Post-translational modifications of the major linear epitope 169-190aa of Ro60 kDa autoantigen alter the autoantibody binding

Ro60 kDa is a member of the Ro/LaRNP ribonucleoprotein complex and its major linear B cell epitope, corresponding to the region 169–190aa, has been found to be the initial target of the autoimmune response in patients with systemic lupus erythematosus. This sequence contains one serine and two arginine amino acid residues, which can potentially be modified post‐translationally by phosphorylation or citrullination, respectively. The aim of this study was to develop an immunoassay for anti‐Ro60 kDa epitope antibody detection and to investigate the changes in the antigenicity of the Ro60 kDa epitope when it is post‐translationally modified, by either citrullination or phosphorylation. Peptide analogues corresponding to the unmodified form of the epitope, its phosphorylated form, and a form with both arginine residues citrullinated were synthesized. The peptide coating conditions were investigated and it was found that the use of highly hydrophilic surfaces increase the efficiency of the coating, as well as the sensitivity of the method for anti‐peptide antibody detection. All peptides were tested by the optimized enzyme‐linked immunosorbent assay (ELISA) against 119 sera from patients with primary Sjögren's syndrome, systemic lupus erythematosus and rheumatoid arthritis with anti‐Ro/SSA reactivity, 20 sera from patients with systemic diseases without anti‐Ro/SSA immune reactivity, as well as against 65 sera from normal individuals. A large proportion of the tested sera reacted against all three peptide analogues, although with a preference for the unmodified form of the epitope. In conclusion, post‐translational modifications of the major Ro60 kDa B cell epitope can alter the autoantibody binding.     

Preferential recognition of the phosphorylated major linear B-cell epitope of La/SSB 349-368 aa by anti-La/SSB autoantibodies from patients with systemic autoimmune diseases

Sera from patients with primary Sjögren Syndrome (pSS) or Systemic Lupus Erythematosus (SLE) often contain autoantibodies directed against La/SSB. The sequence 349–368aa represents the major B‐cell epitope of La/SSB, also it contains, at position 366, a serine aminoacid residue which constitutes the main phosphorylation site of the protein. In this study we investigated the differential recognition of the 349–368aa epitope and its phosphorylated form by antibodies found in sera from patients with systemic autoimmune diseases. Peptides corresponding to the sequence of the unphosphorylated (pep349–368aa) and the phosphorylated form (pep349–368aaPh) of the La/SSB epitope 349–368aa, as well as to a truncated form spanning the sequence 349–364aa and lacking the phosphorylation site (pep349–364aa), were synthesized. Sera from 53 patients with pSS and SLE with anti‐La/SSB specificity, 30 patients with pSS and SLE without anti‐La/SSB antibodies, 25 patients with rheumatoid arthritis and 32 healthy individuals were investigated by ELISA experiments. Autoantibodies to pep349–368aaPh were detected in sera of anti‐La/SSB positive patients with a higher prevalence compared to the pep349–368aa (66%versus 45%). Pep349–368aaPh inhibited the antibody binding almost completely (92%), while pep349–368aa inhibited the binding only partially (45%). Anti‐La/SSB antibodies presented a higher relative avidity for the phosphorylated than the unphosphorylated peptide. Immunoadsorbent experiments using the truncated peptide pep349–364aa indicated that the flowthrough showed a selective specificity for pep349–368aaPh, while the eluted antibodies reacted with both peptide analogues of the La/SSB epitope. These data suggest that sera from pSS and SLE patients with anti‐La/SSB reactivity possess autoantibodies that bind more frequently and with a higher avidity to the phosphorylated major B‐cell epitope of the molecule.     

Nitrite directly vasodilates hypoxic vasculature via nitric oxide-dependent and -independent pathways

Background and purpose:

It is postulated that nitrite requires reduction to nitric oxide in order to exert its relaxant effect upon isolated hypoxic vessels. Herein, we evaluate the relative contribution of nitric oxide and characterize the downstream mechanisms of nitrite-induced vasorelaxation.

