Muscle Involvement in Rheumatoid Arthritis: An Ultrastructural Study

An electron microscopic investigation has been carried out on muscle bioptic samples from patients affected by rheumatoid arthritis (RA). This study was undertaken to seek further ultrastructural alterations affecting striated muscles in RA pathology. Bioptic samples were collected on a total of 30 surgical interventions of hip (10), knee(8), and foot (12). This yielded three muscle types: gluteus maximus, vastus lateralis, and extensor digitorum communis. Muscle samples from 12 patients with no RA stigmata, selected to match RA patients by age and gender, constituted the control group. Tissue samples were prepared both for conventional histochemical methods and according to conventional electron microscopic procedures, including morphometric analysis. Although to a different extent in each sample, in muscles from RA vs. controls the authors observed the simultaneous presence of discrete muscular alterations such as wider separation of myofibrils, myelin figures, dilated sarcotubular system, pleomorphic mitochondria, myofibril flaking, and lipofuscin deposition in the subsarcolemmal region. In addition to a progressive atrophy, the above findings are suggestive of rheumatoid myositis and lend further support to the still poorly documented presence of an idiopathic inflammatory myopathy and inclusion body myositis associated with RA.     

Muscle changes in rheumatoid arthritis

Summary Muscle changes were studied in biopsy material obtained from 100 patients suffering from classical rheumatoid arthritis. The abnormalities consisted of denervation atrophy of type II muscle fibres, degenerative changes in the sarcoplasm including presence of nemaline rods, and changes within the interstitium: namely perivascular nodular myositis, lymphocytic accumulations, different stages of vasculitis and abnormalities within the intramuscular nerves and muscle spindles. The muscles examined were always severely affected. It is considered that the simultaneous presence of these abnormalities is suggestive of rheumatoid arthritis. The importance of histochemical studies is emphasized. The literature concerning muscle changes in rheumatoid arthritis is reviewed.Temporary Research Fellow the Rotterdam Centre for Rheumatic Disease     

Granulomatous myositis. Clinicopathologic study of 12 cases.

Granulomatous inflammation is infrequently encountered in skeletal muscle biopsy material. Of 2,985 muscle biopsy specimens reviewed over 12 years, 12 (0.4%) with granulomatous inflammation were identified. The patients included 9 women who ranged in age from 24 to 76 years (mean 50.3 years). The most common clinical findings included decreased strength or weakness in the extremities (n = 8), muscle pain (n = 5), and weight loss (n = 3). All muscles exhibited nonnecrotizing granulomas; an associated vasculitic process was identified in 2. Endomysial chronic inflammation consisting primarily of lymphocytes and plasma cells was present in 10 muscles, and perivascular chronic inflammation in 8. Degenerating muscle fibers were noted in 10 cases, and regenerating fibers in 11. Evidence of neurogenic atrophy was seen in 8 muscles. Increased endomysial fibrosis was observed in 5 muscles, and type II muscle fiber atrophy in 5 muscles. Stains for acid-fast bacilli and Gomori methenamine silver stain were performed in all but 2 cases and failed to demonstrate organisms. In 3 cases, concomitant sural nerve biopsies were performed, and granulomas were identified in 2 of those cases. Clinicopathologic diagnoses included sarcoidosis (n = 6), vasculitis (n = 2), and granulomatous myositis not otherwise specified (n = 2). In 2 cases, there was insufficient clinical information or follow-up data to determine a cause. In conclusion, granulomatous myositis is infrequently found in muscle biopsy specimens (0.5% of all biopsies in this series); most muscles demonstrate evidence of chronic endomysial or perivascular inflammation accompanied by muscle fiber degeneration and regeneration; and the most common cause for granulomatous myositis was sarcoidosis in this series.     

Can tissue transglutaminase be a marker of idiopathic inflammatory myopathies?

