Fibulin-6 expression and anoikis in human salivary gland epithelial cells: implications in Sjogren's syndrome.

Important changes in acinar and ductal morphology and function, together with pronounced extracellular matrix (ECM) remodelling, are detectable in the labial salivary glands of Sjögren's syndrome (SS) patients. The objective of this work was to determine the effect of treatment with the anti-Ro/SSA auto-antibodies, characterizing SS, on the expression of fibulin-6, a member of the fibulins family of the ECM, in primary human salivary gland epithelial cell (SGEC) cultures established from biopsies of labial minor salivary glands obtained from healthy donors. The induction of cell detachment and anoikis in SGECs treated with anti-Ro/SSA auto-antibodies were also investigated. Changes in fibulin-6 mRNA expression were measured by semi-quantitative reverse transcriptase-PCR and real-time PCR. Fibulin-6 expression in cells treated with anti-Ro/SSA auto-antibodies was evaluated by flow cytometric analysis and confocal laser scanning microscopy. SGECs undergoing death by anoikis were identified and quantified using Calcein blue/YOPRO-1 dyes. Herein, we present the first evidence of fibulin-6 expression in SGEC that results distributed in the cytoplasm surrounding the inner side of the plasma membrane. Fibulin-6 was down-regulated in SGECs treated with anti-Ro/SSA auto-antibodies in which a marked cell detachment and a reduction of cell viability were observed. Notably, a reduction of fibulin-6 expression in SGECs treated with anti-Ro/SSA auto-antibodies corresponds to an increase of anoikis cell death. Our observations demonstrate a dysregulation of fibulin-6 in the pathological processes observed in SS and provide new evidence that disorganization of the ECM environment could damage the architecture and function of the salivary glands.

[New and old strategies against osteoporosis]. / Le terapie dell'osteoporosi: lo stato dell'arte 2009.

Osteoporosis is a skeletal disorder characterized by compromised bone strength predisposing to an increased risk of fracture that affects both cortical bone and trabecular bone. Osteoporosis may be either primary or secondary. Primary osteoporosis can be distinguished into two types: diffuse or local. The treatment can be managed through drugs, exercise and lifestyle. The aim of this paper is to highlight new and old therapeutic strategies.

Autoantibodies from Sjögren's syndrome trigger apoptosis in salivary gland cell line.

The presence of serum autoantibodies has been associated with Sjögren's syndrome (SS), an autoimmune rheumatic disease that targets salivary and lachrymal glands. The association of apoptosis with autoantibodies production seems to play a role in the pathogenesis of glandular damage. The best-defined antibodies in SS are those reacting with the ribonucleoprotein antigens SS-A (Ro) and SS-B (La). Anti-Ro antibodies are found in about 70-90%, and anti-La in approximately the same frequency, of patients with primary SS. The objective of this work was to explore whether anti-Ro and anti-La autoantibodies purified from Sjögren IgG fractions are able to trigger apoptotic process in the human salivary gland cell line A-253. Anti-Ro and anti-La autoantibodies were purified on protein G Sepharose affinity column and used for the A-253 cell treatment. Apoptosis induced by autoantibodies was revealed by FACS analysis, and the active caspase-3 and the cleaved caspase-3 substrate poly (ADP-ribose) polymerase (PARP) was demonstrated by colorimetric assay and Western blot. This report shows that anti-Ro and anti-La autoantibodies, but not healthy IgG, activate the caspase-3 and determine the cleavage of PARP in A-253 cells. Apoptosis triggered by Sjögren autoantibodies could be responsible for the impairment of the secretory function in the salivary glands.

[Hereditary thrombophilia and systemic sclerosis. An unusual case report]. / Una rara associazione di trombofilia ereditaria e sclerosi sistemica.

Authors describe a case of systemic sclerosis with deep venous thrombosis. Great attention must be taken for this case because it represents the condition of hereditary thrombophilia. No similar case was reported in literature; therefore, further studies must go ahead understand the possible relation between the two pathologies.

