Necrotic Muscle Research Articles

First Evaluation of Radioiodinated Flavonoids as Necrosis-Avid Agents and Application in Early Assessment of Tumor Necrosis.

A rapid and accurate identification of necrotic tissues is of great importance to define disease severity, predict prognosis, and monitor responses to therapies. To seek necrosis-avid agents with clinically translational potential, we first evaluated the necrosis avidity of flavonoids in rodent models of muscular, myocardial, and tumoral necrosis. In this study, the necrosis avidity of eight radioiodinated 5,7-dihydroxyflavones was tested by ex vivo gamma counting, histochemical staining, and autoradiography in mouse models of ethanol-induced muscular necrosis. The necrosis avidity of a lead tracer, 131I-5, was further assessed in rat models of myocardial infarction and reperfusion. Therapy response was evaluated by 131I-5 single photon emission computed tomography/computed tomography imaging 24 h after combretastatin A-4 disodium phosphate (CA4P) therapy on rats bearing W256 breast carcinomas. The necrosis avidity mechanism for the tracers was studied by in vitro DNA binding experiments of 12 5,7-dihydroxyflavones and in vivo blocking experiments of 131I-5. In the results, all 131I-5,7-dihydroxyflavones showed intense uptake to necrotic muscles, and 131I-5 emerged as the most potential tracer among them. 131I-5 obtained a necrotic-viable myocardium ratio of 5.0 ± 0.9 in post-mortem biodistribution on reperfused myocardial infarction models and achieved necrosis imaging on CA4P-treated W256 tumors 4 h after tracer injection. DNA binding studies suggested that necrosis avidity was related to DNA binding to a certain extent. The uptake of 131I-5 in necrotic muscle was markedly blocked by excessive ethidium bromide and cold 5 with a 51.95% and 64.29% decline at 1 h after coinjection, respectively. In conclusion, flavonoids are necrosis-avid agents. Furthermore, 131I-5 can serve as a promising necrosis-avid diagnostic tracer for the rapid imaging of necrotic tissues, supporting the further molecular design of radiotracer based on 5.

Clinical research of features of magnetic resonance imaging of high-voltage electrical burns in limbs at early stage

Clinical research of features of magnetic resonance imaging of high-voltage electrical burns in limbs at early stage

Bilateral muscle fiber and nerve influences by TNF-alpha in response to unilateral muscle overuse \u2013 studies on TNF receptor expressions

BackgroundTNF-alpha is suggested to be involved in muscle damage and muscle inflammation (myositis). In order to evaluate whether TNF-alpha is involved in the myositis that occurs in response to muscle overuse, the aim was to examine the expression patterns of TNF receptors in this condition.MethodsA rabbit muscle overuse model leading to myositis in the soleus muscle was used. The expression patterns of the two TNF receptors Tumor Necrosis Factor Receptor type 1 (TNFR1) and Tumor Necrosis Factor Receptor type 2 (TNFR2) were investigated. In situ hybridization and immunofluorescence were utilized. Immunostainings for desmin, NK-1R and CD31 were made in parallel.ResultsImmunoreactions (IR) for TNF receptors were clearly observed in white blood cells, fibroblasts and vessel walls, and most interestingly also in muscle fibers and nerve fascicles in the myositis muscles. There were very restricted reactions for these in the muscles of controls. The upregulation of TNF receptors was for all types of structures seen for both the experimental side and the contralateral nonexperimental side. TNF receptor expressing muscle fibers were present in myositis muscles. They can be related to attempts for reparation/regeneration, as evidenced from results of parallel stainings. Necrotic muscle fibers displayed TNFR1 mRNA and TNFR2 immunoreaction (IR) in the invading white blood cells. In myositis muscles, TNFR1 IR was observed in both axons and Schwann cells while TNFR2 IR was observed in Schwann cells. Such observations were very rarely made for control animals.ConclusionsThe findings suggest that there is a pronounced involvement of TNF-alpha in the developing myositis process. Attempts for reparation of the muscle tissue seem to occur via both TNFR1 and TNFR2. As the myositis process also occurs in the nonexperimental side and as TNF receptors are confined to nerve fascicles bilaterally it can be asked whether TNF-alpha is involved in the spreading of the myositis process to the contralateral side via the nervous system. Taken together, the study shows that TNF-alpha is not only associated with the inflammation process but that both the muscular and nervous systems are affected and that this occurs both on experimental and nonexperimental sides.