In the normal striated muscle, tissue transglutaminase (TG2) content is vestigial. However, this protein's presence has been reported to occur in myoblasts and myotubes during the fetal period. Its increased expression has been also found in the muscle tissue in the course of sporadic inclusion body myositis, as well as in polymyositis (PM) and dermatomyositis (DM), which are considered to be diseases of immunological origin. Based on in vitro studies, a substantial TG2 role in the infiltration of some T cell subsets into inflamed tissues has been suggested lately. In this study, the immunohistochemical reactions in the guinea pig experimental myositis specimens and in the ones from PM/DM patients were compared. The guinea pig tissue specimens were taken from muscles affected by experimental myositis induced by intramuscular injections of: 1/sera from 30 neoplasm patients with no metastases; 2/sera from 10 healthy people; 3/sera from 2 DM patients; 4/neuropeptides (SP, NPY or VIP) and from 5/the muscles affected by the reversed passive Arthus reaction (RPAR). The immunostaining for TG2 revealed substantial presence of this protein in single, damaged muscle fibers and a weak reaction in regenerating fibers appearing in PM/DM patients' specimens. From among experimental myositis specimens, a very intensive reaction appeared only in the damaged and regenerating muscle fibers present in the slides from guinea pig muscles injected with DM patients' sera. Such results suggest some presence of a specific factor(s) (the one(s) responsible for TG2 expression in the damaged muscle fibers) in DM patients' sera. The results suggest that transglutaminase can be a marker of inflammatory myopathies. A probable correlation between TG2 expression in muscles and organismal immunological factors, including the complement activation status, requires additional studies.     

Secretory antibodies to citrullinated peptides in plasma and saliva from rheumatoid arthritis patients and their unaffected first-degree relatives

The aim of this study was to evaluate secretory antibodies to citrullinated proteins (ACPA) in plasma and immunoglobulin (Ig)A ACPA in saliva from patients with rheumatoid arthritis (RA) and their unaffected first-degree relatives (FDRs). Patients with RA (n = 194) and first-degree relatives unaffected by RA (n = 191) were recruited for analysis of secretory antibodies to second-generation cyclic citrullinated peptides (anti-CCP) in plasma. From a subpopulation (25 RA patients, 21 first-degree relatives and 11 controls), saliva samples were obtained for IgA anti-CCP analysis. The presence of secretory ACPA was compared between subject categories, and related to genetic and environmental risk factors. Secretory ACPA occurred in 37 (19%) plasma samples from patients with RA, but only in two (1%) of FDRs. IgA ACPA in saliva was found in three of 25 (12%) patients with RA, but not in any of the 21 FDRs (< 5%). No significant associations were seen between the presence of secretory ACPA and SE or smoking, either among RA patients or among FDRs. Despite occurring in 19% of RA plasma, secretory ACPA was rare in both saliva and plasma among FDRs, even among those positive for conventional ACPA of non-mucosal origin. Longitudinal studies are warranted to determine whether circulating secretory ACPA occurs before or in parallel with the development of clinical arthritis.     

Immunohistochemical localization of HTLV-I p19- and p24-related antigens in synovial joints of patients with rheumatoid arthritis.

In formalin-fixed, paraffin-embedded synovial tissues from patients with early proliferative rheumatoid arthritis (RA), immunoreactivity could be demonstrated utilizing monoclonal IgG antibodies reactive with the p19 and p24 protein of human T cell leukemia virus (HTLV-I). Additionally, surgical specimens of fresh unfixed synovial tissues from patients with RA also demonstrated immunoreactivity. At the light microscopic level, both HTLV-I antigens were detected in approximately 45% of the rheumatoid synovial tissues by the immunocolloidal gold method with silver enhancement (IGSS) and the avidin-biotin-complex technique (ABC), whereas six of eight of the frozen RA specimens stained positive by immunofluorescence. Patients whose synovial tissues were immunoreactive by immunofluorescence were seronegative to HTLV-I antigens as determined by ELISA and immunoblotting. Conversely, cases with osteoarthritis, juvenile rheumatoid arthritis, psoriatic arthritis, Dupuytren's contracture, and gangrene were shown to be nonreactive by immunohistochemistry. The results indicate that expression of antigens is related to or crossreactive with HTLV-I in synovial tissues from patients with rheumatoid arthritis.     