[Wegener's granulomatosis: that is the risk of diagnostic hypervaluation]. / La malattia di Wegener: ovvero il rischio di ipervalutazione diagnostica.

Iter for diagnosis of Wegener's granulomatosis is delineated, underling the role of ANCA, histological test and the risk of diagnostic hypervalutation.

[Pulmonary hypertension during lupus erythematosus]. / Ipertensione polmonare in corso di lupus eritematoso sistemico.

A patient with pulmonary hypertension (PH) during subacute cutaneous lupus erythematosus is described. Since the PH is characteristic of other connective tissue diseases (systemic sclerosis, Sharp syndrome), if a systemic lupus erythemtosus is coexistent, one should make a careful diagnostic screening (capillaroscopy, anti-centromere, or anti-Sc170, or anti-RNP antibodies) to show a possible overlap-syndrome.

[Asymptomatic hyper-creatine-phosphokinasemia: a veritable disease? Observation in some cases]. / L'ipercreatinfosfochinasemia asintomatica può essere considerata una vera malattia? A proposito di alcuni casi osservati.

Myopathy is a pathology of the muscle function, without clinical and instrumental signs that are involved the central nervous system and the peripheral one. In all pathologies causing a damage of the muscle fibres, a release of enzymes as GOT, GPT, LDH, aldolase, CPK occurs. The most significant enzyme for the muscle damage is CPK. Studies state that an increase of the CPKemia is not always an expression of muscle damages, but there are physiological changes in relation to the age, physical activities and racial variations. By the way, it is necessary to focus our attention on this matter. Authors, starting from the observation of lots of cases under their studies, have described two of them, that up to their careful observation could reveal significant.

[RANKL/RANK, OPG and OPT in a group of patients affected by chronic arthritis. Preliminary report]. / Il sistema RANK/RANK, osteoprotegerina e osteopontina in una coorte di soggetti affetti da poliartrite cronica. Studio preliminare.

Recently, receptor activator of nuclear factor-kappaB ligand (RANKL), its receptor RANK, and the decoy receptor osteoprotegerin (OPG), have been identified as paracrine mediator of bone functions. The balance of RANKL/RANK and OPG is critical for osteoclastogenesis modulation and physiological bone remodeling. Osteopontin (OPN) is an extracellular glycosylate bone phosphoprotein and acts both as chemokine and cytokine. It is produced by osteoclast, macrophages, T cells, hematopoietic cells and vascular smooth muscle cells. It is present particularly at high concentration in the lamina limitans and cement line, suggesting its role in the initiation and termination of the bone turnover. Chronic arthritis are a group of rheumatic pathologies characterized by periodical continuous or desultory use of corticosteroids. The main aims of this study are to evaluate OPG, RANKL and OPN serum concentrations in patients affected by chronic arthritis and to compare the above results with those ones obtained by young healthy population. OPG, RANKL and OPN serum concentration has been measured by ELISA method both in 40 patients affected by chronic arthritis then in young healthy population of 81 subjects. The differences between the two considered groups have been analyzed using unpaired T-Student. The difference between the two groups is significant for considered variables: OPG: t = -6.54, p < 0.001; RANKL: t = -2.71, p = 0.008; OPN: t = 2.55, p = 0.01. These results suggest that RANKL/RANK system,OPG and OPN have important role in patients with chronic arthritis.

Tumor necrosis factor inhibitors block apoptosis of human epithelial cells of the salivary glands.