Irreversible electroporation of the uterine cervix in a rabbit model.

The aim of this study was to evaluate the effects of irreversible electroporation (IRE) on the uterine cervix in a rabbit model. IRE ablation was performed in the cervices of 48 New Zealand rabbits, with one ablation lesion in each animal. Gross pathology, transmission electron microscopy, hematoxylin and eosin (H&E), Masson's trichrome (MT) stain, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays were performed at pre-set time points (0h, 12h, 1 d, 2 d, 4 d, 7 d, 14 d, and 28 d post-IRE). All the rabbits tolerated the IRE ablation without serious complications. IRE caused complete cell death of the ablated cervix via cell apoptosis. However, fast recovery of the cervix was observed from 7 d post-IRE, with the signs of collagen fibers hyperplasia, the disappearance of the necrotic cells and muscle fibers, and regeneration and extension of the cervical epithelium. At 28 d post-IRE, the ablated cervices recovered to almost normal. Our study suggested that IRE might be an efficient and safe technology to treat cervical tumors, without causing serious cervical damage.

131I-Evans blue: evaluation of necrosis targeting property and preliminary assessment of the mechanism in animal models

Necrosis is a form of cell death, which is related to various serious diseases such as cardiovascular disease, cancer, and neurodegeneration. Necrosis-avid agents (NAAs) selectively accumulated in the necrotic tissues can be used for imaging and/or therapy of related diseases. The aim of this study was to preliminarily investigate necrosis avidity of 131I-evans blue (131I-EB) and its mechanism. The biodistribution of 131I-EB at 24h after intravenous administration showed that the radioactivity ratio of necrotic to viable tissue was 3.41 in the liver and 11.82 in the muscle as determined by γ counting in model rats. Autoradiography and histological staining displayed preferential uptake of 131I-EB in necrotic tissues. In vitro nuclear extracts from necrotic cells exhibited 82.3% of the uptake in nuclei at 15min, as well as 79.2% of the uptake at 2h after 131I-EB incubation. The DNA binding study demonstrated that evans blue (EB) has strong binding affinity with calf-thymus DNA (CT-DNA) (Ksv=5.08×105L/(mol/L)). Furthermore, the accumulation of 131I-EB in necrotic muscle was efficiently blocked by an excess amount of unlabeled EB. In conclusion, 131I-EB can not only detect necrosis by binding the DNA released from necrotic cells, but also image necrotic tissues generated from the disease clinically.

A true silent compartment syndrome in a competent sensate patient: Case report

A true silent compartment syndrome in a competent sensate patient: Case report

Hierarchical signaling transduction of the immune and muscle cell crosstalk in muscle regeneration

The muscle regeneration is a complicated bioprocess that involved in many cell types, including necrotic muscle cells, satellite cells, mesenchymal cells, pericytes, immune cells, and other cell types present at the injury site. Immune cells involved in both innate and adaptive immune responses regulate the progress of muscle regeneration. In this review, we discussed the roles of different immune cells in muscle regeneration. The immune cells regulate muscle regeneration through cytokine production, cell-cell contacts, and general immune environment regulation. We also describe the current known mechanism of how immune cells regulating muscle regeneration.

Platelet-Derived Growth Factor BB Influences Muscle Regeneration in Duchenne Muscle Dystrophy

Duchenne muscular dystrophy (DMD) is characterized by a progressive loss of muscle fibers, and their substitution by fibrotic and adipose tissue. Many factors contribute to this process, but the molecular pathways related to regeneration and degeneration of muscle are not completely known. Platelet-derived growth factor (PDGF)-BB belongs to a family of growth factors that regulate proliferation, migration, and differentiation of mesenchymal cells. The role of PDGF-BB in muscle regeneration in humans has not been studied. We analyzed the expression of PDGF-BB in muscle biopsy samples from controls and patients with DMD. We performed invitro experiments to understand the effects of PDGF-BB on myoblasts involved in the pathophysiology of muscular dystrophies and confirmed our results invivo by treating the mdx murine model of DMD with repeated i.m. injections of PDGF-BB. We observed that regenerating and necrotic muscle fibers in muscle biopsy samples from DMD patients expressed PDGF-BB. Invitro, PDGF-BB attracted myoblasts and activated their proliferation. Analysis of muscles from the animals treated with PDGF-BB showed an increased population of satellite cells and an increase in the number of regenerative fibers, with a reduction in inflammatory infiltrates, compared with those in vehicle-treated mice. Based on our results, PDGF-BB may play a protective role in muscular dystrophies by enhancing muscle regeneration through activation of satellite cell proliferation and migration.