Experimental approach:

Aortic rings were treated with pharmacological agents and exposed to hypoxia (<1% O₂). Following pre-constriction, nitrite (10 µM final) was added to appropriate baths; isometric tension was recorded throughout.

Key results:

Nitrite (under hypoxic conditions at physiological pH) is capable of exerting physiological effects that cannot be completely inhibited by the inhibitor of soluble guanylate cyclase (sGC), 1H [1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one or a nitric oxide scavenger (carboxy-2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide). Simultaneous blockade of both sGC and cyclooxygenase (COX) completely inhibited the response to nitrite. With regard to the nitric oxide-dependent component, we confirm that aldehyde oxidase, but not xanthine oxidase or endothelial nitric oxide synthase, was important for the actions of nitrite in our model.

Conclusions and implications:

Nitric oxide generated from nitrite is not exclusively responsible for the physiological actions observed in isolated hypoxic vessels. Nitrite operates via different pathways dependent on the presence or absence of endothelium to produce vasorelaxation. In intact vessels, both sGC and COX enzymes appear to be important. Irrespective of this difference in relaxation mechanism, nitrite is capable of producing the same maximum relaxation, regardless of the presence of endothelium. Having investigated possible nitrite reduction sites, we confirm that aldehyde oxidase is important for the actions of nitrite.     

Early coagulopathy in multiple injury: an analysis from the German Trauma Registry on 8724 patients

BACKGROUND: There is increasing evidence for acute traumatic coagulopathy occurring prior to emergency room (ER) admission but detailed information is lacking. PATIENTS AND METHODS: A retrospective analysis using the German Trauma Registry database including 17,200 multiple injured patients was conducted to determine (a) to what extent clinically relevant coagulopathy has already been established upon ER admission, and whether its presence was associated (b) with the amount of intravenous fluids (i.v.) administered pre-clinically, (c) with the magnitude of injury, and (d) with impaired outcome and mortality. Eight thousand seven hundred and twenty-four patients with complete data sets were screened. RESULTS: Coagulopathy upon ER admission as defined by prothrombin time test (Quick's value) <70% and/or platelets <100,000 microl(-1), was present in 34.2% of all patients. There was an increasing incidence for coagulopathy with increasing amounts of i.v. fluids administered pre-clinically. Coagulopathy was observed in >40% of patients with >2000 ml, in >50% with >3000 ml, and in >70% with >4000 ml administered. Ten percentage of patients presented with clotting disorders although pre-clinical resuscitation was limited to 500 ml of i.v. fluids maximum. The mean ISS score in the coagulopathy group was 30 (S.D. 15) versus 21 (S.D. 12) (p<0.001). Twenty-nine percentage of patients with coagulopathy developed multi organ failure (p<0.001). Early in-hospital mortality (<24h) was 13% in patients with coagulopathy (p<0.001) and overall in-hospital mortality totalled 28% (p<0.001). CONCLUSION: There is a high frequency of established coagulopathy in multiple injury upon ER admission. The presence of early traumatic coagulopathy was associated with the amount of intravenous fluids administered pre-clinically, magnitude of injury, and impaired outcome.     

Update on Sjögren's syndrome autoimmune epithelitis: from classification to increased neoplasias

Sjögren's syndrome is a chronic autoimmune disease that affects not only the exocrine glands but also various extraglandular sites. The syndrome can be found alone (primary) or in association with other autoimmune rheumatic diseases (secondary). The diagnosis is based on a combination of subjective and objective criteria, proposed by the American–European Study Group; the differential diagnosis includes diseases producing dry mouth, KCS, and major salivary gland enlargement. The initial clinical picture determines the outcome of patients Thus the periepithelial extraglandular manifestations are the result of lymphocytic invasion in epithelial tissues of lungs, kidneys, and liver; they appear early in the disease and have a benign course. The extraepithelial manifestations, such as skin vasculitis, peripheral neuropathy, and glomerulonephritis, with low C4 levels, are associated with increased morbidity and high risk for lymphoma. A strong predisposition seems to exist in these patients for the development of lymphoproliferation, especially for low-grade salivary gland MZ lymphomas. Therefore, the clinical follow-up of Sjögren's syndrome patients should include routine complement determination and serum immunoelectrophoresis to detect the possible emergence of a monoclonal B-cell population, susceptible to lymphoma development.

