Recognition of new citrulline-containing peptide epitopes by autoantibodies produced in vivo and in vitro by B cells of rheumatoid arthritis patients.

Anti-citrullinated peptide/protein antibodies (ACPAs) are highly sensitive and specific markers of rheumatoid arthritis (RA). Identification of peptide epitopes that may detect different subgroups of RA patients might have diagnostic and prognostic significance. We have investigated citrulline- and arginine-containing peptide pairs derived from filaggrin, collagen or vimentin, and compared this citrulline-peptide panel with the serological assays conventionally used to detect ACPAs. Furthermore, we studied if the same citrulline-peptides identify antibody-secreting cells in in vitro cultures of RA B cells. Recognition of citrulline- and arginine-containing filaggrin, vimentin and collagen peptide epitopes were tested by Multipin ELISA system, by indirect ELISA and by a peptide-specific microarray. B cells were purified from blood by negative selection; antibody-producing cells were enumerated by ELISPOT assay. The panel composed of citrulline-peptide epitopes of filaggrin, collagen and vimentin was recognized by RA sera with a sensitivity and specificity comparable with the currently used tests. Moreover, the combined citrulline-peptide panel including the new short epitope peptide of filaggrin, fil311-315, also identified nearly one-third of RA cases that were negative for antibodies against cyclic citrullinated peptides, mutated citrullinated vimentin or for rheumatoid factor. The results with the peptide-specific microarray have shown that although most ACPAs recognizing the four citrulline peptides are IgG, some of them specifically recognizing citrulline-containing filaggrin peptides (fil311-315 and fil306-326) are IgM, and so may be produced either by newly formed activated B cells or by unswitched B memory cells. Furthermore, the citrulline-peptides of filaggrin and vimentin detect ACPA-producing cells, and so could also be applied to study the B cells of RA patients.

Specific expression of PAD4 and citrullinated proteins in lung cancer is not associated with anti-CCP antibody production.

Anti-citrullinated protein antibodies (ACPAs), produced against citrullinated proteins, are diagnostic and prognostic markers of rheumatoid arthritis (RA). The underlying mechanism that explains the connection of smoking, citrullination [catalyzed by peptidyl arginine deiminases (PADs)] and ACPAs is still unclarified in RA. Thus, we searched for a non-arthritic model in which an increased cell death allows the formation of autoantibodies. Data supporting that lung cancer might be a good candidate are as follows: (i) smoking plays a role in its pathogenesis, (ii) the disease is frequently accompanied by paraneoplastic syndrome, (iii) smoking increases citrullination in the lung, (iv) various types of malignancies are associated with increased citrullination and (v) lung cancer tissue shows similarities with RA synovium. Serum PAD4, rheumatoid factor (RF) and ACPA levels were measured in 42 lung cancer patients; expression of cytokeratin 7 (CK7), PAD4 and citrullinated proteins was visualized in 113 lung cancer tissues. All parameters were analyzed in correlation with smoking history. None of the patients had polyarthritis or autoimmune disease. Significantly increased RF levels were associated with higher PAD4 levels in smoker lung cancer patients compared with non-smokers. Both PAD4 and citrullination immunostaining strongly correlated with that of CK7 in lung cancer, however, did not differ according to smoking history. Two of 30 smoker lung cancer patients had high anti-cyclic citrullinated peptide levels. In conclusion, PAD4 and citrullination may be helpful in distinguishing lung cancer from healthy tissue. Smoking, abnormal serum PAD4 and RF levels may not be sufficient for the production of ACPAs and development of autoimmunity.

Regeneration of reinnervated rat soleus muscle is accompanied by fiber transition toward a faster phenotype.

The functional recovery of skeletal muscles after peripheral nerve transection and microsurgical repair is generally incomplete. Several reinnervation abnormalities have been described even after nerve reconstruction surgery. Less is known, however, about the regenerative capacity of reinnervated muscles. Previously, we detected remarkable morphological and motor endplate alterations after inducing muscle necrosis and subsequent regeneration in the reinnervated rat soleus muscle. In the present study, we comparatively analyzed the morphometric properties of different fiber populations, as well as the expression pattern of myosin heavy chain isoforms at both immunohistochemical and mRNA levels in reinnervated versus reinnervated-regenerated muscles. A dramatic slow-to-fast fiber type transition was found in reinnervated soleus, and a further change toward the fast phenotype was observed in reinnervated-regenerated muscles. These findings suggest that the (fast) pattern of reinnervation plays a dominant role in the specification of fiber phenotype during regeneration, which can contribute to the long-lasting functional impairment of the reinnervated muscle. Moreover, because the fast II fibers (and selectively, a certain population of the fast IIB fibers) showed better recovery than did the slow type I fibers, the faster phenotype of the reinnervated-regenerated muscle seems to be actively maintained by selective yet undefined cues.

Early detection of cardiotoxicity by 2D and 3D deformation imaging in patients receiving chemotherapy.

