Major vault protein/lung resistance related protein: a novel biomarker for rheumatoid arthritis.

OBJECTIVES: Rheumatoid arthritis (RA) can lead to joint destruction and early institution of effective treatment can preserve joint function. Biomarkers can establish early diagnosis and predict effect of treatment. Vault particles, large cytoplasmic ribonucleoprotein particles that participate in inflammation, might serve as biomarkers. The aim of this study was to assess the diagnostic and the prognostic value of major vault protein (MVP) and their antibodies in RA. METHODS: Serum samples from 159 RA patients, 26 early RA (ERA) patients, 21 patients with osteoarthritis (OA) and 30 healthy individuals were tested for MVP, anti-cyclic citrullinated peptide (anti-CCP) and C-reactive protein (CRP) using enzyme-linked immunosorbent assays (ELISA). Rheumatoid factor (RF) was tested by nephelometry, and anti-MVP antibodies were detected by anti-MVP peptide ELISA using an in-house protocol. RESULTS: MVP levels were higher in RA and ERA, compared to OA and healthy controls (p<0.00001). A combination of MVP with RF or anti-CCP showed an improved diagnostic accuracy compared to RF or anti-CCP alone in RA and ERA. MVP exhibited similar AUC levels to anti-CCP and RF in RA whereas in ERA, MVP exhibited the same or slightly higher AUC levels, compared to anti-CCP and RF, respectively. High MVP levels were associated with lack of response to treatment. Levels of anti-MVP peptide 2 antibodies were significantly higher in RA compared to healthy controls (t= 2.73, p=0.007). CONCLUSIONS: MVP and autoantibodies against MVP may have the potential to serve as diagnostic and prognostic biomarkers in RA.

Evaluation of Protective Efficacy of Selected Immunodominant B-Cell Epitopes within Virulent Surface Proteins of Streptococcus pneumoniae.

Four previously identified immunodominant B-cell epitopes, located within known virulent pneumococcal proteins CbpD, PhtD, PhtE, and ZmpB, had shown promising in vivo immunological characteristics, indicating their potential to be used as vaccine antigens. In this study, we further evaluated the opsonophagocytic activity of antibodies against these epitopes and their capacity to protect mice from pneumococcal sepsis. An opsonophagocytic killing assay (OPKA) revealed that OPKA titers of human anti-peptide antibodies against pneumococcal serotypes 1, 3, and 19A were significantly higher (P < 0.001) than those of the control sera, suggesting their functional potential against virulent clinical isolates. Data obtained from mice actively immunized with any of the selected epitope analogues or with a mixture of these (G_Mix group) showed, compared to controls, enhanced survival against the highly virulent pneumococcal serotype 3 (P < 0.001). Moreover, passive transfer of hyperimmune serum from G_Mix to naive mice also conferred protection to a lethal challenge with serotype 3, which demonstrates that the observed protection was antibody mediated. All immunized murine groups elicited gradually higher antibody titers and avidity, suggesting a maturation of immune response over time. Among the tested peptides, PhD_pep19 and PhtE_pep40 peptides, which reside within the zinc-binding domains of PhtD and PhtE proteins, exhibited superior immunological characteristics. Recently it has been shown that zinc uptake is of high importance for the virulence of Streptococcus pneumoniae; thus, our findings suggest that these epitopes deserve further evaluation as novel immunoreactive components for the development of a polysaccharide-independent pneumococcal vaccine.

In silico analysis and in vitro evaluation of immunogenic and immunomodulatory properties of promiscuous peptides derived from Leishmania infantum eukaryotic initiation factor.

