Mitochondria are involved in bronchial smooth muscle remodeling in severe preschool wheezers.

BACKGROUND: Bronchial remodeling is a key feature of asthma that is already present in preschoolers with wheezing. Moreover, bronchial smooth muscle (BSM) remodeling at preschool age is predictive of asthma at school age. However, the mechanism responsible for BSM remodeling in preschoolers with wheezing remains totally unknown. In contrast, in adult asthma, BSM remodeling has been associated with an increase in BSM cell proliferation related to increased mitochondrial mass and biogenesis triggered by an altered calcium homeostasis. Indeed, BSM cell proliferation was decreased in vitro by the calcium channel blocker gallopamil. OBJECTIVE: Our aim was to investigate the mechanisms involved in BSM cell proliferation in preschoolers with severe wheezing, with special attention to the role of mitochondria and calcium signaling. METHODS: Bronchial tissue samples obtained from 12 preschool controls without wheezing and 10 preschoolers with severe wheezing were used to measure BSM mass and establish primary BSM cell cultures. BSM cell proliferation was assessed by manual counting and flow cytometry, ATP content was assessed by bioluminescence, mitochondrial respiration was assessed by using either the Seahorse or Oroboros technique, mitochondrial mass and biogenesis were assessed by immunoblotting, and calcium response to carbachol was assessed by confocal microscopy. The effect of gallopamil was also evaluated. RESULTS: BSM mass, cell proliferation, ATP content, mitochondrial respiration, mass and biogenesis, and calcium response were all increased in preschoolers with severe wheezing compared with in the controls. Gallopamil significantly decreased BSM mitochondrial biogenesis and mass, as well as cell proliferation. CONCLUSION: Mitochondria are key players in BSM cell proliferation in preschoolers with severe wheezing and could represent a potential target to treat BSM remodeling at an early stage of the disease.

Thigh muscle MRI findings in myopathy associated with anti-mitochondrial antibody.

INTRODUCTION: Myopathy associated with anti-mitochondrial antibody (AMA) has recently been characterized as a distinct type of idiopathic inflammatory myopathy. The purpose of this study is to evaluate the pattern of involvement in thigh muscles in AMA myopathy using MRI. METHODS: Six patients with AMA myopathy were identified and their muscle MRI findings evaluated. RESULTS: On thigh muscle MRI, all six patients showed high signal intensity with short-tau inversion recovery that reflected disease activity mostly in the adductor magnus, called a "cuneiform sign." Fatty degeneration was also prominent in the adductor magnus, as well as the semimembranosus muscles. DISCUSSION: These characteristic changes on MRI contrast with those of other inflammatory myopathies. From these observations, we concluded that the localization pattern of the inflammatory changes in muscle MRI can contribute to the diagnosis of AMA myopathy.

The clinical and histopathological features of idiopathic inflammatory myopathies with asymmetric muscle involvement.

The objective is to determine the frequency of idiopathic inflammatory myopathies (IIMs) with asymmetric muscle involvement (IIMs-A) as initial manifestations in total IIMs and to compare the demographic, clinical, histopathological and radiological characteristics of IIMs-A with classical IIMs (IIMs-C). We retrospectively reviewed the clinical, laboratory, muscle images, histopathological features and treatment response of patients at the Qilu hospital who were diagnosed as IIMs from April 2005 to August 2017. We found among 134 IIMs patients, 13(9.2%) patients with IIMs-A were identified, of which 7 patients were diagnosed as dermatomyositis (DM), 2 as polymyositis (PM), 4 as immune-mediated necrotizing myopathy (IMNM) using the European Neuromuscular Centre (ENMC) criteria. The mean age of our group was 59.1 years old. The duration from the initial symptoms to the first examination was less than 12 months in 12 patients (92.3%). 46.2% patients accompanied with weakness of distal limbs and bulbar symptoms. Finger flexion involvement was found in 5 patients (38.5%). There was no patient that finger flexion was weaker than shoulder abduction. The creatine kinase (CK) level in the serum ranged from 41 IU/L to 9125 IU/L (average: 3192.7 ±â€¯2769.9 IU/L). Serum positive anti-mitochondrial antibodies (AMAs) were found in four patients (30.8%). Endomysial fibrosis and inflammatory cell infiltration were detected in 92.3%, 84.6% patients respectively. Mitochondrial abnormalities in histopathological finding of muscle biopsy were seen in 100% cases. The major histocompatibility complex class I (MHC-I) (84.6%) and class II (MHC-II) (92.3%) expressed on muscle biopsies from almost all cases of our patients. MAC antibody, however, was detected in only 20-40% patients. Eight patients (61.5%) had favorable outcomes. The conclusion was that IIMs-A presented mainly in DM, generally with mitochondrial abnormality and highly positive AMAs. The relationship between the presence of AMAs and the asymmetric muscle involvement in DM needs to be further clarified. We should also consider the diagnosis of IIMs when the patient has features of positive AMAs and asymmetric muscle involvement.