Novel mutation (c.8725T>C) in two siblings with late-onset LAMA2-related muscular dystrophy

Objective: LAMA2 mutation-related muscular dystrophy is classified into two clinical phenotypes: severe early-onset congenital muscular dystrophy and mild late- onset muscular dystrophy. According to several reported cases, late-onset LAMA2-related muscular dystrophy clinically shows the limb girdle muscular dystrophy pattern and has been considered to be associated with the partial expression of laminin α2. Methods: We describe here a patient initially suspected of having limb girdle muscular dystrophy, but was later diagnosed with late-onset LAMA2-related muscular dystrophy. Results: We found compound heterozygous variations on the patient's LAMA2 gene, a heterozygous nonsense variation in exon 49 (c.6955C>T/p.Arg2319*), previously reported as a pathogenic variation, and a heterozygous missense variation in exon 62 (c.8725T>C/p.Cys2909Arg), which is a novel variation. Her and her sister's brain magnetic resonance images (MRI) revealed diffuse symmetric high signal unmyelinated changes in bilateral cerebral white matter. Muscle tissue sections were stained with hematoxylin and eosin, and showed mild muscle fiber size variation, a few necrotic and regenerative muscle fibers, and endomysial edema Immunohistochemical staining for laminin α2 showed a reduction compared with the control. Conclusion: We suggest that this novel missense mutation is the likely pathogenic mechanism through the genotype–phenotype correlation and the annotation process.

Integrated Diagnosis Project for Inflammatory Myopathies: An association between autoantibodies and muscle pathology.

Inflammatory myopathies are a heterogeneous group of immune-mediated diseases that involve skeletal muscle as well as many other organs. The classification of inflammatory myopathies has been based on clinical diagnoses, pathological diagnoses, and autoantibodies, independently. The clinical phenotypes of inflammatory myopathies are characterized by various autoantibodies that are originally detected by RNA or protein immunoprecipitation. However, since the correlation between histological features and autoantibodies had not been fully elucidated, we created the "Integrated Diagnosis Project for Inflammatory Myopathies" in October 2010. Based on our work and previous studies, the three major subsets of inflammatory myopathies defined by autoantibodies are immune-mediated necrotizing myopathy (IMNM), antisynthetase syndrome, and dermatomyositis. IMNM is the pathological entity, characterized by significant necrotic and regeneration muscle fibers with minimal or no inflammatory cell infiltration. The detection of autoantibodies against signal recognition particles or 3-hydroxy-3-methylglutaryl-coenzyme A reductase is important for the diagnosis of IMNM. Antisynthetase syndrome, characterized by myositis, interstitial lung disease, skin rash, arthropathy, and Raynaud phenomenon, is the clinical entity based on the presence of aminoacyl transfer RNA synthetase antibodies. Perifascicular necrosis is a distinctive hallmark of antisynthetase syndrome in muscle pathology. The diagnosis of dermatomyositis is usually based on clinical features of typical skin rash. Several autoantibodies are associated with specific subsets of dermatomyositis. Myxovirus resistance A expression in the myofiber cytoplasm has a better sensitivity for the diagnosis of dermatomyositis compared to perifascicular atrophy. The screening of autoantibodies has clinical relevance for managing patients with inflammatory myopathies.