Dinucleotide repeat polymorphism in intron II of human Toll-like receptor 2 gene and susceptibility to rheumatoid arthritis

Human Toll-like receptors (TLRs) participate in innate immune response and signal the activation of adaptive immunity. The presence of a functional intronic polymorphism consisting of guanine-thymine repeats in TLR2 gene was recently reported. Here, we investigated a dinucleotide repeat polymorphism in intron II of TLR2 in Korean patients with rheumatoid arthritis (RA). The numbers of guanine-thymine [(GT)(n)] repeats in intron II of the TLR 2 gene were counted in 183 patients with RA and in 148 healthy controls, using the gene scanning technique. We classified alleles into two subclasses for further analysis, 12-16 GT repeats (S allele) and 17-28 repeats (L allele). By subgroup analysis, we also examined whether the S allele is associated with the presence of shared epitope (SE), rheumatoid factor (RF), joint erosion and extra-articular complications. S-allele frequency was significantly increased in patients with RA than in healthy controls [30.3% vs. 23.0%, P = 0.03, or 1.46, 95% confidence interval (CI) 1.03-2.07], and genotypes containing S alleles were more frequent in patients with RA than in healthy controls (54.4% vs. 46.5%. P = 0.04, or 1.57, 95% CI 1.01-2.42). A skewed S-allele distribution was not found to be related to the presence of SE. Subgroup analysis showed no genotypic or allele frequency differences between patients with/without RF, joint erosion, or extra-articular complications. Genotype containing shorter GT repeats in intron II of the TLR2 gene may confer susceptibility to RA in Koreans.     

Inclusion body myositis and paramyxoviruses.

Inclusion body myositis (IBM) is a distinct type of muscle disease. The characteristic electron microscopic findings, intranuclear or intracytoplasmic inclusions composed of microtubular filaments, morphologically resemble paramyxovirus nucleocapsids. These findings and the reported immunoreactivity of the inclusions with mumps virus antibodies have suggested that inclusion body myositis is a chronic virus infection. We analyzed skeletal muscle specimens from three patients with characteristic light microscopic features and electron microscopically verified inclusions of IBM by immunocytochemistry using antibodies raised against members of the paramyxovirus group, and by in situ hybridization with a cRNA probe representing the mumps virus nucleocapsid gene. The specificity of the reactions was demonstrated with infected and uninfected cultured cells. No immunocytochemical staining or hybridization signal was observed in biopsy specimens from IBM patients. These findings speak against a paramyxovirus etiology of IBM.     

Ubiquitin immunostaining and inclusion body myositis: Study of 30 patients with inclusion body myositis

Distinction of inclusion body myositis (IBM) from other forms of inflammatory myopathy is significant from prognostic and therapeutic standpoints. This study retrospectively examines ubiquitin expression by paraffin immunohistochemistry in muscle biopsy material from 30 patients with IBM. Patients included 19 men and 11 women (ages 29 to 80 years; mean, 64 years). All biopsies were characterized by endomysial chronic inflammation, muscle fiber degeneration and regeneration, rimmed vacuoles, and angular atrophic esterase-positive muscle fibers. Ragged red fibers were identified in biopsies of five patients and a partial cytochrome C-oxidase deficiency by enzyme histochemistry in biopsies of 10 patients. Evidence of intranuclear or cytoplasmic tubulofilamentous structures confirming a diagnosis of IBM was observed in all 30 cases. Paracrystalline mitochondrial inclusions were noted in five patients. Discrete myocyte intranuclear ubiquitin-positive inclusions were noted in 14 patients (47%). Discrete intracytoplasmic ubiquitin-positive inclusions were noted in 24 (80%) patients. Positive staining of rimmed vacuoles by ubiquitin was observed in 25 (83%) patients. Diffuse staining of scattered muscle fibers was observed in 21 (70%) patients. In a control group including patients with polymyositis (n = 3), dermatomyositis (n = 3), necrotizing vasculitis (n = 1), and granulomatous myositis (n = 1), discrete intranuclear or cytoplasmic ubiquitin-positive inclusions were not observed. Rimmed vacuoles were not seen either by light microscopy or ubiquitin immunostaining in any of the eight cases. Occasional myofibers from all eight cases showed diffuse, positive muscle fiber staining. Although not present in all cases, evidence of ubiquitinpositive myocytic intranuclear or cytoplasmic inclusions or positive-staining rimmed vacuoles in the setting of an inflammatory myopathy may be suggestive of a diagnosis of inclusion body myositis. Use of ubiquitin immunohistochemistry may be useful in cases in which frozen tissue or tissue processed for electron microscopy is not available, and IBM is suspected. Light or electron microscopic evidence of mitochondrial abnormalities were noted in a significant subset of patients (13 of 30; 43%) of patients with IBM.     

