Anti-carbamylated protein antibodies in systemic lupus erythematosus patients with articular involvement.

Objective Several studies have evaluated the prevalence of rheumatoid factor (RF) and anti-citrullinated proteins antibodies (ACPA) in systemic lupus erythematosus (SLE) patients but no data are available on the anti-carbamylated proteins (anti-CarP), a new biomarker for rheumatoid arthritis (RA). We evaluated the anti-CarP prevalence in SLE patients with joint involvement and the associations with different phenotypes. Methods Seventy-eight SLE patients with joint involvement were enrolled (F/M 73/5; mean ± SD age 47.6 ± 11.2 years; mean ± SD disease duration 214.3 ± 115.6 months). As control groups, we evaluated SLE patients without joint manifestations ( N = 15), RA ( N = 78) and healthy individuals (HS, N = 98). Anti-CarP were assessed by home-made ELISA in all patients and controls, RF and ACPA in SLE patients with joint involvement (commercial ELISA kit). Results The prevalence of anti-CarP in SLE patients with joint involvement was similar to RA ( p = NS) and significantly higher compared with SLE without joint involvement and HS ( p < 0.0001, p < 0.0001, respectively). Four patients were positive for all three antibodies: seventy-five percent of these showed Jaccoud arthropathy. Fourty-five percent of ACPA-ve/RF-ve patients were anti-CarP + ve. Conclusions The evaluation of anti-CarP in SLE joint involvement demonstrated a prevalence of almost 50%, similar to RA and significantly higher than SLE without joint involvement and HS.

Anti-carbamylated protein antibodies in unaffected first-degree relatives of rheumatoid arthritis patients: lack of correlation with anti-cyclic citrullinated protein antibodies and rheumatoid factor.

OBJECTIVES: To investigate the prevalence of anti-carbamylated protein antibodies (anti-CarP) in the healthy first-degree relatives (HFDRs) of patients with rheumatoid arthritis (RA). METHODS: We enrolled 141 HFDRs of 63 patients with RA diagnosed accordingly to the 2010 ACR/EULAR criteria. Fifty-six normal healthy subjects (NHS), sex- and age-matched, served as controls. Anti-CarP IgG, anti-cyclic citrullinated peptide antibody (anti-CCP) IgG and rheumatoid factors (RF) isotypes (IgG, IgA, IgM) were assessed by solid-phase ELISA. RESULTS: Anti-CarP were detectable in 13 HFDRs (9.2%), anti-CCP in 9 (6.3%), IgG-RF in 10 (7%), IgA-RF in 17 (12%), and IgM-RF in 13 (9.2%) HFDRs. Twenty-nine (46%) RA patients were positive for anti-CarP, 31 (49.2%) for anti-CCP, and 34 (53.9%) for RF. One NHS (1.7%) resulted positive for anti-CarP, none for anti-CCP and RF. Anti-CarP showed significantly higher serum levels in RA and HFDRs than in NHS (p<0.0001 and p=0.0012, respectively). A significant correlation between anti-CCP and RF were found among RA patients (p=0.0002), whereas no correlations were reported between autoantibodies tested in the HFDRs. CONCLUSIONS: Anti-CarP can be found in the sera of HFDRs of RA patients and their prevalence is significantly higher than in NHS. No correlation of anti-CarP with anti-CCP and RF antibodies in RA HFDRs was found.

Olanzapine-induced neutropenia in a patient with systemiclupus erythematosus: a role of FcγRIIIb polymorphism?

In this study, we report the case of a Chinese patient with systemic lupus erythematosus (SLE) who developed neutropenia after treatment by olanzapine for the SLE-related psychiatric symptoms. The relationship between agranulocytosis, SLE and olanzapine is still unknown. Fcγ receptor IIIb (FcγRIIIb) is a low-affinity receptor, constitutively expressed only by neutrophils; NA1 and NA2 have been identified as representing polymorphisms of FcγRIIIb. NA1 is associated with the incidence of autoimmune neutropenia and is particularly frequent in Asiatic ethnic groups. The Chinese patient resulted to be homozygous for NA1. We suggest that the presence of NA1 allele may be a predisposing factor to olanzapine-induced agranulocytosis in patients with SLE. Hence, the analysis of FcγRIIIb polymorphism should be investigated in other cases of antipsychotic-induced agranulocytosis.