The aim of the present study was to find out whether early cardiac changes in patients receiving chemotherapy can be detected by the conventional and deformation parameters of 2D and 3D echocardiography. Twenty-five healthy subjects with normal regional left ventricular function (group 1) and 25 patients receiving chemotherapy (group 2) underwent 2D and 3D transthoracic echocardiography (Toshiba Artida Medical System). All patients (group 2) were examined before and during cardiotoxic chemotherapy at a 3-month follow-up. Left ventricular volumes, ejection fraction, muscle mass, global longitudinal, global radial, global circumferential strain, and rotation were analyzed with 2D and 3D echocardiography, while twist and time-to-peak-intervals were analyzed with 3D echocardiography. For left ventricular volumes and muscle mass, no significant differences were seen between the two study groups (P<0.05). According to our results, myocardial dysfunction induced by cardiotoxic chemotherapy can be detected by 2D global radial strain. Detecting myocardial dysfunction by global longitudinal and circumferential strain requires more than 3 months follow-up. Changes in rotation, twist or time-to-peak intervals could not be verified at the 3-month follow-up in the present study. 2D global radial strain seems to be the most sensitive and robust parameter to detect early myocardial damage during chemotherapy. 3D echocardiography is not yet an established method to detect myocardial damage in clinical practice due to lower spatial and temporal resolution.

Citrullination under physiological and pathological conditions.

Citrullination, one of the enzymatic posttranslational modifications has become a hot topic of recent research as it is involved in various physiological and pathological processes. Antibodies against citrullinated proteins called anti-citrullinated protein antibodies, are the hallmark (diagnostic and prognostic factors) of rheumatoid arthritis, and are specific for the disease. Citrullination has an important role in the normal function of the immune system, skin keratinization, the insulation of neurons and the plasticity of the central nervous system including its essential role in gene regulation. Abnormal citrullination has been proposed to play a role in multiple sclerosis and Alzheimer's disease, and recent research has drawn attention to its implication in tumorigenesis. Still, it is unclear whether citrullination is the cause or the consequence of these pathological alterations. Here, we discuss crucial aspects of citrullination during both physiological and pathological conditions.

Increased serum concentration of immune cell derived microparticles in polymyositis/dermatomyositis.

Microparticles are recently recognized players of intercellular communication. They are involved in signal transduction, cell activation and apoptosis. Their importance is also suggested in autoimmune diseases such as rheumatoid arthritis and systemic sclerosis. We investigated the role of microparticles in polymyositis/dermatomyositis, a group of rare autoimmune diseases, characterized by specific skin lesions and muscle weakness. The plasma concentration of monocyte and lymphocyte derived microparticles of 20 patients with polymyositis/dermatomyositis and 20 healthy controls were determined by flow cytometry. The structure of microparticles was visualized by electron microscopy. Significantly elevated numbers of monocyte (CD14 positive), T-lymphocyte (CD3 positive) and B-lymphocyte (CD19 positive) derived microparticles were found in the plasma samples of polymyositis/dermatomyositis patients, compared to healthy controls (p=0.001, 0.01 and 0.006, respectively). Furthermore, the plasma levels of monocyte and B-lymphocyte derived microparticles correlated with the manual muscle strength test (r=0.497, p=0.027; r=0.508, p=0.023; respectively). Patients with anti-Jo-1 antibody and lung involvement had significantly higher numbers of T- and B-lymphocyte and monocyte derived MPs (p=0.006, 0.012 and 0.007, respectively, for anti-Jo-1; p=0.013, 0.016 and 0.025, respectively, for lung involvement). After ultracentrifugation, CK activity could be detected only in traces in the resuspended pellet containing microparticles of healthy and diseased individuals. The electron microscopy revealed slightly different microparticles in the samples of patients with polymyositis/dermatomyositis. These results suggest that immune cell derived microparticles may contribute to the inflammatory process in polymyositis/dermatomyositis, however, CK-positive, possibly muscle derived microparticles do not seem to be present in the blood of patients with polymyositis/dermatomyositis.

Rheumatoid arthritis and smoking: putting the pieces together.

Besides atherosclerosis and lung cancer, smoking is considered to play a major role in the pathogenesis of autoimmune diseases. It has long been known that there is a connection between rheumatoid factor-positive rheumatoid arthritis and cigarette smoking. Recently, an important gene-environment interaction has been revealed; that is, carrying specific HLA-DRB1 alleles encoding the shared epitope and smoking establish a significant risk for anti-citrullinated protein antibody-positive rheumatoid arthritis. We summarize how smoking-related alteration of the cytokine balance, the increased risk of infections (the possibility of cross-reactivity) and modifications of autoantigens by citrullination may contribute to the development of rheumatoid arthritis.

Androgens negatively regulate myostatin expression in an androgen-dependent skeletal muscle.

Myostatin is an important negative regulator of skeletal muscle growth, while androgens are strong positive effectors. In order to investigate the possible interaction between myostatin and androgen pathways, we followed myostatin expression in the androgen-dependent levator ani (LA) muscle of the rat as a function of androgen status. By testosterone deprivation (castration), we induced LA growth arrest in young male rats, whilst atrophy in adult ones, however, both processes could be reversed by testosterone supplementation. After castration, a significant up-regulation of active myostatin protein (and its propeptide) was found, whereas the subsequent testosterone treatment reduced myostatin protein levels to normal values in both young and adult rats. Similarly, a testosterone-induced suppression of myostatin mRNA levels was observed in castrated adult but not in young animals. Altogether, androgens seem to have strong negative impact on myostatin expression, which might be a key factor in the weight regulation of LA muscle.