Inhibition of tumor necrosis factor-alpha (TNF-alpha) in organ-specific autoimmune disease is proving efficacious for a large number of patients. A wide array of biological agents has been designed to inhibit TNF-alpha, such as adalimumab (fully humanized) and etanercept (soluble TNF-alpha receptor fusion constructs p75 subunit). Recently, we suggested that anti-Ro and anti-La autoantibodies (Abs) isolated from patients with Sjögren's syndrome, an autoimmune rheumatic disease, are able to trigger cell death through extrinsic apoptotic mechanisms in human salivary gland epithelial cells (SGEC). We analyzed if primary human SGEC cultures, established from biopsy of labial minor salivary glands, are able to produce TNF-alpha, an inductor of the extrinsic apoptotic pathway, when treated with anti-Ro autoantibodies. A comparative study was performed to test the efficacy of adalimumab and etanercept to block TNF-alpha-mediated apoptosis. ELISA assay and RT-PCR were employed to visualize TNF-alpha production, and apoptosis was evaluated by DNA ladder and flow cytometry. We found that cell treatment with anti-Ro autoantibodies determines TNF-alpha production that reaches a maximum at 16 h and is decreased (P < 0.05) at 24 and 48 h. Adalimumab seems to be more efficacious than etanercept in blocking TNF-alpha-mediated apoptosis. The YOPRO-1 (+) and propidium iodide (-) method revealed 60% of apoptotic cells after 24 h of incubation with anti-Ro compared with 15% of apoptotic cells treated with anti-Ro plus adalimumab and 25% of apoptotic cells treated with anti-Ro plus etanercept. The antiapoptotic effect of adalimumab and etanercept was supported by inhibition of DNA laddering induced by anti-Ro Abs. These data validate the therapeutic efficacy of the anti-TNF reagents in the treatment of autoimmune disorders.

Modulation of the Fcgamma receptors induced by anti-Ro and anti-La autoantibodies: observations in salivary gland cells.

Only few reports have shown protein expression of the Fcgamma receptors (FcgammaRs) molecules on human salivary gland cells. In this study we investigate a possible upregulation of FcgammaRs following anti-Ro and anti-La autoantibodies treatment. Anti-Ro and anti-La autoantibodies were purified from IgG fractions obtained from 14 patients with primary Sjögren's syndrome (SS), using Sepharose 4B-Ro and Sepharose 4B-La affinity columns. Flow cytometry and RT-PCR were used to study the FCgammaRI, FCgammaRII and FCgammaRIII receptors expression and upregulation by anti-Ro and anti-La on a salivary gland cell line. The present data document that the anti-Ro and anti-La autoantibodies determine an increase of the FcgammaRs expression on salivary gland cells, and provide evidence that both the high affinity FcgammaRI and the low affinity FcgammaRII and FcgammaRIII are overexpressed. Treatment with IgG isolated from healthy donors had no effect on the basal FCgammaRs expression.

Fcgamma receptors mediate internalization of anti-Ro and anti-La autoantibodies from Sjögren's syndrome and apoptosis in human salivary gland cell line A-253.

BACKGROUND: The presence of serum anti-Ro and anti-La autoantibodies directed against the ribonucleoproteins Ro and La has been associated with Sjögren's syndrome (SS), an autoimmune rheumatic disease that targets salivary and lachrymal glands. There is increasing evidence of the direct involvement of autoantibodies in the pathogenesis of tissue injury and correlation of their presence with clinical manifestations in SS. The focus of this work was to explore the cellular apoptotic pathway triggered by binding and penetration of anti-Ro and anti-La autoantibodies in human salivary gland cell line A-253 and to identify the membrane receptors through which anti-Ro and anti-La could exert their effect. METHODS: Anti-Ro and anti-La autoantibodies were purified from IgG fractions, obtained from eleven healthy volunteers and patients with primary Sjögren's syndrome, using Sepharose 4B-Ro and Sepharose 4B-La affinity columns. Flow cytometry, RT-PCR, western blot and confocal microscopy analysis were used to visualize the FCgammaRI, FCgammaRII and FCgammaRIII receptors on the A-253 cell membrane. DNA laddering and western blot analysis of caspases activation were studied to evaluate in A-253 cells treated with anti-Ro and anti-La autoantibodies. RESULTS: The results yeilded the evidence of the presence of members of the Fcgamma receptors (FcgammaRs) family on the cell membrane of the human salivary gland cell line A-253. Furthermore, we demonstrated that, in the A-253 cell line, anti-Ro and anti-La autoantibodies can access the cells probably through Fcgamma receptors, and trigger apoptotis. CONCLUSIONS: We conclude that anti-Ro and anti-La autoantibodies have pathogenic effects that could depend on binding to Fcgamma receptors.