Increased muscle activity to stabilise mobile bearing knees in patients with rheumatoid arthritis

The aim of this study was to assess the differences in muscle activity (surface EMG) between a posterior stabilised (PS) total knee design and a mobile bearing (MB) posterior cruciate ligament retaining design in rheumatoid arthritis (RA) patients during a step-up task. Four patients with a PS total knee prosthesis and three patients with a MB total knee prosthesis were selected based on pain score, knee function, range of motion and joint stability. Clinical scores and functional scores were comparable between the two groups pre-operatively and at the 1-year follow-up. Visual analysis of the EMG activity of the main flexor and extensor muscles showed that the activity of both extensor and flexor muscles of the MB group was on average higher compared to the PS group. When the maximum activities of the muscles were compared, the patients in the MB group showed a significant higher maximum peak activity (p<0.05) of the Vastus Medialis (VM), Vastus Lateralis (VL) and Semitendinosus (ST) during step-up than the patients in the PS group. Also the instance of activation of the Vastus Medialis and the Vastus Lateralis was significant earlier in the MB group compared to the PS group. Since the differences between the PS and the MB group did not only show an increase of muscle activity but also an earlier activation of the flexor muscles, this may express compensation by coordination. Rehabilitation programs for RA patients should include besides muscle strength training, elements of muscle-coordination training.     

Immunopathologic and morphologic studies of skeletal muscle in Chagas'' disease.

Skeletal muscle biopsies from 21 individuals infected with Trypanosoma cruzi were studied my means of immunofluorescence, ultrastructural immunochemical, light and electron microscopic, and histochemical procedures. In 12 cases, definite morphologic alterations were found. These alterations were coincident with the presence of circulating antibodies against the plasma membrane of striated muscle fibers and endothelial cells (EVI antibodies). In almost all cases the lesions also presented autologous immunoglobulins bound to the plasma membrane of muscle fibers and endothelial cells. Interstitial inflammatory exudate was not observed in the diseased muscle. On the basis of these observations, it is suggested that the EVI antibody is related to some of the pathogenetic mechanism of skeletal muscle damage in Chagas' disease.     

Skeletal muscle fibers express major histocompatibility complex class II antigens independently of inflammatory infiltrates in inflammatory myopathies

The aim of our study was to address the question of whether muscle fibers express major histocompatibility complex (MHC) class II in inflammatory myopathies. For this purpose we performed a systematic study of MHC class II antigen expression on muscle fiber membranes in muscle tissue from polymyositis and dermatomyositis patients in various stages of disease activity. Thirty-two patients with classical clinical signs of myositis were divided into subgroups depending on duration of clinical signs of myositis and presence or absence of inflammatory infiltrates in muscle tissue. Immunohistochemistry as well as double-immunofluorescence stainings were used to identify the presence of MHC class II in muscle tissue. MHC class I was included for comparison. Quantification of positive staining was performed using an image analysis system in addition to evaluation by manual microscopic scoring and laser confocal microscopy. It was demonstrated that a significant proportion of skeletal muscle fibers in inflammatory myopathies express MHC class II as well as MHC class I and that MHC antigen expression is independent of the inflammatory cell infiltration. Furthermore, there were no differences in staining pattern between polymyositis and dermatomyositis patients. Our results indicate that MHC class II and MHC class I molecules may be involved in initiating and maintaining the pathological condition in myositis rather than only being a consequence of a preceding local inflammation.     

Patterns of neuromuscular disease. As related to stages of normal embryogenesis in voluntary muscle.