It is generally considered as imperative the ability to control leishmaniasis through the development of a protective vaccine capable of inducing long-lasting and protective cell-mediated immune responses. In this current study, we demonstrated potential epitopes that bind to H2 MHC class I and II molecules by conducting the in silico analysis of Leishmania infantum eukaryotic Initiation Factor (LieIF) protein, using online available algorithms. Moreover, we synthesized five peptides (16-18 amino acids long) which are part of the N-terminal portion of LieIF and contain promising MHC class I and II-restricted epitopes and afterwards, their predicted immunogenicity was evaluated in vitro by monitoring peptide-specific T-cell responses. Additionally, the immunomodulatory properties of these peptides were investigated in vitro by exploring their potential of inducing phenotypic maturation and functional differentiation of murine Bone-Marrow derived Dendritic Cells (BM-DCs). It was revealed by our data that all the synthetic peptides predicted for H2 alleles; present the property of immunogenicity. Among the synthetic peptides which contained T-cell epitopes, the peptide 52-68 aa (LieIF_2) exhibited immunomodulatory properties with the larger potential. LieIF_2-pulsed BM-DCs up-regulated the expression of the co-stimulatory surface molecules CD80 and CD86, as well as the production of the proinflammatory cytokine TNF-α and of the Th1-polarizing cytokines IL-12 and IFN-γ. The aforementioned data suggest that selected parts of LieIF could be used to develop innovative subunit protective vaccines able to induce effective immunity mediated by MHC class I-restricted as well as class II-restricted T-cell responses.

Association Between Soluble Cell Adhesion Molecules (sP-Selectin, sE-Selectin, and sICAM-1) and Antibodies Against the Antigens of Proteus mirabilis in Rheumatoid Arthritis Patients.

Objective The purpose of this study was to examine the association between the serum concentration of soluble cell adhesion molecules (CAMs) and antibodies against antigens of Proteus mirabilis (P. mirabilis) in rheumatoid arthritis (RA) patients, taking into consideration the implication of P. mirabilis in the etiopathogenesis of RA. Methods The serum levels of soluble P-selectin (sP-selectin), soluble E-selectin (sE-selectin), and soluble intercellular adhesion molecule-1 (sICAM-1) were determined by sandwich enzyme-linked immunosorbent assay (ELISA) in 59 RA patients and 36 healthy controls. Using the same ELISA method, the serum levels of class-specific antibodies against hemolysin (HpmB), urease C (UreC), and urease F (UreF) enzymes of P. mirabilis were also measured.  Results In this study, increased levels of sP-selectin and sICAM-1 were observed in RA patients, while the levels of sE-selectin were increased in comparison with healthy controls but did not present a statistically significant difference. Moreover, increased levels of antibodies against HpmB, UreC, and UreF of P. mirabilis were found. Additionally, it was observed that the sE-selectin levels presented a significant correlation with IgG antibodies against the UreF antigen (there is no corresponding antigen in human tissue) in all the RA patients. A statistically significant correlation was observed between levels of soluble CAMs and antibodies against P. mirabilis in the different subgroups. Conclusion The observed correlation between soluble CAMs and antibodies against antigens of P. mirabilis, specifically in the subgroup of biologic therapy, indicates that P. mirabilis exists and provokes refractory in the treatment of RA.

Serum ß-Defensin 2, A Novel Biomarker for the Diagnosis of Acute Infections.

BACKGROUND: Defensins are natural antimicrobial peptides that the human body secretes to protect itself from an infection. Thus, they are ideal molecules to serve as biomarkers for infection. This study was conducted to evaluate the levels of human ß-defensins in patients with inflammation. METHODS: CRP, hBD2 and procalcitonin were measured in 423 sera of 114 patients with inflammation and healthy individuals using nephelometry and commercial ELISA assays. RESULTS: Levels of hBD2 in the serum of patients with an infection were markedly elevated compared to those of hBD2 in patients with inflammation of non-infectious etiology (p < 0.0001, t = 10.17) and healthy individuals. ROC analysis demonstrated that hBD2 showed the highest detection performance for infection (AUC 0.897; p < 0.001) followed by PCT (AUC 0.576; p = ns) and CRP (AUC 0.517; p = ns). In addition, analysis of hBD2 and CRP in patients' sera collected at different time points showed that hBD2 levels could help differentiate inflammation of infectious and non-infectious etiology during the first 5 days of hospitalization, while CRP levels could not. CONCLUSIONS: hBD2 has the potential to serve as a diagnostic biomarker for infection. In addition, the levels of hBD2 may reflect the efficacy of antibiotic treatment.

Investigating the Role of Anti-TPO Antibodies in HIV-Associated Thrombocytopenia before and after Initiation of HAART: A Case-Control Longitudinal Study.