Effects of reactive oxygen species and interplay of antioxidants during physical exercise in skeletal muscles.

A large number of researches have led to a substantial growth of knowledge about exercise and oxidative stress. Initial investigations reported that physical exercise generates free radical-mediated damages to cells; however, in recent years, studies have shown that regular exercise can upregulate endogenous antioxidants and reduce oxidative damage. Yet, strenuous exercise perturbs the antioxidant system by increasing the reactive oxygen species (ROS) content. These alterations in the cellular environment seem to occur in an exercise type-dependent manner. The source of ROS generation during exercise is debatable, but now it is well established that both contracting and relaxing skeletal muscles generate reactive oxygen species and reactive nitrogen species. In particular, exercises of higher intensity and longer duration can cause oxidative damage to lipids, proteins, and nucleotides in myocytes. In this review, we summarize the ROS effects and interplay of antioxidants in skeletal muscle during physical exercise. Additionally, we discuss how ROS-mediated signaling influences physical exercise in antioxidant system.

Ly6G+ neutrophil-derived miR-223 inhibits the NLRP3 inflammasome in mitochondrial DAMP-induced acute lung injury.

MicroRNA (miRNA) mediates RNA interference to regulate a variety of innate immune processes, but how miRNAs coordinate the mechanisms underlying acute lung injury/acute respiratory distress syndrome (ALI/ARDS) in patients with pulmonary inflammatory injury is still unknown. In this study, we demonstrated that miR-223 limits the number of Ly6G+ neutrophils and inhibits the activity of the NLRP3 inflammasome to alleviate ALI induced by mitochondrial damage-associated molecular patterns (DAMPs) (MTDs). miR-223 expression is increased in the lungs of MTD-induced mice or ARDS patients following trauma/transfusion or following the physiological remission of ALI/ARDS. miR-223-/+ mice exhibited more severe ALI and cytokine dysregulation. Other studies have shown that MTD-induced increases in miR-223 expression are mainly contributed by Ly6G+ neutrophils from the haematopoietic system. miR-223 blocks bone marrow-derived Ly6G+ neutrophil differentiation and inhibits peripheral cytokine release. In addition, MTD-induced miR-223 expression activates a negative feedback pathway that targets the inhibition of NLRP3 expression and IL-1ß release; therefore, miR-223 deficiency can lead to the sustained activation of NLRP3-IL-1ß. Finally, elimination of peripheral Ly6G+ neutrophils and pharmacological blockade of the miR-223-NLRP3-IL-1ß signalling axis could alleviate MTD-induced ALI. In summary, miR-223 is essential for regulating the pathogenesis of DAMP-induced ALI.

Preventive effects of electrical stimulation on inflammation-induced muscle mitochondrial dysfunction.

