Objectives Autoantibodies reactive with Ro52 are often found in sera of patients with Sjögren's syndrome (SS). 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 immunised with Ro52 in alum adjuvant. Control mice were immunised either with maltose-binding protein 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 analysed in vivo and in vitro by immunofluorescence. Sera from immunised mice were passively transferred into untreated or alum injected NZM2758 mice. Results By day 30 post-immunisation, Ro52 immunised mice generated immunoprecipitating anti-Ro52 antibodies and they had the maximum drop in saliva production. Both Ro52 immunised and control mice showed evidence of mild sialoadenitis. However, only Ro52 immunised 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 internalised by a SG cell line and this uptake was inhibited by cytochalasin D treatment. Conclusions Our data show 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.

Diabetes mellitus is the fifth most common cause of death worldwide. Due to its chronic nature, diabetes is a debilitating disease for the patient and a relevant cost for the national health system. Type 2 diabetes mellitus is the most common form of diabetes mellitus (90% of cases) and is characteristically multifactorial, with both genetic and environmental causes. Diabetes patients display a significant increase in the risk of developing cardiovascular disease compared to the rest of the population. This is associated with increased blood clotting, which results in circulatory complications and vascular damage. Platelets are circulating cells within the vascular system that contribute to hemostasis. Their increased tendency to activate and form thrombi has been observed in diabetes mellitus patients (i.e., platelet hyperactivity). The oxidative damage of platelets and the function of pro-oxidant enzymes such as the NADPH oxidases appear central to diabetes-dependent platelet hyperactivity. In addition to platelet hyperactivity, endothelial cell damage and alterations of the coagulation response also participate in the vascular damage associated with diabetes. Here, we present an updated interpretation of the molecular mechanisms underlying vascular damage in diabetes, including current therapeutic options for its control.

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 (A_2A and A_2B) of the four known adenosine receptor subtypes (A₁, A_2A, A_2B, and A₃). Agonization of these receptors results in an enhanced intracellular cAMP and the inhibition of platelet activation and aggregation. Therefore, adenosine receptors A_2A and A_2B 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 A_2A and A_2B receptors reported in the literature. This group includes agonists with good selectivity for A_2A or A_2B receptors, as well as non-selective compounds that activate more than one type of adenosine receptor. Chemically, most A_2A and A_2B 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 A_2A and A_2B receptors and review the available literature data on their effects on platelet function.

Objective: The serologic hallmark of primary Sjögren's syndrome (SS) is the presence of IgG antibodies specific for Ro (SSA) and La (SSB). The molecular characteristics of gland‐derived B cells at the site of primary SS inflammation have been described previously; however, parallels between glandular antibody‐secreting cells (ASCs) and serologic antibody specificities have not been evaluated. We used recombinant monoclonal antibody (mAb) technology to study the specificities of salivary gland (SG)–derived ASCs, evaluate their molecular characteristics, and identify IgG antibody specificity.Methods: Human antibodies were generated from glandular IgG ASCs. Heavy chain and light chain use and immunoglobulin subclass were analyzed by sequencing. Enzyme‐linked immunosorbent assay, indirect immunofluorescence, enzyme immunoassay, and ³⁵S‐labeled protein immunoprecipitation analysis were used to determine antibody specificity.Results: Evaluation of single ASCs in SG biopsy specimens from a patient with primary SS and a patient with SS and overlapping systemic lupus erythematosus revealed significant concordance between serum autoantibody and glandular ASC specificities. Gland‐derived ASC heavy chains and light chains were extensively somatically hypermutated, which is indicative of antigen‐driven responses. Specifically, we produced the first fully human mAb derived from SGs.Conclusion: In patients with SS, the SGs are a site for the production of antibodies that extend beyond the canonical Ro and/or La SS specificities. Glandular antibody production strongly reflected the serologic humoral response in the 2 patients whom we studied.

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.

Platelets contain high levels of amyloid β (Aβ) peptides and have been suggested to participate in the deposition of amyloid plaques in Alzheimer’s disease. This study aimed to determine whether human platelets release pathogenic Aβ peptides Aβ_1-42 and Aβ_1-40 and to characterize the mechanisms regulating this phenomenon. Enzyme-linked immunosorbent assays (ELISAs) and single platelet immunostaining were utilised to study the release and subcellular localisation of Aβ, respectively. ELISAs revealed that thrombin and the pro-inflammatory molecule lipopolysaccharide (LPS) induce platelet release of both Aβ_1-42 and Aβ_1-40. Notably, LPS preferentially induced the release of Aβ_1-42, which was potentiated by the reduction of oxygen from atmospheric levels to physiological hypoxia. The selective β secretase (BACE) inhibitor LY2886721 showed no effect on the release of either Aβ_1-40 or Aβ_1-42 in our ELISA experiments. This suggested a store-and-release mechanism that was confirmed in immunostaining experiments showing co-localisation of cleaved Aβ peptides with platelet alpha granules. Taken together, our data suggest that human platelets release pathogenic Aβ peptides as a result of a store-and-release mechanism rather than a de novo proteolytic event. Although further studies are required to fully characterize this phenomenon, we suggest the possibility of a role for platelets in the deposition of Aβ peptides and the formation of amyloid plaques. Interestingly, the combination of hypoxia and inflammation that we simulated in vitro with reduced oxygen tension and LPS may increase the release of fibrillogenic Aβ_1-42 and, consequently, exacerbate amyloid plaque deposition in the brain of patients with Alzheimer’s disease.