increased in healthy individuals following exposure to subway air, whereas asthmatics evidenced decreases or no change.Several of the altered oxylipins have known or suspected bronchoprotective or anti-inflammatory effects, suggesting a possible reduced anti-inflammatory response in asthmatics following exposure to subway air. These observations may have ramifications for sensitive subpopulations in urban areas