This longitudinal, case-control study aimed to investigate the role of thrombopoietin (TPO) and anti-TPO antibodies in HIV-associated thrombocytopenia, focusing on the changes seen before and after the initiation of highly active antiretroviral therapy (HAART). Patients were assessed before and at least six months after the initiation of HAART. In total, 75 PLWHIV (age/sex-matched and randomized at 2:1, according to thrombocytopenia status) were included in this study. The baseline assessment revealed significantly higher TPO levels in thrombocytopenic patients (140.45 vs. 106.8 mg/mL, p = 0.008). Furthermore, anti-TPO-positive patients displayed lower platelet counts (109,000 vs. 139,000/L, p = 0.002) and TPO levels (114.7 vs. 142.7 mg/mL, p = 0.047). Longitudinally, HAART initiation reduced the frequency of thrombocytopenia from 75.47% to 33.96% (p < 0.001) and elevated the median platelet counts from 131,000 to 199,000 (p < 0.001). No significant difference in median platelet counts was found post-HAART among the anti-TPO subgroups (p = 0.338), a result contrasting with pre-HAART findings (p = 0.043). Changes in anti-TPO status corresponded with significant platelet count alterations (p = 0.036). Notably, patients who became anti-TPO negative showed a median increase of 95,000 platelets (IQR: 43,750-199,500). These marked differences between subgroups underscore the potential role of anti-TPO antibodies in modulating the hematological response to HAART. Further research is needed to elucidate the complex interplay between HIV infection, HAART, and thrombocytopenia.

{beta}2 Glycoprotein I ({beta}2GPI) binds platelet factor 4 (PF4): implications for the pathogenesis of antiphospholipid syndrome.

Antiphospholipid syndrome (APS) is an autoimmune thrombophilia characterized by arterial/venous thrombosis and/or pregnancy morbidity in the presence of antiphospholipid antibodies that mainly recognize beta2 glycoprotein I (beta2GPI). To investigate potential platelet ligands of beta2GPI, platelet membrane proteins from healthy persons and patients with APS were passed through a beta2GPI-affinity column. By using mass spectrometry, platelet factor 4 (PF4) appeared as the dominant beta2GPI binding protein. PF4 could bind in vitro, with high-affinity, recombinant beta2GPI, and the binding was abrogated by soluble beta2GPI. Coprecipitation experiments further confirmed this interaction. In silico molecular docking showed that PF4 tetramers can bind 2 beta2GPI molecules simultaneously. Size exclusion chromatography confirmed that anti-beta2GPI antibodies selectively interact with complexes composed of (beta2GPI)(2)-(PF4)(4). In addition, as shown by the beta2GPI antigenicity evaluation, the reactivity of APS sera was higher against PF4-beta2GPI complex than against beta2GPI alone. On complex formation, anti-beta2GPI-beta2GPI-PF4 significantly induced platelet p38MAPK phosphorylation and TXB2 production, mainly through F(ab')(2) fragments of antibodies. In summary, this study makes evident that beta2GPI forms stable complexes with PF4, leading to the stabilization of beta2GPI dimeric structure that facilitates the antibody recognition. This interaction can probably be involved in the procoagulant tendency of APS.

Association of the idiotype:antiidiotype antibody ratio with the efficacy of intravenous immunoglobulin treatment for the prevention of recurrent autoimmune-associated congenital heart block.