Evaluation of Radioiodinated 1,4-Naphthoquinones as Necrosis Avid Agents for Rapid Myocardium Necrosis Imaging

Identifying necrotic myocardium in ischemic regions is of great importance for risk stratification and clinical decision-making. However, rapid noninvasive imaging of necrotic myocardium is still challenging. This study sought to evaluate the potential of 1,4-naphthoquinones to rapidly visualize necrotic myocardium and the possible mechanisms of necrosis avidity. Six 1,4-naphthoquinones were radiolabeled with iodine-131 and the necrosis avidity was estimated in mouse models with muscular necrosis by gamma counting and autoradiography. The necrotic myocardium imaging property and biodistribution of [131I]naphthazarin (6) were determined in rat models with re-perfused myocardial infarction. A possible mechanism of necrosis avidity was explored by in vitro DNA-binding and in vivo blocking experiments. The radiochemical purities of the six radiotracers were greater than 95%. The uptakes in necrotic muscles of all six radiotracers were higher than those in viable muscles, and [131I]naphthazarin (6) showed the highest necrotic-to-viable ratio and necrosis-to-blood ratio at all tested time points. The necrotic myocardium could be clearly visualized by single-photon emission computed tomography/x-ray computed tomography (SPECT/CT) using [131I]naphthazarin (6) as early as 3h post-injection. Post-mortem biodistribution showed the uptake of [131I]naphthazarin (6) in necrotic myocardium was 11.67-fold higher than that in viable myocardium. Absorption spectra and emission spectra suggested naphthazarin (6) could bind to DNA through intercalation. The uptake of [131I]naphthazarin (6) in necrotic muscle could be significantly blocked by excessive ethidium bromide (a typical DNA intercalator) and cold naphthazarin (6) with 63.49 and 71.96% decline at 3h post-injection in vivo, respectively. 1,4-Naphthoquinones retained necrosis avidity and [131I]naphthazarin (6) rapidly visualized necrotic myocardium. The necrosis avidity mechanism of [131I]naphthazarin (6) may be attributed to its binding with exposed DNA in necrotic tissues.

Zoonotic necrotizing myositis caused by Streptococcus equi subsp. zooepidemicus in a farmer

BackgroundStreptococcus equi subsp. zooepidemicus is a beta-hemolytic group C streptococcus mainly causing infections in domesticated animals. Here we describe the first case of zoonotic necrotizing myositis caused by this bacterium.Case presentationThe patient was a 73-year-old, previously healthy farmer with two asymptomatic Shetland ponies in his stable. After close contact with the ponies while feeding them, he rapidly developed erythema of his left thigh and sepsis with multiple organ failure. The clinical course was severe and complicated, requiring repetitive surgical excision of necrotic muscle, treatment with vasopressors, mechanical ventilation and continuous venovenous hemofiltration, along with adjunctive hyperbaric oxygen therapy. The patient was discharged from hospital at day 30, without obvious sequelae.The streptococcal isolate was identified as Streptococcus equi by MALDI-ToF MS, and was later assigned subspecies identification as S. equi subsp. zooepidemicus. Multilocus sequence typing identified the strain as a novel sequence type (ST 364), closely related to types previously identified in horses and cattle. A focused proteomic analysis revealed that the ST 364 expressed putative virulence factors similar to that of Streptococcus pyogenes, including homologues of the M protein, streptodornases, interleukin 8-protease and proteins involved in the biosynthesis of streptolysin S.ConclusionThis case illustrates the zoonotic potential of S. equi subsp. zooepidemicus and the importance of early clinical recognition, rapid and radical surgical therapy, appropriate antibiotics and adequate supportive measures when necrotizing soft tissue infection is suspected. The expression of Streptococcus pyogenes-like putative virulence determinants in ST 364 might partially explain the fulminant clinical picture.

Recovery of Vibrio harveyi from scale drop and muscle necrosis disease in farmed barramundi, Lates calcarifer in Vietnam

Symptoms of scale drop and muscle necrosis have been considered as an emerging problem in farmed barramundi (Lates calcarifer) in Vietnam since 2013. Naturally diseased fish exhibited remarkable external clinical signs of scale loss, muscle degradation and eventually died. The objective of this study was to determine the infectious causative agent of the clinically diseased fish collected from barramundi caged culture in central Vietnam in 2015. Histological examination from naturally sick fish revealed signs of severe necrotic muscles with infiltration of massive immune-related cells, severe hemorrhage and blood congestion in the brain, collapsed kidney tubules and epithelial cells sloughing into the lumen. Five different bacterial species were recovered from diseased fish and putatively identified as Vibrio harveyi, Vibrio tubiashii, Tenacibaculum litopenaei, Tenacibaculum sp. and Cytophaga sp. based on homology of 16S rDNA sequences and biochemical characteristics. Experimental infection revealed that only V. harveyi killed the fish with similar clinical signs and histological changes compared to naturally diseased fish. Additionally, several unculturable bacteria including T. maritimum were also uncovered from DNA extracted from necrotic muscles by species-specific PCR and 16S rDNA clone library sequencing, but their roles in disease manifestation need further investigation.

