Chaperone patterns in vernal keratoconjunctivitis are distinctive of cell and Hsp type and are modified by inflammatory stimuli.

BACKGROUND: Vernal keratoconjunctivitis (VKC) is a severe ocular allergy with pathogenic mechanism poorly understood and no efficacious treatment. The aims of the study were to determine quantities and distribution of Hsp chaperones in the conjunctiva of VKC patients and assess their levels in conjunctival epithelial and fibroblast cultures exposed to inflammatory stimuli. METHODS: Hsp10, Hsp27, Hsp40, Hsp60, Hsp70, Hsp90, Hsp105, and Hsp110 were determined in conjunctiva biopsies from nine patients and nine healthy age-matched normal subjects, using immunomorphology and qPCR. Conjunctival epithelial cells and fibroblasts were cultured and stimulated with IL-1ß, histamine, IL-4, TNF-α, or UV-B irradiation, and changes in Hsp levels were determined by Western blotting. RESULTS: Hsp27, Hsp40, Hsp70, and Hsp90 levels increased in the patients' conjunctiva, whereas Hsp10, Hsp60, Hsp100, and Hsp105 did not. Double immunofluorescence demonstrated colocalization of Hsp27, Hsp40, Hsp70, and Hsp90 with CD68 and tryptase. Testing of cultured conjunctival cells revealed an increase in the levels of Hsp27 in fibroblasts stimulated with IL-4; Hsp40 in epithelial cells stimulated with IL-4 and TNF-α and in fibroblasts stimulated with IL-4, TNF-α, and IL-1ß; Hsp70 in epithelial cells stimulated with histamine and IL-4; and Hsp90 in fibroblasts stimulated with IL-1ß, TNF-α, and IL-4. UV-B did not induce changes. CONCLUSIONS: VKC conjunctiva displays distinctive quantitative patterns of Hsps as compared with healthy controls. Cultured conjunctival cells respond to cytokines and inflammatory stimuli with changes in the Hsps quantitative patterns. The data suggest that interaction between the chaperoning and the immune systems drives disease progression.

Clinical implications of autoantibody screening in patients with autoimmune myositis.

OBJECTIVE: To evaluate the clinical usefulness of serum autoantibody profiling in patients with autoimmune myositis. METHODS: We retrospectively studied 74 consecutive patients: 68 had definite or probable myositis according to Bohan-Peter criteria, six suffered from antisynthetase syndrome with subclinical myopathy. Myositis specific antibodies (MSA) (anti-ARS, -SRP, -Mi-2) were determined by RNA immunoprecipitation or immunoblot, myositis associated antibodies (MAA) (anti-RoRNP, -U1RNP, -PM/Scl, -Ku) by immunoblot. RESULTS: Forty-three patients (58%) were positive for MSA: anti-Jo-1 in 15/27 polymyositis (PM) (55%), 4/33 dermatomyositis (DM) (12%), 1/8 overlap (12%) and 2/6 antisynthetase syndrome (33%); anti-ARS non-Jo-1 in 1/27 PM (4%), 2/33 DM (6%) and 4/6 antisynthetase syndrome (67%); anti-Mi-2 in 1/27 PM (4%) and 11/33 DM (33%); anti-SRP in 3/27 PM (11%) and 1/33 DM (3%). One patient was anti-Jo-1/Mi-2 positive, one anti-Jo-1/SRP positive. Moreover, 27 patients (36%) were positive for MAA: anti-Ro/SSA in 8/27 PM (30%), 7/33 DM (21%), 1/8 overlap (12%), and 3/6 antisynthetase syndrome (50%); anti-U1RNP in 1/27 PM (3.7%), 1/33 DM (3%), and 2/8 overlap (25%); anti-PM/Scl in 2/8 overlap (25%), anti-Ku in 2/8 overlap (25%). Anti-Jo-1 was predominantly associated with PM, anti-Mi-2 was almost exclusively found in DM patients. Anti-ARS antibodies were closely associated with interstitial lung disease and polyarthritis; notably, anti-ARS non-Jo-1 was more frequent in patients without overt muscle alterations. Anti-Ro/SSA antibody was not associated with any disease subset, but significantly more frequent in antisynthetase syndrome. CONCLUSIONS: Searching for MSA and MAA in patients with autoimmmune myositis is recommended because of its diagnostic and clinical value. Anti-ARS non-Jo-1 antibodies seem to preferentially target patients with pulmonary fibrosis without overt myopathy.

[Myositis specific and myositis associated autoantibodies in idiopathic inflammatory myopathies: a serologic study of 46 patients]. / Gli anticorpi miosite specifici e miosite associati nelle miopatie infiammatorie idiopatiche: studio sierologico di 46 pazienti.

