Objectives Autoantibodies reactive with Ro52 are often found in sera of Sjögren’s syndrome (SS) patients. This study was undertaken to investigate the role of Ro52-induced immune responses in pathogenesis of SS. Methods New Zealand Mixed (NZM) 2758 mice were immunized with Ro52 in alum adjuvant. Control mice were immunized either with Maltose binding protein (MBP) or injected with alum alone. Mice were monitored for anti-Ro52 antibody, sialoadenitis and pilocarpine induced salivation. Antibody binding to salivary gland (SG) cells was analyzed in vivo and in vitro by immunofluorescence. Sera from immunized mice were passively transferred into untreated or alum injected NZM2758 mice. Results By day 30 post-immunization, Ro52 immunized mice generated immunoprecipitating anti-Ro52 antibodies and they had the maximum drop in saliva production. Both Ro52 immunized and control mice showed evidence of mild sialoadenitis. However, only Ro52 immunized mice had antibody deposition in their SG. Passive transfer of Ro52-immune sera induced SG dysfunction in recipient mice, only if the recipients were primed with alum. In vitro, antibodies from Ro52-immune sera were internalized by a SG cell line and this uptake was inhibited by Cytochalasin D treatment. Conclusion Our data shows for the first time that antibodies induced by Ro52 are capable of inducing SG dysfunction, and that this phenomenon is dependent on the activation of innate immunity. The mouse model described in this study implies that autoantibody deposition in the SG might be an important step in the induction of xerostomia and pathogenesis of SS.

No abstract

Objectives The serologic hallmark of primary Sjögren’s syndrome (pSS) is IgG antibodies specific for Ro (SSA) and La (SSB). Molecular characteristics of glandular-derived B cells at the site of pSS inflammation have been described; however, parallels between glandular antibody-secreting cells (ASC) and serologic antibody specificities have not been evaluated. We utilized recombinant monoclonal antibody (hmAb) technology to study salivary gland-(SG) derived ASC specificities, evaluating their molecular characteristics and identified IgG antibody specificity. Methods hmAbs were generated from glandular IgG ASC. Heavy and light chain usage and immunoglobulin subclass were analyzed by sequencing. ELISA, indirect immunofluorescence, enzyme immunoassay and 35S immunoprecipitation analysis were used to determine antibody specificity. Results Evaluation of single ASCs from SG biopsies of patients with primary SS or with SS and overlapping SLE revealed significant concordance between serum autoantibody and glandular ASC specificities. Glandular-derived ASC heavy and light chains were extensively somatically hypermutated, indicative of antigen-driven responses. Specifically, we produced the first fully human monoclonal autoantibodies derived from salivary glands in this study. Conclusions Salivary glands in SS patients are a site for antibody production, which extend beyond the canonical Ro and/or La SS specificities. Furthermore, we demonstrate that glandular antibody production strongly reflects the serological humoral response in the two patients studied herein.

Adenosine receptors are a subfamily of highly-conserved G-protein coupled receptors. They are found in the membranes of various human cells and play many physiological functions. Blood platelets express two (A2A and A2B) of the four known adenosine receptor subtypes (A1, A2A, A2B, and A3). Agonization of these receptors results in an enhanced intracellular cAMP and the inhibition of platelet activation and aggregation. Therefore, adenosine receptors A2A and A2B could be targets for anti-platelet therapy, especially under circumstances when classic therapy based on antagonizing the purinergic receptor P2Y12 is insufficient or problematic. Apart from adenosine, there is a group of synthetic, selective, longer-lasting agonists of A2A and A2B receptors reported in the literature. This group includes agonists with good selectivity for A2A or A2B receptors, as well as non-selective compounds that activate more than one type of adenosine receptor. Chemically, most A2A and A2B adenosine receptor agonists are adenosine analogues, with either adenine or ribose substituted by single or multiple foreign substituents. However, a group of non-adenosine derivative agonists has also been described. This review aims to systematically describe known agonists of A2A and A2B receptors and review the available literature data on their effects on platelet function.

Objective: Using 3KO (triple NOX [NADPH oxidase] knockout) mice (ie, NOX1 −/− /NOX2 −/− /NOX4 −/− ), we aimed to clarify the role of this family of enzymes in the regulation of platelets in vitro and hemostasis in vivo. Approach and Results: 3KO mice displayed significantly reduced platelet superoxide radical generation, which was associated with impaired platelet aggregation, adhesion, and thrombus formation in response to the key agonists collagen and thrombin. A comparison with single-gene knockouts suggested that the phenotype of 3KO platelets is the combination of the effects of the genetic deletion of NOX1 and NOX2, while NOX4 does not show any significant function in platelet regulation. 3KO platelets displayed significantly higher levels of cGMP—a negative platelet regulator that activates PKG (protein kinase G). The inhibition of PKG substantially but only partially rescued the defective phenotype of 3KO platelets, which are responsive to both collagen and thrombin in the presence of the PKG inhibitors KT5823 or Rp-8-pCPT-cGMPs, but not in the presence of the NOS (NO synthase) inhibitor L-NG-monomethyl arginine. In vivo, triple NOX deficiency protected against ferric chloride–driven carotid artery thrombosis and experimental pulmonary embolism, while hemostasis tested in a tail-tip transection assay was not affected. Procoagulatory activity of platelets (ie, phosphatidylserine surface exposure) and the coagulation cascade in platelet-free plasma were normal. Conclusions: This study indicates that inhibiting NOXs has strong antithrombotic effects partially caused by increased intracellular cGMP but spares hemostasis. NOXs are, therefore, pharmacotherapeutic targets to develop new antithrombotic drugs without bleeding side effects.