OBJECTIVE: Congenital heart block (CHB), a manifestation of neonatal lupus, is associated with maternal anti-Ro/SSA and anti-La/SSB autoantibodies and recurs in ∼18% of subsequent pregnancies. This study was undertaken to investigate the effect of the idiotype:antiidiotype (Id:anti-Id) antibody ratio in the ability of intravenous immunoglobulin (IVIG) administered during subsequent pregnancies to prevent CHB. METHODS: We studied 16 anti-Ro/SSA and anti-La/SSB-positive pregnant women from the Preventive IVIG Therapy for Congenital Heart Block study who had previously given birth to a child with neonatal lupus. In 3 of the mothers, the study pregnancy resulted in the birth of a child with neonatal lupus (2 with CHB and 1 with rash). Sequential serum samples were obtained from all mothers immediately before the administration of IVIG during pregnancy and were evaluated for antibodies against the major B cell epitope 349-364aa of La/SSB (idiotype) and its antiidiotypic antibodies. RESULTS: Following IVIG treatment, serum titers of anti-La(349-364) (Id antibodies) decreased in 80% of the mothers, and in 60% an increase in anti-Id antibodies against anti-La(349-364) was observed. The Id:anti-Id ratio was significantly higher in mothers whose offspring developed neonatal lupus compared to mothers who gave birth to a healthy child (P<0.0001). Removal of anti-Id antibodies substantially increased the reactivity against La(349-364) in sera from 5 of 7 mothers tested. All IVIG preparations were examined for Id and anti-Id antibody activity. IVIG from batches administered to mothers who gave birth to a healthy child had an Id:anti-Id activity ratio of <1, in contrast to that given to mothers who gave birth to a child with neonatal lupus. Addition of the IVIG preparations to the maternal sera further enhanced antiidiotypic activity (by up to 4.7-fold) in 11 of 13 patients studied. CONCLUSION: This is the first study in humans to demonstrate that IVIG influences the Id-anti-Id network of a specific pathogenic autoantibody. Specifically, we showed that IVIG enhanced the anti-Id antibody response in pregnant women with anti-La/SSB antibodies. A high Id:anti-Id ratio in both the IVIG preparation and the maternal serum may explain the absence of an effect of IVIG in preventing recurrent neonatal lupus in some cases.

Fine epitope specificity of anti-erythropoietin antibodies reveals molecular mimicry with HIV-1 p17 protein: a pathogenetic mechanism for HIV-1-related anemia.

BACKGROUND: Circulating autoantibodies to endogenous erythropoietin (anti-Epo) are detected in human immunodeficiency virus type 1 (HIV-1)-infected patients and represent a risk factor for anemia. The aim of this study was to map the B-cell epitopes on the Epo molecule. METHODS: Serum samples from HIV-1-positive patients and healthy individuals were tested against overlapping peptides covering the entire sequence of Epo. RESULTS: Serum samples from anti-Epo-positive patients exhibited significant binding to Epo epitopes spanning the following sequences: amino acids 1-20 (Ep1), amino acids 54-72 (Ep5), and amino acids 147-166 (Ep12). Structural analysis of erythropoietin revealed that the immunodominant epitopes, Ep1 and Ep12, comprise the interaction interface with Epo receptor (EpoR). Autoantibodies binding to this specific region are anticipated to inhibit the Epo-EpoR interaction, resulting in blunted erythropoiesis; this phenomenon is indicated by the significantly higher Epo levels and lower hemoglobin levels of anti-Ep1-positive patients compared with anti-Ep1-negative individuals. The region corresponding to the Ep1 epitope exhibited a 63% sequence homology with the ³4LVCASRELERFAVNPGLLE5² fragment of the HIV-1 p17 matrix protein. CONCLUSIONS: These results suggest that the main body of anti-Epo is directed against a functional domain of Epo, and that the presence of anti-Epo can be considered to be a result of a molecular mimicry mechanism, which is caused by the similarity between the Ep1 region and the p17 protein.

Autopathogenic correlation of periodontitis and rheumatoid arthritis.

Recently, a number of studies have pointed to a potential relationship between periodontitis (PO) and RA and vice versa. Both diseases are characterized by chronic inflammation, osseous destruction, damage of the supporting soft tissues, similar cellular immune responses and common immunogenetic findings. Although a definite, methodological report associating these diseases is missing from the literature, it is possible that both diseases share a common aetiopathogenic background. This background includes the post-translation modification citrullination, which guides the conversion of the amino acid arginine to citrulline in certain self-proteins, generating neo-epitope structures. This results in reduced self-tolerance, development of autoimmunity and the production of ACPAs. The current hypothesis suggests that certain oral bacteria induce the citrullination of proteins under the action of the enzyme peptidyl arginine deiminase (PAD), which exists in both Porphyromonas gingivalis and inflammatory cells. Antibodies against citrullinated proteins and peptides constitute a common serological finding in both RA and PO. The aim of this review is to map the immunological and serological profiles of PO, and to unveil the parameters that connect PO with the appearance of RA at clinical, prognostic and pathogenetic levels. Until now, there have been no reports sufficiently mapping the immunological profile of PO and defining its aetiopathogenic connection with RA, although a similarity between the immunological profile of PO and RA is highly expected.