Skeletal muscle samples from the upper and lower extremities of 38 human fetuses (6 to 18 weeks' gestation) of both sexes were studied by histologic, histochemical, and electron microscopic methods. Ultrastructural morphometry was applied. In the different stages of normal development are found regressive changes, ranging from reversible dystrophic to irreversible necrotic alterations, which are characteristic of either primary myogenic myopathies or primary neurogenic muscle atrophies in older age. Several pathologic patterns of so-called congenital myopathies also presented. On the basis of their findings, the authors conclude that in a certain group of congenital myopathies full normal development of muscle is hindered or blocked and that groups of muscles or the whole voluntary musculature is arrested at certain stages of maturation. For example, insufficient or absent innervation of the fetal muscle may be a factor in Werdnig-Hoffmann or Kugelberg-Welander syndromes. The authors' findings suggest that pathologic patterns in muscle diseases have been used physiologically in fetal development to eliminate unnecessary overproduction of muscle fibers.     

The Presence of Anti-Protein A Antibodies in Patients with Rheumatoid Arthritis

Sixteen patients with rheumatoid arthritis (RA) were examined for the presence of anti-protein A antibodies. The F(ab')2 preparations from five RA patients showed significant binding to IgG-free protein A on ELISA. The protein A binding was further examined by immunoblotting. The F(ab')2 preparations of high protein A-binding protein gave a specific reaction with IgG-free protein A on nitrocellulose paper. This demonstrates the presence of anti-protein A antibodies in patients with RA. Those RA patients with anti-protein A antibodies had more active disease as judged by the Lansbury's activity index. The level of serum rheumatoid factor (RAHA) was significantly higher in patients with anti-protein A antibodies than in those without anti-protein A antibodies.     

Neutrophils of rheumatoid arthritis patients on anti-TNF-α therapy and in disease remission present reduced adhesive functions in association with decreased circulating neutrophil-attractant chemokine levels

Neutrophils participate in the initiation and progression of rheumatoid arthritis (RA) although the exact mechanisms responsible for neutrophil accumulation in rheumatoid joints are not understood. This study compared the adhesive and chemotactic functions of neutrophils from RA patients in activity (DAS28 > 3.2) and not in activity (DAS28 < 2.6) and observed the effects of different treatment approaches on these functions. Neutrophils were isolated from healthy controls (CON), and patients with active or inactive RA in use of therapy not specific for RA (NSAIDs), in use of DMARDs and in use of anti-TNF-α therapy. Adhesive and chemotactic properties were evaluated using in vitro assays; adhesion molecule expression was assessed by flow cytometry and real-time PCR and circulating chemokines were determined by ELISA. No significant alterations in the adhesive and chemotactic properties of neutrophils from active RA were observed when compared to CON neutrophils, independently of treatment regimen. In contrast, neutrophils from RA patients in disease remission presented reduced adhesive properties and a lower spontaneous chemotactic capacity, in association with decreased adhesion molecule expression, although profiles of alterations differed for those patients on DMARDs and those on anti-TNF-α therapy. Circulating levels of the major neutrophilic chemokines, IL-8 and epithelial neutrophil activating peptide-78, were also significantly decreased in those patients demonstrating a clinical response. Remission of RA appears to be associated with ameliorations in aspects important for neutrophil adhesion and chemotaxis; whether these alterations contribute to decrease neutrophil migration to the synovial fluid, with consequent improvements in the clinical manifestations of RA, remains to be determined.     

Glomerulonephritis in patients with rheumatoid arthritis (RA). Report of five cases and review of the literature

We reported five cases of glomerulonephritis developed in the course of rheumatoid arthritis (RA), which had no apparent relation with therapeutic agents. Systemic angiitis was observed in patient 1 and overlapping of systemic lupus erythematosus (SLE) with RA was implied in patient 2, while the other three patients did not show these changes. Renal biopsies were performed in all of the cases and glomerular deposition of immune complexes was suggested by both the immunofluorescent and electron microscopic findings, though the amount was variable. Furthermore, cases of glomerulonephritis in patients with RA were reviewed in the literature and classified into three groups: occurring in association with angiitis, overlapping with SLE or other collagen diseases, and developing without the above two factors. Group 3 was further divided into two subgroups according to immunologic abnormalities. All cases included in the three groups can be regarded as glomerulonephritis of RA. The cases of group 2, however, can more preferentially be considered to be caused by SLE or some other collagen disease that has overlapped with RA. Deposition of immune complexes was suggested in most of the cases examined with immunofluorescence. Various changes were seen in the glomerulonephritis of RA presented here (patients 1, 3, 4, and 5) and reviewed in the literature (groups 1 and 3). Immune complexes might be involved in the pathogenesis of glomerulitis.     