• Sjögren's syndrome is a chronic and, in the majority of patients, is a disease with benign course.

• Sjögren's syndrome can occur alone or in association with other autoimmune diseases.

• Characteristic autoantibodies are anti-Ro/SSA and La/SSB.

• The differential diagnosis includes all disease presenting with dry mouth, dry eye, or major salivary gland enlargement.

• The clinical picture at the presentation of the patient determines the outcome. Thus, periepithelial extraglandular manifestations appear early in the disease and have a benign course. Extraepithelial manifestations, with low C4 levels, are associated with increased morbidity and high risk of lymphoma.

• A physician examining a patient with Sjögren's syndrome for the first time should consider the presence of NHL if the patient displays clinical signs such as significant enlargement of the salivary glands, lymphadenopathy, splenomegaly, skin vasculitis, and peripheral neuropathy.

• NHLs in Sjögren's syndrome fall into two main categories: the first relates to the majority of patients who develop an indolent extranodal MZ lymphomas; the second relates to those developing high-grade aggressive lymphomas, such as de novo or secondary DLBCLs, which are only occasionally encountered in Sjögren's syndrome.

• Patients with Sjögren's syndrome with indolent salivary MALT lymphomas usually have a quite uncomplicated clinical course with a median overall survival of 6.4 years. The combination of rituximab plus CHOP has a significant clinical effect in DLBCL.

• At the initial presentation, it is important to identify clinical features that possibly predispose to histologic progression and parameters that address the efficacy of treatment in presenting high-grade transformation.

Neuroendocrine dysfunction in Sjogren's syndrome

Interactions among the immune, nervous and endocrine systems, which are mediated by hormones, neuropeptides, neurotransmitters, cytokines and their receptors, appear to play an important role in modulating host susceptibility and resistance to inflammatory disease. The neuroendocrine system has two main components: the central and the peripheral. The central compartment is located in the locus ceruleus, the brainstem centers of the autonomic system and the paraventricular nucleus; the peripheral mainly consists of the sympathetic/adrenomedullary system, the hypothalamic-pituitary-adrenal axis (HPA), the hypothalamic-pituitary-gonadal (HPG) axis, and the neuroendocrine tissue located in several organs throughout the body. Hormones and neuropeptides may influence the activities of lymphoid organs and cells via endocrine and local autocrine/paracrine pathways or alter the function of different cell types in target organs. Recent studies highlighted alterations of the neuroendocrine system in systemic autoimmune diseases, including rheumatoid arthritis, systemic lupus erythematosus and Sjogren's syndrome (SS). SS, a prototype autoimmune disorder, has a wide clinical spectrum, extending from organ involvement (autoimmune exocrinopathy) to systemic disease and B cell lymphoma. In SS, several functions of the neuroendocrine system are impaired. First, the HPA axis appears to be disturbed, since significantly lower basal ACTH and cortisol levels were found in patients with SS and were associated with a blunted pituitary and adrenal response to ovine corticotropin-releasing factor compared to normal controls. Second, HPG axis is also involved, since lack of estrogens is associated with human disease and the development of autoimmune exocrinopathy in several experimental models. Finally, exocrine glands are enriched with neuroendocrine-related molecules, adjacent to local autoimmune lesions. Certain clinical manifestations of the disease, including the sicca manifestations, easy fatigue, fibromyalgia and psychological disturbances can be very well explained by mechanisms directly related to disturbances of the neuroendocrine axis. On the other hand, the molecular and biochemical effects of the inflammatory molecules or cell-to-cell interaction, observed during the local or systemic autoimmune injury with cells and mediators of the neuroendocrine system, are largely unexplored.