Peripheral neuropathies in Sjogren syndrome: a new reappraisal.

BACKGROUND: The prevalence of peripheral neuropathy in patients with Sjögren syndrome remains unclear owing to conflicting results in the published series, with numbers ranging from 2% to over 60% of Sjögren syndrome patients. Whether peripheral neuropathy is a feature of the systemic or glandular disease or whether it is related to a circulating antineuronal antibody remains also uncertain. METHODS: The authors reviewed the records of patients with primary Sjögren syndrome (pSS), fulfilling the Revised European-American Classification Criteria, seen in their department from 1992 to 2009. The patients with previously recorded neuropathic features were re-examined clinically and electrophysiologically. Other causes of polyneuropathy were excluded. The authors also searched for circulating antineural antibodies using immunofluorescence and western blot and for antibodies against muscarinic and nicotinic acetylcholine receptors as potential biomarkers. RESULTS: 509 cases met the diagnostic criteria for pSS. Among these, 44 patients were recorded as having neuropathic symptoms. After completing the evaluation, however, only nine (1.8%) had polyneuropathy with objective clinical signs and abnormal electrophysiological findings. The neuropathy was axonal in all, in five pure sensory and in four sensorimotor. The patients with peripheral neuropathy had extraglandular manifestations such as palpable purpura and vasculitis. No evidence of antineural autoimmunity was found, and no candidate biomarkers were identified. CONCLUSION: Polyneuropathy is a rare manifestation of pSS occurring in 1.8% of patients. In the majority of patients, it is a late event and frequently associated with systemic disease or risk factors for lymphoma development.

Diagnostic performance of rapid antigen tests (RATs) for SARS-CoV-2 and their efficacy in monitoring the infectiousness of COVID-19 patients.

The most widely used test for the diagnosis of SARS-CoV-2 infection is a PCR test. PCR has very high sensitivity and is able to detect very low amounts of RNA. However, many individuals receiving a positive test result in a context of a PCR-based surveillance might be infected with SARS-CoV-2, but they are not contagious at the time of the test. The question arises regards if the cost effective, portable rapid antigen tests (RATs) have a better performance than PCR in identification of infectious individuals. In this direction, we examined the diagnostic performance of RATs from 14 different manufacturers in 400 clinical samples with known rRT-PCR cycles threshold (cT) and 50 control samples. Substantial variability was observed in the limit of detection (LOD) of different RATs (cT = 26.8-34.7). The fluorescence-based RAT exhibited a LOD of cT = 34.7. The use of the most effective RATs leads to true positive rates (sensitivities) of 99.1% and 90.9% for samples with cT ≤ 30 and cT ≤ 33, respectively, percentages that can guarantee a sensitivity high enough to identify contagious patients. RAT testing may also substantially reduce the quarantine period for infected individuals without compromising personal or public safety.

RNA recognition motif (RRM) of La/SSB: the bridge for interparticle spreading of autoimmune response to U1-RNP.