Tofacitinib Decreases Autophagy of Fibroblast-Like Synoviocytes From Rheumatoid Arthritis Patients.

The pathway of Janus tyrosine kinases (JAKs) has a central role in the pathogenesis of Rheumatoid Arthritis (RA) by regulating multiple immune functions and cytokine production. The JAK inhibitor tofacitinib is effective in RA patients not responding to methotrexate or TNF-inhibitors. Since hyperactive autophagy has been associated with impaired apoptosis of RA fibroblast-like synoviocytes (FLS), we aimed to investigate the role of tofacitinib in modulating autophagy and apoptosis in these cells. FLS isolated from RA biopsies were cultured with tofacitinib in presence of autophagy inducer rapamycin and in serum deprivation condition. Levels of autophagy, apoptosis, and citrullinated proteins were analyzed by western blot, flow cytometry, immunocytofluorescence, and Real-Time PCR. Rapamycin induced an increase in RA-FLS autophagy while the levels of autophagy marker LC3-II were reduced after in vitro treatment with tofacitinib. The analysis of autophagic flux by specific fluorescence dye confirmed the reduction of autophagy in RA FLS. The treatment with tofacitinib did not influence apoptosis of RA FLS. Modulation of the autophagic process by tofacitinib did not significantly change citrullination. The results of this study demonstrate that tofacitinib is able to modulate autophagy of FLS contributing to its effectiveness in RA patients.

Reduction of autophagy and increase in apoptosis correlates with a favorable clinical outcome in patients with rheumatoid arthritis treated with anti-TNF drugs.

BACKGROUND: Autophagy has emerged as a key mechanism in the survival and function of T and B lymphocytes, and its activation was involved in apoptosis resistance in rheumatoid arthritis (RA). To investigate whether the relationship between autophagy and apoptosis may impact the response to the therapy, we analyzed ex vivo spontaneous autophagy and apoptosis in patients with RA subjected to treatment with anti-tumor necrosis factor (TNF) drugs and in vitro the effects of TNFα and anti-TNF drugs on cell fate. METHODS: Peripheral blood mononuclear cells (PBMCs) from 25 RA patients treated with anti-TNF drugs were analyzed for levels of autophagy marker LC3-II by western blot and for the percentage of annexin V-positive apoptotic cells by flow cytometry. The same techniques were used to assess autophagy and apoptosis after in vitro treatment with TNFα and etanercept in both PBMCs and fibroblast-like synoviocytes (FLS) from patients with RA. RESULTS: PBMCs from patients with RA responsive to treatment showed a significant reduction in LC3-II levels, associated with an increased apoptotic activation after 4 months of therapy with anti-TNF drugs. Additionally, the expression of LC3-II correlated with DAS28. TNFα was able to induce autophagy in a dose-dependent manner after 24 h of culture in RA PBMCs and FLS. Moreover, etanercept caused a significant reduction of autophagy and of levels of citrullinated proteins. CONCLUSIONS: Our results show how the crosstalk between autophagy and apoptosis can sustain the survival of immune cells, thus influencing RA progression. This suggests that inhibition of autophagy represents a possible therapeutic target in RA.

Anti-D4GDI antibodies activate platelets in vitro: a possible link with thrombocytopenia in primary antiphospholipid syndrome.