Cachexia is a complex metabolic syndrome associated with underlying chronic diseases and is characterized by the overexpression of pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF-α), which impair muscle oxidative metabolism. We hypothesized that electrical stimulation (ES) would prevent decrement in muscle oxidative metabolism by suppressing the phosphorylation of p38 MAPK, a critical regulator of inflammatory response. Therefore, the purpose of the present study was to verify the effects of ES on inflammatory-induced decrement of oxidative metabolism in mice tibialis anterior muscles. ICR mice were randomly divided into three groups: control, lipopolysaccharide (LPS) injection for 4days, and LPS injection plus ES (LPS+ES). Cachexia was induced in the animals in the LPS groups via LPS injection (10mg/kg body weight/day, i.p.) during the intervention period. The animals in the LPS+ES group were stimulated electrically (carrier frequency, 2500Hz; modulation frequency, 100Hz; duration, 240s/day; type of contraction, isometric) during the intervention period. LPS injection resulted in decreased body and muscle wet weight and increased expression of TNF-α in plasma and skeletal muscle. In addition, LPS injection decreased indicators of mitochondrial function such as succinate dehydrogenase (SDH) and citrate synthase (CS) activity as well as the expression of PGC-1ɑ, and increased the phosphorylation of p38 MAPK. On the other hand, the intervention of ES attenuated the changes in muscle wet weight, SDH activity, CS activity, p38 MAPK, and PGC-1ɑ. These results suggest that ES could prevent decrement in muscle oxidative metabolism induced by pro-inflammatory cytokines in cachexia.

[Clinical and histopathological features of myositis associated with anti-mitochondrial antibodies].

Anti-mitochondrial antibodies (AMA) are known to be characteristic markers of primary biliary cirrhosis (PBC). The association of PBC with myositis has been reported mainly as case reports, and comprehensive studies of the clinical and histopathological features of patients with myositis and AMAs or PBC have not been conducted thus far. We retrospectively reviewed 212 patients with inflammatory myopathies in our laboratory and found 24 patients with AMA-positive myositis (11%) (seven patients with PBC and 17 patients without PBC). The analysis of clinical and histopathological features revealed that myositis associated with AMAs frequently include patients with a clinically chronic disease course, muscle atrophy, cardiopulmonary involvement and granulomatous inflammation, regardless of the presence or absence of PBC. We also reviewed and analyzed the clinical features of previously reported patients. The analysis of 75 patients, which have been described in previous case reports including the ones of meeting abstracts, also showed the similar results about clinical features of myositis associated with AMAs and supported our findings. Our study suggests that myositis associated with AMAs form a characteristic subgroup.

Neurophysiological and mitochondrial abnormalities in MuSK antibody seropositive myasthenia gravis compared to other immunological subtypes.

OBJECTIVE: To compare the electrophysiological and histopathological features of immunological myasthenia gravis (MG) subtypes. METHODS: Fifty MG patients underwent clinical examination, MuSK-Ab and AChR-Ab analysis. The majority underwent quantitative and single-fiber electromyography (QEMG, SFEMG), repetitive nerve stimulation and deltoid muscle biopsy. From muscle specimens with histological mitochondrial dysfunction, we amplified mitochondrial DNA (mtDNA). In specimens with mtDNA deletions, the nuclear gene POLG1 was sequenced. RESULTS: Five AChR-Ab seropositive [AChR(+)] and 5 seronegative [AChR(-)] patients were MuSK-Ab seropositive [MuSK(+)]. Five of 7 neurophysiologically examined MuSK(+) patients (71%) had proximal myopathic pattern, compared to 7 of 31 MuSK(-)/AChR(+) patients (23%) (P=0.012). SFEMG was abnormal in all examined MuSK(+) patients. All 7 biopsied MuSK(+) and 32 MuSK(-) patients (89%) had cytochrome c oxidase (COX) negative fibers. Three of five MuSK(+) and 13 of 20 MuSK(-) patients analyzed had multiple mtDNA deletions but no POLG1 mutations. CONCLUSIONS: Similar degree of SFEMG abnormalities was present in proximal muscles among MuSK(+) and AChR(+) patients. Proximal myopathy was over-represented in MuSK(+) patients; however, both MuSK(+) and MuSK(-) patients had mild myopathy with frequent mitochondrial abnormalities. SIGNIFICANCE: The weakness in MuSK(+) patients is most likely due to disturbed neuromuscular transmission. The frequently encountered mitochondrial dysfunction in MG warrants further study.

[Current understanding of primary biliary cirrhosis].

