ObjectivesRheumatoid arthritis (RA)-specific anti-citrullinated protein/peptide antibodies (ACPAs) appear before disease onset and are associated with bone destruction. We aimed to dissect the role of ACPAs in osteoclast (OC) activation and to identify key cellular mediators in this process.MethodsPolyclonal ACPA were isolated from the synovial fluid (SF) and peripheral blood of patients with RA. Monoclonal ACPAs were isolated from single SF B-cells of patients with RA. OCs were developed from blood cell precursors with or without ACPAs. We analysed expression of citrullinated targets and peptidylarginine deiminases (PAD) enzymes by immunohistochemistry and cell supernatants by cytometric bead array. The effect of an anti-interleukin (IL)-8 neutralising antibody and a pan-PAD inhibitor was tested in the OC cultures. Monoclonal ACPAs were injected into mice and bone structure was analysed by micro-CT before and after CXCR1/2 blocking with reparixin.ResultsProtein citrullination by PADs is essential for OC differentiation. Polyclonal ACPAs enhance OC differentiation through a PAD-dependent IL-8-mediated autocrine loop that is completely abolished by IL-8 neutralisation. Some, but not all, human monoclonal ACPAs derived from single SF B-cells of patients with RA and exhibiting distinct epitope specificities promote OC differentiation in cell cultures. Transfer of the monoclonal ACPAs into mice induced bone loss that was completely reversed by the IL-8 antagonist reparixin.ConclusionsWe provide novel insights into the key role of citrullination and PAD enzymes during OC differentiation and ACPA-induced OC activation. Our findings suggest that IL8-dependent OC activation may constitute an early event in the initiation of the joint specific inflammation in ACPA-positive RA.

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Oxidation-associated malondialdehyde (MDA) modification of proteins can generate immunogenic neo-epitopes that are recognized by autoantibodies. In health, IgM antibodies to MDA-adducts are part of the natural antibody pool, while elevated levels of IgG anti-MDA antibodies are associated with inflammatory and autoimmune conditions. Yet, in human autoimmune disease IgG anti-MDA responses have not been well characterized and their potential contribution to disease pathogenesis is not known. Here, we investigate MDA-modifications and anti-MDA-modified protein autoreactivity in rheumatoid arthritis (RA). While RA is primarily associated with autoreactivity to citrullinated antigens, we also observed increases in serum IgG anti-MDA in RA patients compared to controls. IgG anti-MDA levels significantly correlated with disease activity by DAS28-ESR and serum TNF-alpha, IL-6, and CRP. Mass spectrometry analysis of RA synovial tissue identified MDA-modified proteins and revealed shared peptides between MDA-modified and citrullinated actin and vimentin. Furthermore, anti-MDA autoreactivity among synovial B cells was discovered when investigating recombinant monoclonal antibodies (mAbs) cloned from single B cells, and 3.5% of memory B cells and 2.3% of plasma cells were found to be anti-MDA positive. Several clones were highly specific for MDA-modification with no cross-reactivity to other antigen modifications such as citrullination, carbamylation or 4-HNE-carbonylation. The mAbs recognized MDA-adducts in a variety of proteins including albumin, histone 2B, fibrinogen and vimentin. Interestingly, the most reactive clone, originated from an IgG1-bearing memory B cell, was encoded by near germline variable genes, and showed similarity to previously reported natural IgM. Other anti-MDA clones display somatic hypermutations and lower reactivity. Importantly, these anti-MDA antibodies had significant in vitro functional properties and induced enhanced osteoclastogenesis, while the natural antibody related high-reactivity clone did not. We postulate that these may represent distinctly different facets of anti-MDA autoreactive responses.

ObjectivesRheumatoid arthritis (RA)-specific anti-citrullinated protein/peptide antibodies (ACPAs) might contribute to bone loss and arthralgia before the onset of joint inflammation. We aimed to dissect additional mechanisms by which ACPAs might contribute to development of joint pathology.MethodsFibroblast-like synoviocytes (FLS) were isolated from the synovial membrane of patients with RA. The FLS cultures were stimulated with polyclonal ACPAs (anti-CCP-2 antibodies) purified from the peripheral blood of patients with RA or with monoclonal ACPAs derived from single synovial fluid B cells. We analysed how ACPAs modulate FLS by measuring cell adhesion and mobility as well as cytokine production. Expression of protein arginine deiminase (PAD) enzymes and protein citrullination were analysed by immunofluorescence, and signal transduction was studied using immunoblotting.ResultsChallenge of FLS by starvation-induced stress or by exposure to the chemokine interleukin-8 was essential to sensitise the cells to ACPAs. These challenges led to an increased PAD expression and protein citrullination and an ACPA-mediated induction of FLS migration through a mechanism involving phosphoinositide 3-kinase activation. Inhibition of the PAD enzymes or competition with soluble citrullinated proteins or peptides completely abolished the ACPA-induced FLS migration. Different monoclonal ACPAs triggered distinct cellular effects in either fibroblasts or osteoclasts, suggesting unique roles for individual ACPA clones in disease pathogenesis.ConclusionWe propose that transient synovial insults in the presence of a certain pre-existing ACPA repertoire might result in an ACPA-mediated increase of FLS migration.