Molecular Recognition Mechanism of Hematopoietic Prostaglandin D Synthase with its Cofactor and Substrate

Hematopoietic prostaglandin (PG) D synthase (H-PGDS) is a Sigma class member of the glutathione S-transferase (GST) superfamily, and requires glutathione (GSH) as a cofactor. H-PGDS catalyzes the isomerization of prostaglandin H2 (PGH2) to prostaglandin D2 (PGD2), which acts as an allergic and inflammatory mediator. The overproduction of PGD2 by H-PGDS causes allergic and inflammatory reactions in the necrotic muscle fibers of Duchenne muscular dystrophy (DMD) patients. Therefore, H-PGDS inhibitors are thought to have potential for use in drug therapy for DMD.The catalytic mechanism of H-PGDS remains unclear, since essential information, such as the binding affinity and stoichiometry of GSH and PGH2 to H-PGDS, have not been precisely determined yet. Therefore, to clarify the issues related to the cofactor and substrate recognition of H-PGDS, we investigated the interaction between H-PGDS and PGH2 in the presence and absence of GSH.For this purpose, isothermal titration calorimetry (ITC) was performed using a substrate mimetics U-44069 in the place of PGH2 since PGH2 is quite unstable in most solutions. Recombinant H-PGDS was prepared using an E. coli expression system and used for the ITC measurements. The results showed that U-44069 binds to H-PGDS in the presence of the cofactor, GSH, but the binding was not significant in the absence of GSH. Therefore, these results suggest that the GSH binding promotes the interaction between H-PGDS and PGH2, and that GSH plays an important role, not only for the catalytic reaction but also for substrate binding.

Effects of Chitosan and Hyaluronic Acid in Healing of Chemically Induced Oral Ulcer in Rabbits

Twelve healthy, local bread male rabbits were used in this study, weighting (1.5-2) kg , divided equally into three groups: in First group (Control group), the ulcers were induced by local application of Sulfuric Acid in concentration 38% in rabbit cheek mucosa ,animals were left without treatment. In second group, same procedure was performed then 0.03 gm of chitosan raw was applied locally on ulcer site twice daily at 1st, 2nd, and 3rd day post induce ulcer while animal under general anesthesia. In third group, same procedure was performed, then saturated gauze with 5 drop of hyfresh drug contain hyalronic acid 2.0 mg\ml were applied locally on ulcer site twice daily at 1st, 2nd, and 3rd day post induces ulcer while animal under general anesthesia.Grossly finding at 3rd day shows limited area of necrotic mucus membrane (m.m) with presence of sluphing area in the second group, while in the control and third groups at same period showed presence of necrotic area of (m.m) surrounded by elevated congestion (m.m), as well as at day 7 evidence regression of necrotic area with definite re-epithelization area in the second group, while in the third group showed advance in regression of necrotic area when compared with earlier period and beginning of re-epithelization, in control group record decreased in necrotized area surrounded by congested area and also small ulcer area was found. Histopathological results in control group at 3rd day showed inflammatory cells particularly neutrophils infiltration between necrotic muscle fibers with hemorrhage in subepithelial layer while in the second group showed granulation tissue consisting from congested blood vessels and marked fibroblast proliferation with collagen fiber deposition, but in the third group showed presence of inflammatory cells particularly neutrophils and macrophages between necrotic muscle fiber. At day 7 in control group showed severe inflammatory cells particularly neutrophils infiltration between marked necrotic muscle fibers, while In the second group at the same period showed thickness collagen fibers with macrophages and lymphocytes infiltration with congested blood vessels, in third group showed inflammatory cells with congested blood vessels, edema, necrotic debris with severe neutrophils infiltration between marked necrotic muscle fiber.

Radiolabeled Rhein as Small-Molecule Necrosis Avid Agents for Imaging of Necrotic Myocardium.