AIM: To examine characteristics of the course of primary biliary cirrhosis (PBC). MATERIAL AND METHODS: The study of 150 PBC patients aged 26-82 years used clinical, biochemical, immunological and morphological methods. RESULTS: Antimitochondrial antibodies of type M2 (AMAM2) were detected in 85.4% patients with PBC. A positive correlation was found between clinicobiochemical activity of the disease and number of AMAM2. AMAM2-positive patients more frequently had extrahepatic manifestations, overlap syndrome, higher biochemical and immunological activity vs AMAM2-negative patients. CONCLUSION: The following variants of PBC course were identified: classic AMAM2-positive, AMAM2-negative, overlapping of PBC and autoimmune hepatitis. Concentration of AMAM2 has diagnostic and prognostic value in PBC.

Muscle damage and autoantibody fixation in an experimental model of autoimmune myopathy.

An experimental model of autoimmune myopathy was designed using parental antigens (muscle mitochondrial fraction) in F1 hybrid rats (male Wistar x female Sprague-Dawley). The immune response was modulated by spleen fragment transplant from either Wistar (W) or F1. Antibody fixation and inflammatory reaction were studied in Extensor digitorum longus and soleus muscles. Immunization without spleen transplant resulted in antibody fixation mainly in capillaries and incompletely around muscle fibers; whorled fibers were found in 1/3 of F1 rats immunized with antigen from W rats. Spleen transplants from Sprague Dawley (SD) rats were usually accepted by F1; in some animals, antibodies surrounded completely muscle fibers and the percentage of animals showing soleus muscle lesions was increased. Spleen transplants from non immunized F1 were usually rejected by immunized F1; antibody reaction was found inside fibers of most of the rats, muscle damage was present in 40% of the animals immunized with W, but absent in those immunized with SD antigen. In conclusion, this model can be used to study immunological responses to alloantigens (parental to F1). Spleen fragment transplant modulates the immune response. There was discrepancy between antibody fixation and muscle damage. The immunological response was different according to muscle fiber type composition and/or microcirculatory characteristics.

[Prevalence and characteristics of anti-tissue antibodies in chronic hepatitis caused by hepatitis C virus]. / Prévalence et caractéristiques des anticorps anti-tissus au cours des hépatites chroniques dues au virus de l'hépatite C.

OBJECTIVES: The prevalence and significance of antiorganelle antibodies in the serum of patients with chronic hepatitis C is a subject of controversy. We studied prospectively these characteristics in patients with chronic hepatitis C. METHODS AND RESULTS: Among 156 patients (age: 55 +/- 14 years; 83 females), 30 (19%) had significant titers of antiorganelle antibodies: anti-nuclear antibodies in 18, anti-smooth muscle antibodies in 8 (no anti-actin or anti-vimentine subtypes), anti-LKM1 in 2, type 2 anti-mitochondrial antibodies in 2 patients. Anti-organelle antibodies were not detected in 126 patients. Patients with anti-organelle antibodies were significantly older but no difference was found between the two groups for sex ratio, serum amino-transferases or gammaglobulins, histopathological liver activity or prevalence of lymphocytic sialadenitis. The presence of anti-organelle antibodies was not related to HLA phenotype, especially B8 DR3, or DR4. Response to alpha interferon, estimated by serum aminotransferase levels after six months of treatment, was the same in both groups. CONCLUSIONS: These results suggest that serum anti-organelle antibodies are prevalent in during chronic hepatitis C but do not indicate a distinct autoimmune mechanism. Furthermore, the typing of anti-smooth muscle antibodies might help distinguish chronic hepatitis C from type 1 autoimmune chronic hepatitis.

Alpha B-crystallin in oxidative muscle fibers and its accumulation in ragged-red fibers: a comparative immunohistochemical and histochemical study in human skeletal muscle.

The alpha B-crystallin gene is abundantly expressed in the vertebrate lens and at lower levels in various non-lenticular tissues. Among the non-lenticular tissues, alpha B-crystallin is present at high levels in the heart and skeletal muscle. Using a specific antibody against alpha B-crystallin, the cellular localization of alpha B-crystallin was studied in biopsies of human skeletal muscles. Expression of alpha B-crystallin was observed in normal oxidative muscle fibers that show positive reactions for NADH-tetrazolium reductase and cytochrome c oxidase. In muscle diseases increased immunoreactivity for alpha B-crystallin was found in ragged-red fibers, which stained darkly with histochemistry for succinate dehydrogenase. Since alpha B-crystallin is related to small heat-shock proteins and can be induced by various stress conditions, the increased alpha B-crystallin immunoreactivity of ragged-red fibers could result from profound oxidative stress produced by the abnormal mitochondrial metabolism.