BACKGROUND: Thrombocytopenia is a manifestation associated with primary antiphospholipid syndrome (PAPS), and many studies have stressed the leading role played by platelets in the pathogenesis of antiphospholipid syndrome (APS). Platelets are highly specialized cells, and their activation involves a series of rapid rearrangements of the actin cytoskeleton. Recently, we described the presence of autoantibodies against D4GDI (Rho GDP dissociation inhibitor beta, ARHGDIB) in the serum of a large subset of SLE patients, and we observed that anti-D4GDI antibodies activated the cytoskeleton remodeling of lymphocytes by inhibiting D4GDI and allowing the upregulation of Rho GTPases, such as Rac1. Proteomic and transcriptomic studies indicate that D4GDI is very abundant in platelets, and small GTPases of the RHO family are critical regulators of actin dynamics in platelets. METHODS: We enrolled 38 PAPS patients, 15 patients carrying only antiphospholipid antibodies without clinical criteria of APS (aPL carriers) and 20 normal healthy subjects. Sera were stored at - 20 °C to perform an ELISA test to evaluate the presence of anti-D4GDI antibodies. Then, we purified autoantibodies anti-D4GDI from patient sera. These antibodies were used to conduct in vitro studies on platelet activation. RESULTS: We identified anti-D4GDI antibodies in sera from 18/38 (47%) patients with PAPS, in sera from 2/15(13%) aPL carriers, but in no sera from normal healthy subjects. Our in vitro results showed a significant 30% increase in the activation of integrin αIIbß3 upon stimulation of platelets from healthy donors preincubated with the antibody anti-D4GDI purified from the serum of APS patients. CONCLUSIONS: In conclusion, we show here that antibodies anti-D4GDI are present in the sera of PAPS patients and can prime platelet activation, explaining, at least in part, the pro-thrombotic state and the thrombocytopenia of PAPS patients. These findings may lead to improved diagnosis and treatment of APS.

Microparticles and autophagy: a new frontier in the understanding of atherosclerosis in rheumatoid arthritis.

Microparticles (MPs) are small membrane vesicles released by many cell types under physiological and pathological conditions. In the last years, these particles were considered as inert cell debris, but recently many studies have demonstrated they could have a role in intercellular communication. Increased levels of MPs have been reported in various pathological conditions including infections, malignancies, and autoimmune diseases, such as rheumatoid arthritis (RA). RA is an autoimmune systemic inflammatory disease characterized by chronic synovial inflammation, resulting in cartilage and bone damage with accelerated atherosclerosis increasing mortality. According to the literature data, also MPs could have a role in endothelial dysfunction, contributing to atherosclerosis in RA patients. Moreover many researchers have shown that a dysregulated autophagy seems to be involved in endothelial dysfunction. Autophagy is a reparative process by which cytoplasmic components are sequestered in double-membrane vesicles and degraded on fusion with lysosomal compartments. It has been shown in many works that basal autophagy is essential to proper vascular function. Taking into account these considerations, we hypothesized that in RA patients MPs could contribute to atherosclerosis process by dysregulation of endothelial autophagy process.

Role of alpha-synuclein in autophagy modulation of primary human T lymphocytes.

It has been demonstrated that α-synuclein can aggregate and contribute to the pathogenesis of some neurodegenerative diseases and it is capable of hindering autophagy in neuronal cells. Here, we investigated the implication of α-synuclein in the autophagy process in primary human T lymphocytes. We provide evidence that: (i) knocking down of the α-synuclein gene resulted in increased autophagy, (ii) autophagy induction by energy deprivation was associated with a significant decrease of α-synuclein levels, (iii) autophagy inhibition by 3-methyladenine or by ATG5 knocking down led to a significant increase of α-synuclein levels, and (iv) autophagy impairment, constitutive in T lymphocytes from patients with systemic lupus erythematosus, was associated with abnormal accumulation of α-synuclein aggregates. These results suggest that α-synuclein could be considered as an autophagy-related marker of peripheral blood lymphocytes, potentially suitable for use in the clinical practice.

Autoantibodies to the adenosine triphosphate synthase play a pathogenetic role in Alzheimer's disease.

It has become evident that an autoimmune component could play a role in Alzheimer's disease (AD) onset and/or progression. The aim of this study was to identify neuronal antigenic targets specifically recognized by serum autoantibodies and to investigate their cellular effects and their possible pathogenetic role. We identified, by an immunoproteomic approach using mouse brain proteins, the adenosine triphosphate (ATP) synthase ß subunit as a new autoantigen in AD. Using an ELISA assay we found that serum anti-ATP synthase autoantibodies were present in 38% of patients with AD, but in no age-matched healthy subjects or in patients with Parkinson's disease or atherosclerosis. Analytical cytology studies, using SH-SY5Y neuroblastoma cell line, showed that ATP synthase autoantibodies were capable of inducing the inhibition of ATP synthesis, alterations of mitochondrial homeostasis and cell death by apoptosis. These findings suggest that autoantibodies specific to ATP synthase can exert a pathogenetic role via a mechanism that brings into play the impairment of the extracellular ATP homeostasis and the alteration of mitochondrial function triggering cell death by apoptosis.