A rapid and accurate identification of necrotic myocardium is of great importance for diagnosis, risk stratification, clinical decision-making, and prognosis evaluation of myocardial infarction. Here, we explored technetium-99m labeled rhein derivatives for rapid imaging of the necrotic myocardium. Three hydrazinonicotinic acid-linker-rhein (HYNIC-linker-rhein) derivatives were synthesized, and then, these synthetic compounds were labeled with technetium-99m using ethylenediaminediacetic acid (EDDA) and tricine as coligands [99mTc(EDDA)-HYNIC-linker-rhein]. The necrosis avidity of the three 99mTc-labeled rhein derivatives was tested in a mouse model of ethanol-induced muscular necrosis by gamma counting, histochemical staining, and autoradiography. A lead tracer for visualization of necrotic myocardium was assessed by single photon emission computed tomography/computed tomography (SPECT/CT) imaging in a rat model with reperfused myocardial infarction. The necrosis avidity mechanism of the tracer was explored by DNA binding studies in vitro and blocking experiments in vivo. Results showed that the uptake in necrotic muscles of the three 99mTc-compounds was higher than that in viable muscles (P < 0.001). Autoradiography and histochemical staining results were consistent with selective uptake of the radiotracer in the necrotic regions. Among the these tracers, 99mTc(EDDA)-HYNIC-ethylenediamine-rhein [99mTc(EDDA)-HYNIC-2C-rhein] displayed the best distribution profiles for imaging. The necrotic myocardium lesions were clearly visualized by SPECT/CT using 99mTc(EDDA)-HYNIC-2C-rhein at 1 h after injection. The necrotic-to-viable myocardium and necrotic myocardium-to-blood uptake ratios of 99mTc(EDDA)-HYNIC-2C-rhein were 4.79 and 3.02 at 1 h after injection. DNA binding studies suggested HYNIC-linker-rhein bound to DNA through intercalation. The uptake of 99mTc(EDDA)-HYNIC-2C-rhein in necrotic muscle was significantly blocked by excessive unlabeled rhein, with 77.61% decline at 1 h after coinjection. These findings suggested 99mTc(EDDA)-HYNIC-2C-rhein emerged as a "hot spot" imaging probe that has a potential for rapid imaging of necrotic myocardium. The necrosis avidity mechanism of 99mTc(EDDA)-HYNIC-linker-rhein may be due to its interaction with exposed DNA in necrotic tissues.

Autoantibodies of Inflammatory Myopathies: Update

Inflammatory myopathies are a heterogeneous group of immune-mediated diseases that involve the skeletal muscle as well as many other organs. In addition to a histological diagnosis at muscle biopsy, the clinical phenotypes of inflammatory myopathies can be defined by the presence of various autoantibodies that are originally detected by RNA or protein immunoprecipitation. However, the correlation between histological features and autoantibodies has not been fully elucidated. Immune-mediated necrotizing myopathy (IMNM), which is characterized by significant necrotic and regeneration muscle fibers with minimal or no inflammatory cell infiltration, is associated with the presence of autoantibodies. IMNM is now classified as a distinct category of inflammatory myopathies, separate from polymyositis, dermatomyositis, and sporadic inclusion body myositis. Here, we divided the autoantibodies of inflammatory myopathies into the following categories: those associated with IMNM, those with activity against aminoacyl transfer RNA synthetase, those associated with dermatomyositis, and those related to other disorders, including overlap syndrome, inclusion body myositis, and primary biliary cirrhosis. The detection of autoantibodies against signal recognition particle or 3-hydroxy-3-methylglutaryl-coenzyme A reductase is useful for the diagnosis of IMNM. The screening of autoantibodies has clinical relevance for managing patients with inflammatory myopathies.

Histopathological changes and biochemical analysis of sulfadiazine injected in egg in chicken embryo pectoral muscles

This study aimed to investigate the effects of different doses of sulfadiazine on embryonic chicken pectoral muscles. In total, 100 fertile eggs were obtained and divided into five groups of control (no injection) and sulfadiazine injection at doses of 2, 10, 30, and 70 mg/kg. After hatching, pectoral muscle tissues were harvested from the newly hatched chickens for histopathological examination and measurement of oxidative stress parameters. Microscopic examination of pectoral muscle samples indicated that sulfadiazine administration changed the histopathological structure of chicken pectoral muscles only at very high doses (30 and 70 mg/kg). Major histopathologic events associated with sulfadiazine cytotoxicity were multifocal degeneration, necrotic tissue changes, and inflammatory cell infiltration (predominantly mononuclear cells) around degenerated and necrotic muscle fibers. Moreover, sulfadiazine at doses of 10, 30 and 70 mg/kg increased malondialdehyde level and decreased glutathione, ferric reducing antioxidant power, and total carotenoid, which indicated oxidative damage in broiler skeletal muscles. Therefore, it could be concluded that in-egg administration of up to 10 mg/kg of sulfadiazine is safe for chicken embryo, whereas dosage of 30 mg/kg (or above) is considered highly toxic.