Anti-M9 antibodies in sera from patients with primary biliary cirrhosis recognize an epitope of glycogen phosphorylase.

Anti-M9 antibodies in sera from patients with primary biliary cirrhosis (PBC) were previously found to recognize two antigenic determinants at 98 and 59 kD, using a purified antigen fraction derived from rat liver mitochondria in the Western blot. Here we show that these antibodies are directed against an epitope of the enzyme glycogen phosphorylase. By Western blotting, a determinant at 98 kD was obtained testing anti-M9 positive sera against phosphorylase from skeletal muscle, and after plasmin treatment a degradation product appeared at 59 kD. Both determinants were identical to the M9-specific determinants 98 and 59 kD as shown by absorption studies. When these antibodies were eluted from the 98 and 59 kD determinants of the M9 antigen after immunoblotting, they again recognized the same epitopes on plasmin-treated phosphorylase. Furthermore, phosphorylase enzyme activity could be also demonstrated in the purified M9 fraction, and anti-M9-positive/anti-M2-negative but not anti-M9-negative/anti-M2-positive sera could be shown to stimulate phosphorylase activity. Testing sera from 1189 patients with different hepatic and non-hepatic disorders against M9 and phosphorylase from skeletal muscle by ELISA, 20% were positive with phosphorylase and only 2% with the M9 fraction. These data indicate that the commercially available phosphorylase from skeletal muscle cannot be recommended as M9 source. It may still contain non-PBC-specific epitopes which are probably recognized by naturally occurring antibodies directed against this highly conserved protein.

A mitochondrial encephalomyopathy with specific deficiencies of two respiratory chain polypeptides and a circulating autoantibody to a mitochondrial matrix protein.

A 15-yr-old boy with mitochondrial encephalomyopathy and NADH CoQ reductase (Complex I) deficiency is presented. Immunoblotting demonstrated specific deficiencies of the 24 kDa FeS protein of Complex I and subunit II of Complex IV. The patient's serum contained an antibody to a specific mitochondrial matrix polypeptide of apparent Mr 41 kDa. The specific polypeptide deficiencies involve products of nuclear (24 kDa FeS protein) and mitochondrial (subunit II) genes and suggest some intergenomic regulation. The relevance of the circulating antibody to the pathogenesis of the patient's Complex I deficiency is discussed.

Ragged-red fibres detected in paraffin sections by a monoclonal antibody to inner mitochondrial membrane.

An immunohistochemical method is reported using the M-II68 monoclonal antibody, which detects mitochondrial accumulations ("ragged-red fibres") in routinely processed (formalin-fixed, paraffin-embedded) muscle tissue. Ten cases with electron-microscopically and histochemically proven mitochondrial myopathy featured 4% to 24% ragged-red fibres. In a series of 50 muscle biopsies without mitochondrial myopathy, scattered ragged-red fibres (less than 0.1%) were present in a few normal and pathological muscles. The immunohistochemical method is specific for mitochondria, does not require frozen tissue and permits rapid examination of large areas.

[Changes in humoral immunity in ischemic cardiomyopathy]. / Modificazioni dell'immunità umorale in corso di cardiopatia ischemica.

The present study has been performed with the aim of assessing the incidence and the possible implications of the changes in humoral immunity in patients with coronary heart disease. Serial determinations of the immunoglobulins (Ig) G, A and M, of specific anti-heart antibodies and of some non-organ-specific antibodies have been carried out in the venous blood of 15 patients with acute myocardial infarction (AMI), of 30 subjects with angina pectoris (AP) and of 30 controls. The occurrence of anti-smooth muscle and anti-nuclear antibodies resulted negligible in all subjects, while anti-mitochondrial antibodies were found in a relatively high percentage of cases, which is probably due to chance. Only in 13.3% of AMI patients, and in 16.7% of AP subject, were anti-heart antibodies detectable, and their presence was not related to the occurrence of Dressler's syndrome, nor to any clinical finding. The mean IgG curve in the AMI patients showed a triphasic time-course in the first 20 days of disease. In the AP patients an inverse correlation has been found between monthly frequencies of anginal attacks and serum concentrations of IgG (r = 0.382; p less than 0.05). In the control group serum IgA were directly correlated to age (r = 0.493; p less than 0.01); furthermore, in patients with exertional or mixed angina serum IgA were often higher than those of patients with only rest angina (x2 = 3.906; p less than 0.05). These data suggest the working hypothesis that a possible link (of secondary or primary type) between serum concentrations of IgA and severity of atherosclerosis may exist.