An increased repertoire of potential osteoclast (OC) precursors could accelerate the development of bone-erosive OCs and the consequent bone damage in rheumatoid arthritis (RA). Immature dendritic cells (DCs) can develop into OCs, however, the mechanisms underlying this differentiation switch are poorly understood. We investigated whether protein citrullination and RA-specific anti-citrullinated protein Abs (ACPAs) could regulate human blood-derived DC-OC transdifferentiation. We show that plasticity toward the OC lineage correlated with peptidyl arginine deiminase (PAD) activity and protein citrullination in DCs. Citrullinated actin and vimentin were present in DCs and DC-derived OCs, and both proteins were deposited on the cell surface, colocalizing with ACPAs binding to the cells. ACPAs enhanced OC differentiation from monocyte-derived or circulating CD1c + DCs by increasing the release of IL-8. Blocking IL-8 binding or the PAD enzymes completely abolished the stimulatory effect of ACPAs, whereas PAD inhibition reduced steady-state OC development, as well, suggesting an essential role for protein citrullination in DC-OC transdifferentiation. Protein citrullination and ACPA binding to immature DCs might thus promote differentiation plasticity toward the OC lineage, which can facilitate bone erosion in ACPA-positive RA.

Oxidized low-density lipoprotein (ox-LDL)-induced endothelial damage contributes to the initiation and pathogenesis of atherosclerosis. Salidroside can alleviate atherosclerosis and attenuate endothelial cell injury induced by ox-LDL. However, the mechanisms involved in this process are not fully understood. Therefore, the purpose of the present study was to investigate the role of the adenosine monophosphate-activated protein kinase (AMPK)/sirtuin (SIRT)1 pathway in the protection of salidroside against ox-LDL-induced human umbilical vein endothelial cells (HUVECs) injuries. The results revealed that salidroside reverses ox-LDL-induced HUVECs injury as demonstrated by the upregulation of cell viability and downregulation of LDH release. In addition, salidroside increased the expression of the SIRT1 protein in ox-LDL-treated HUVECs. Next, it was demonstrated that SIRT1 knockdown induced by transfection with small interfering (si)RNA targeting SIRT1 (siSRT1) abolished the protection of salidroside against ox-LDL-induced HUVECs injuries. This was illustrated by a decrease in cell viability and an increase in LDH release, caspase-3 activity and apoptosis rate. Furthermore, salidroside mitigated ox-LDL-induced reactive oxygen species production, upregulated malondialdehyde content and NADPH oxidase 2 expression and decreased superoxide dismutase and glutathione peroxidase activities, while these effects were also reversed by siSIRT1 transfection. In addition, it was demonstrated that salidroside suppressed ox-LDL-induced mitochondrial dysfunction as demonstrated by the increase in mitochondrial membrane potential and decreases in cytochrome c expression, and Bax/Bcl-2 reductions. However, these effects were eliminated by SIRT1 knockdown. Finally, it was demonstrated that salidroside significantly upregulated the phosphorylated-AMPK expression in ox-LDL-treated HUVECs and AMPK knockdown induced by transfection with AMPK siRNA (siAMPK) leads to elimination of the salidroside-induced increase in cell viability and the decrease in LDH release. Notably, siAMPK transfection further decreased the expression of SIRT1. In conclusion, these results suggested that salidroside protects HUVECs against ox-LDL injury through inhibiting oxidative stress and improving mitochondrial dysfunction, which were dependent on activating the AMPK/SIRT1 pathway.

In this study, we performed a study on 106 children with epilepsy who were treated with sodium valproate (the VPA group, n = 37), oxcarbazepine (the OXC group, n = 34), or levetiracetam (the LEV group, n = 35). In addition, the clinical data of epileptic children who were newly diagnosed in the same period without antiepileptic drug (AED) treatment (the untreated group, n = 35) and normal children who received physical examination in our hospital (the healthy group, n = 35) were selected as controls. We analyzed the efficacy and safety of different AEDs, used blood ammonia and homocysteine levels as the observation indicators, and calculated the incidence of hyperammonemia (VAH) and hyperhomocysteinemia (HHcy) treated with different AEDs. And, based on the effect of epilepsy status on the cognitive function of patients, we also analyzed the effect of different AED treatments on children’s cognitive function. Our results show that sodium valproate, oxcarbazepine, and levetiracetam are all effective in the treatment of children with epilepsy and can be used as the first-line choice of antiepileptic treatment for children with epilepsy. However, compared with sodium valproate, levetiracetam and oxcarbazepine have a lower incidence of adverse drug reactions and do not cause an increase in blood ammonia and Hcy levels, so they have higher safety of drug treatment. In addition, compared with sodium valproate, levetiracetam and oxcarbazepine have better recovery of cognitive function in children with epilepsy and so they have better application value.

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