The ubiquitin ligase tripartite-motif-protein 32 is induced in Duchenne muscular dystrophy

Activation of the proteasome pathway is one of the secondary processes of cell damage, which ultimately lead to muscle degeneration and necrosis in Duchenne muscular dystrophy (DMD). In mdx mice, the proteasome inhibitor bortezomib up-regulates the membrane expression of members of the dystrophin complex and reduces the inflammatory reaction. However, chronic inhibition of the 26S proteasome may be toxic, as indicated by the systemic side-effects caused by this drug. Therefore, we sought to determine the components of the ubiquitin-proteasome pathway that are specifically activated in human dystrophin-deficient muscles. The analysis of a cohort of patients with genetically determined DMD or Becker muscular dystrophy (BMD) unveiled a selective up-regulation of the ubiquitin ligase tripartite motif-containing protein 32 (TRIM32). The induction of TRIM32 was due to a transcriptional effect and it correlated with disease severity in BMD patients. In contrast, atrogin1 and muscle RING-finger protein-1 (MuRF-1), which are strongly increased in distinct types of muscular atrophy, were not affected by the DMD dystrophic process. Knock-out models showed that TRIM32 is involved in ubiquitination of muscle cytoskeletal proteins as well as of protein inhibitor of activated STAT protein gamma (Piasγ) and N-myc downstream-regulated gene, two inhibitors of satellite cell proliferation and differentiation. Accordingly, we showed that in DMD/BMD muscle tissue, TRIM32 induction was more pronounced in regenerating myofibers rather than in necrotic muscle cells, thus pointing out a role of this protein in the regulation of human myoblast cell fate. This finding highlights TRIM32 as a possible therapeutic target to favor skeletal muscle regeneration in DMD patients.

Microvascular alterations and the role of complement in dermatomyositis.

Different mechanisms have been proposed to explain the pathological basis of perifascicular muscle fibre atrophy in dermatomyositis. These include ischaemia due to immune-mediated microvascular injury, enhanced expression of type 1 interferon-induced gene transcripts in perifascicular capillaries and muscle fibres, and occlusion of larger perimysial blood vessels. Microvascular complement deposition is a feature of dermatomyositis pathology but the trigger for complement activation, the predominant complement pathway involved, or its role in the pathogenesis of the disease, has not been clearly defined. In the first step of this study we examined the density of capillaries and transverse vessels and searched for occlusion or depletion of larger perimysial blood vessels in 10 patients with dermatomyositis. This revealed an invariable association of perifascicular atrophy with capillary and transverse vessel depletion. The capillary and transverse vessel densities in non-atrophic fibre regions were not significantly different from those in muscle specimens of 10 age-matched controls. Next, in the same 10, as well as in 40 additional dermatomyositis patients, we searched for vascular deposits of IgG, IgM, and the C5b-9 complement membrane attack complex. Thirty-one of 50 dermatomyositis specimens contained C5b-9 reactive endomysial microvessels but none of these or other vessels reacted for IgG. Ten of 50 specimens harboured IgM-positive capillaries but only a few of these reacted for C5b-9. Finally, we analysed and compared different pathways of complement activation in dermatomyositis, lupus nephritis, and necrotic muscle fibres in Duchenne dystrophy. In lupus nephritis, C5-b9 deposits co-localized with IgG, IgM, C1q, and C4d, consistent with immune complex dependent activation of the classical complement pathway. In both dermatomyositis and Duchenne dystrophy, C5-b9 deposits co-localized with C1q and C4d and rarely with IgM indicating activation of the classical complement pathway. We conclude that: perifascicular atrophy in dermatomyositis is consistently associated with focal microvascular depletion, and that microvascular membrane attack complex deposits in dermatomyositis result from activation of the classical complement pathway triggered by direct binding of C1q to injured endothelial cells.