Fatal infantile mitochondrial myopathy and renal dysfunction caused by cytochrome c oxidase deficiency: immunological studies in a new patient.

A 3-month-old female infant had profound generalized weakness, de Toni-Fanconi-Debre syndrome, and lactic acidosis. She required assisted ventilation and died at the age of 8 months. Muscle biopsy showed accumulation of mitochondria, glycogen, and lipid droplets. Histochemical reaction and immunocytochemical stain for cytochrome c oxidase showed very weak results, but both reactions were normal in intrafusal fibers of the muscle spindle. In crude extracts of the patient's muscle, cytochrome c oxidase activity was undetectable and enzyme-linked immunosorbent assay showed decreased reaction at all dilutions of antiserum. These data indicate that the amount of immunoreactive enzyme protein is markedly decreased in muscle of patients with fatal infantile cytochrome c oxidase deficiency and renal dysfunction.

Serum IgE and smooth muscle antibodies in pre-eclampsia.

The serum IgE concentrations and the occurrence of autoantibodies were measured in 20 women with preeclampsia and in 17 women with normal pregnancy. All women studied were primigravid and matched according to the age and the duration of pregnancy. The serum IgE concentration was higher in pre-eclampsia. Antinuclear and antimitochondrial antibodies were found only occasionally, whereas smooth muscle antibodies were found in preeclamptic women more often than in normal pregnancy. These results are in agreement with the findings of disturbed immune function in pre-eclampsia, but do not clarify the etiology of this disorder.

Elevated levels of smooth muscle autoantibodies in patients with acromegaly.

Screening of sera from patients with central nervous system (CNS) tumors for serum antibodies to tumor and normal tissue antigens revealed that the sera from a significant percentage of patients with pituitary adenoma demonstrated reactivity for smooth muscle antibodies (SMA) at a serum titer (1/25) at which other CNS tumors are devoid of this reaction. The sera were assessed by an indirect immunofluorescent antibody assay on fresh cryostat sections of rat kidney, liver, diaphragm, and stomach tissue. Absorption of SMA-positive sera with extracts containing smooth muscle tissue abolished the reaction. The overall incidence of SMA among patients harboring pituitary tumors was 30% (12/40). Assessment of the functional types of the tumor revealed a distinct predilection for such findings among patients with clinical acromegaly. Among patients with hypersecretion of growth hormone (CA) 90% (9/10) have SMA (both IgG and IgM type) whereas SMA was positive in only 10% (3/30) of corresponding group of patients with pituitary tumors resulting in hypercortisolemia or those that did not result in a hyperfunctional endocrine state.

Smooth-muscle antibodies in rheumatoid arthritis.

Smooth-muscle antibodies (SMA) detected by the indirect immunofluorescence method were found more often in rheumatoid arthritis (RA) patients (15.3%) than in normal controls (7.6%) (0.02 > p > 0.01). The increased frequency was due to IgG-antibodies which occurred in 12.2% of RA patients, in 4.1% of normal controls and in 7.3% of patients with other arthritic diseases. Eight of 9 IgG-SMA-positive RA sera reacted with F-actin, and one serum contained non-actin antibodies. In RA, ANA were found in 35.7%, glomerular antibodies in 1.0%, parietal cell antibodies in 4.1% and mitochondrial antibodies in 2.0%. SMA were associated with the occurrence of rheumatoid factors and mitochondrial antibodies (0.02% > p and 2p = 0.04, respectively). In the SMA-positive group the erythrocyte sedimentation rate was higher and elevated serum alkaline phosphatase values were found more often than in the SMA-negative group.