Levels of the soluble forms of CD80, CD86, and CD83 are elevated in the synovial fluid of rheumatoid arthritis patients

The release of soluble forms of CD80 (sCD80), CD86 (sCD86), and CD83 (sCD83) provide a potentially powerful immunoregulatory mechanism. We therefore investigated the potential presence and relative levels of these molecules in the synovial fluid (SF) and serum of patients with rheumatoid arthritis (RA) and osteoarthritis (OA). Serum and SF levels were measured by enzyme-linked immunosorbent assay. Serum levels of sCD80, sCD86, and sCD83 in RA and OA patients were similar to those present in normal donor serum (NDS) and the SF of OA patients. In contrast, when compared with NDS and OA SF levels, almost all RA SF samples had elevated sCD83 levels (32/35, >0.63 ng/ml) and a substantial proportion had elevated sCD80 (13/29, >0.22 ng/ml) or sCD86 (16/33, >2.31 ng/ml) levels. Analysis of matched pairs of serum and SF from RA patients demonstrated that the SF/serum ratio for sCD80 (95% CI = 1.7-3), sCD86 (95% CI = 1.5-3.1), and sCD83 (95% CI = 3.6-7.8) levels was >1 in almost all patients. In conclusion, this study shows that the SF from almost all RA patients contain elevated levels of sCD83 and the majority of these samples also contain elevated levels of sCD80 and/or sCD86. These molecules may play a role in modulating immune responses within the rheumatoid joint.     

Polymerization of myosin on activation of rat anococcygeus smooth muscle

The in vivo state of assembly ofmyosin in vertebrate smooth muscle is controversial. In vitrostudies on purified smooth muscle myosin show that it ismonomeric (10S) under relaxing conditions and filamentous undercontraction conditions. Electron microscopic and antibodylabelling studies of intact smooth muscles, on the other hand,suggest that myosin is filamentous in the relaxed as well as thecontracting state and that 10S myosin occurs only in traceamounts. However, birefringence, conventional EM and X-raydiffraction evidence suggests that in certain smooth muscles invivo (e.g. rat anococcygeus), while myosin filaments exist in therelaxed state, their number increases on contraction. Here, wehave used low temperature electron microscopic techniques (rapidfreezing followed by freeze-substitution), which preserve labilecomponents in close to their in vivo state, to detect any changein filament number on contraction. The results from ratanococcygeus have been compared with those from guinea pig taeniacoli, in which other techniques have revealed no change infilament number. In the anococcygeus, we find evidence for a 23%increase in filament density in transverse sections ofcontracting muscle compared with relaxed muscle. In the taeniacoli we find no change. These results are in qualitativeagreement with earlier findings. They provide evidence forpolymerization of myosin in contracting rat anococcygeus, andsuggest that this process is subtle and occurs only in somesmooth muscles     

NETs analysed by novel calprotectin-based assays in blood donors and patients with multiple myeloma or rheumatoid arthritis: A pilot study

Two novel enzyme-linked immunosorbent assays (ELISAs), designed to detect complexes containing DNA, leucocyte calprotectin and S100A12 proteins, were generated for improved specificity and rapid measurement of neutrophil extracellular traps (NETs). The assays were applied on plasma and serum samples from blood donors for establishment of reference values, and from patients with multiple myeloma (MM) or rheumatoid arthritis (RA) in order to examine putatively increased values in the two different inflammatory conditions. Although NETs were hardly detectable in healthy individuals, NET levels were as expected highly and statistically significantly increased in RA patients. The detection of statistically significantly increased NET levels in MM is a novel finding.