OBJECTIVE: To characterize serum autoantibody profiles of patients with idiopathic inflammatory myopathies (IIM) by searching for myositis-specific (MSA) and myositis-associated (MAA) antibodies with sensitive and specific laboratory tests. METHODS: We tested the sera from 46 Caucasian patients diagnosed as affected with IIM at the Division of Rheumatology of Padova University (21 polimyositis, PM; 22 dermatomyositis, DM; 3 myositis overlap syndrome). All patients had definite IIM according to the criteria of Bohan-Peter. MSA including anti-tRNA synthetase (anti-Jo-1 and others) and anti-Mi-2 were determined by RNA immunoprecipitation and a modified immunoblot test, respectively. MAA (-U1RNP, -U2RNP, RoRNP, PM/Scl, Ku) were detected by counterimmunoelectrophoresis and immunoblot. RESULTS: Serum MSA and/or MAA were found in 30/46 (65%) patients with IIM. Twenty-three patients (50%) were positive for at least one MSA: anti-Jo-1 in 15 (33%), anti-Mi-2 in 6 (13%), and other anti-tRNA synthetase in 3 (6%). One patient was anti-Jo-1/Mi-2 positive. Moreover, 18 patients (39%) were positive for at least one MAA: anti-Ro/SSA in 13 (28%), anti-U1RNP in 4 (9%), anti-PM/Scl in 1 (2%) and anti-Ku in 1 (2%). Coexisting MSA and MAA were observed in 8 patients (17%), anti-Jo-1/SSA positive in most cases. Anti-Jo-1 was predominantly associated with PM (57% in PM vs 14% in DM), whereas anti-Mi-2 was exclusively found in DM patients (27%). Anti-synthetase antibodies were closely associated with interstitial lung disease and polyarthritis; anti-Mi-2 positive DM patients did not have lung involvement. Notably, anti-Ro/SSA antibody was frequently observed and almost equally detected in either PM or DM (about 30%): in more than 50% of cases the antibody was associated with one MSA. CONCLUSIONS: By means of analytically reliable methods, MSA was detected in 50% of our IIM patients. Searching for MSA in patients with IIM is recommended because of its diagnostic and prognostic value.

Pregnancy in rare autoimmune rheumatic diseases: UCTD, MCTD, myositis, systemic vasculitis and Beçhet disease.

The physiological adaptation of the immune system to pregnancy can potentially affect the course of all autoimmune rheumatic diseases (ARD), conversely the autoimmune processes characteristic of these conditions may compromise the foetal outcome. Unfortunately, very few reports on pregnancy outcome in patients with rare ARD are available. In this paper, we briefly review the data published until now on these disorders. Some general guidelines which were elaborated for more prevalent ARD seem to be valid also for such rare conditions: 1) patients should be correctly informed on the risk of becoming pregnant; 2) pregnancies should be planned when the disease is in remission since it increases the probability of successful maternal and foetal outcome; 3) patients should be regularly monitored during gestation and postpartum by a multidisciplinary team including rheumatologist, obstetrician, and neonatologist; 4) in the case of disease relapse an adequate treatment, even aggressive if necessary, should be recommended since active disease can be more detrimental for foetus than drugs; 5) pregnancies complicated by the onset of rare ARD have a particularly severe prognosis; in these cases a prompt treatment and very close clinical surveillance are indicated.

Antinucleosome antibodies in SLE: a two-year follow-up study of 101 patients.

We prospectively analyzed the diagnostic sensitivity and specificity as well as the clinical relevance of antinucleosome antibodies in SLE. One hundred and one consecutive SLE patients were followed for 3 years. Three serial serum samples from each patient were tested for antinucleosome antibodies by ELISA (optimum cut-off value 10 U/ml), and for anti-dsDNA antibody (by ELISA and IIF on Crithidia luciliae), and anti-dsDNA avidity (by Scatchard plot analysis). Sera from 100 healthy individuals (HI), 35 patients with systemic sclerosis (SSc), 30 with primary Sjögren's syndrome (SS), 20 with rheumatoid arthritis (RA) and 48 with infectious diseases (ID), were assayed as controls. SLE activity and damage were evaluated using the ECLAM score and the SLICC/ACR index. At baseline, antinucleosome antibodies were found in 87 patients with SLE (86.1%), in 8 patients with SSc (22.8%), in 2 HI (2%), and in 1 ID (2.1%). The sensitivity and specificity of antinucleosome testing for SLE were 86.1% and 95.3%, respectively. The prevalence of antinucleosome antibodies in SLE was significantly higher than that of anti-dsDNA antibodies, with a correlation between them. No relevant relationship was found between antinucleosome antibodies and disease features, including renal involvement, disease activity, and disease damage. During follow-up, no significant variation was observed in antinucleosome level, nor in anti-dsDNA antibody level or avidity. We conclude that antinucleosome antibodies are commonly found in SLE. Low antibody levels can be detected in SSc, whereas medium/high levels are highly specific for SLE. Their clinical relevance during the disease course and utility for monitoring the individual patient seem to be poor.

Ecto-ATPase activity of alpha-sarcoglycan (adhalin).

alpha-Sarcoglycan is a component of the sarcoglycan complex of dystrophin-associated proteins. Mutations of any of the sarcoglycan genes cause specific forms of muscular dystrophies, collectively termed sarcoglycanopathies. Importantly, a deficiency of any specific sarcoglycan affects the expression of the others. Thus, it appears that the lack of sarcoglycans deprives the muscle cell of an essential, yet unknown function. In the present study, we provide evidence for an ecto-ATPase activity of alpha-sarcoglycan. alpha-Sarcoglycan binds ATP in a Mg2+-dependent and Ca2+-independent manner. The binding is inhibited by 3'-O-(4-benzoyl)benzoyl ATP and ADP. Sequence analysis reveals the existence of a consensus site for nucleotide binding in the extracellular domain of the protein. An antibody against this sequence inhibits the binding of ATP. A dystrophin.dystrophin-associated protein preparation demonstrates a Mg-ATPase activity that is inhibited by the antibody but not by inhibitors of endo-ATPases. In addition, we demonstrate the presence in the sarcolemmal membrane of a P2X-type purinergic receptor. These data suggest that alpha-sarcoglycan may modulate the activity of P2X receptors by buffering the extracellular ATP concentration. The absence of alpha-sarcoglycan in sarcoglycanopathies leaves elevated the concentration of extracellular ATP and the persistent activation of P2X receptors, leading to intracellular Ca2+ overload and muscle fiber death.