Systemic lupus erythematosus (SLE) is characterized by the production of grouped sets of autoantibodies targeting mainly the U1 ribonucleoprotein (RNP) and/or Ro/La RNP particles. Intraparticle diversification of the autoimmune response is believed to occur via epitope spreading. So far, it is not known how the autoimmune response "jumps" from one particle to another. To the extent that the majority of nuclear autoantigens in SLE are RNA binding proteins and major epitopes were previously mapped within their RRM (RNA recognition motifs), conserved sequences within RRM could be involved in the intermolecular and inter-particle diversification process of the autoimmune response. We investigated the potential of RRM of the La/SSB autoantigen to induce antibodies that cross-recognize components of the U1-RNP particle and therefore its capacity to produce interparticle epitope spreading. We immunized New Zealand white rabbits with a peptide corresponding to the epitope 145-164 of La/SSB (belonging to the RRM of La/SSB), attached in four copies on a scaffold carrier. Sera were drawn from 20 sera of patients with SLE and anti-U1-RNP antibodies and 26 sera of primary Sjögren syndrome patients with anti-La/SSB antibodies. All sera were evaluated for reactivity against the major epitope of La/SSB (pep349-364), the RNP antigen and the RRM-related epitope of La/SSB (pep145-164). Specific antibodies against pep145-164 were purified with immunoaffinity columns from selected sera. After the immunization of the animals with pep145-164, a specific IgG antibody response was detected, directed against the La/SSB autoantigen (wks 3-7), the immunizing peptide (wks 3-27), and the RNP autoantigen (wks 7-20). This response gradually decreased to low levels between postimmunization wks 27-42. Purified antibodies against pep145-164 recognized La/SSB and a 70-kD autoantigen in Western blot and exhibited significant reactivity in anti-U1-RNP ELISA. Depletion of anti-pep145-164 antibodies eliminated anti-U1-RNP reactivity from immunized rabbit sera but not from human sera. In addition, pep145-164 was recognized to a greater extent by autoimmune sera with anti-RNP reactivity compared with anti-La/SSB-positive sera, in contrast to pep349-364 of La/SSB, which was recognized almost exclusively by sera with anti-La/SSB reactivity. These data suggest that the RRM region of La/SSB can trigger interparticle B-cell diversification to U1-RNP-70 autoantigen via molecular mimicry. Identification of key sequences that trigger and perpetuate the autoimmune process is particularly important for understanding pathogenetic mechanisms in autoimmunity.

A novel mechanism of thrombosis in antiphospholipid antibody syndrome.

Antiphospholipid antibody syndrome (APS) is an autoimmune thrombophilia mediated by autoantibodies directed against phospholipid-binding plasma proteins, mainly ß2 Glycoprotein I (ß2GPI)-a plasma apolipoprotein and prothrombin (PT). A subgroup of these antibodies termed "Lupus Anticoagulant" (LA) elongate in vitro the clotting times, this elongation not corrected by adding normal plasma in the detection system. The exact mechanism by which these autoantibodies induce thrombosis is not well understood. Resistance to natural anticoagulants such as protein C, impaired fibrinolysis, activation of endothelial cells to a pro-coagulant phenotype and activation of platelets, are among the mechanisms partially supported by experimental evidence. Artificially dimerized ß2GPI binds tightly to platelet membrane activating them. We search for mechanisms of natural dimerization of ß2GPI by proteins of the platelet membranes and found that platelet factor 4 (PF4) assembled in homotetramers binds two molecules of ß2GPI and this complex is recognized by anti-ß2GPI antibodies, the whole complexes being thrombogenic in terms of activating platelets as confirmed by p38MAP kinase phosphorylation and thromboxane B2 production. Of note PF4/heparin complexes are also immunogenic triggering the production of anti-PF4/heparin antibodies which activate also platelets (the so-called "heparin-induced thrombocytopenia and thrombosis syndrome", HITT). The anti-ß2GPI antibodies activate platelets by their F(ab)2, while the anti-PF4/heparin by their Fc fragments. Thus PF4 is a common denominator in the pathogenesis of APS and HITT which share also clinical characteristics such as thrombocytopenia and thrombosis.

B-cell epitopes of the intracellular autoantigens Ro/SSA and La/SSB: tools to study the regulation of the autoimmune response.

A common serologic finding in systemic autoimmune diseases is the presence of autoantibodies against intracellular autoantigens. Although their pathogenesis is not fully understood, autoantibodies are important tools for establishing diagnosis, classification and prognosis of autoimmune diseases. In Systemic Lupus Erythematosus (SLE) and Sjögren's syndrome (SS) autoantibodies mainly target multicomponent ribonucleoprotein complex Ro/La RNP. The last years, the main characteristics, the clinical significance of the anti-Ro/SSA and anti-La/SSB autoantibodies, their biologic function, as well as their B-cell antigenic determinants (epitopes) have been addressed. More specifically, the structural characteristics and clinical associations of epitopes along with their utility as tools to investigate the autoimmune response have been investigated in detail. New insights for the pathogenetic role of epitopes in initiation, propagation and regulation of systemic autoimmunity have been emerged. In this regard, the role of epitope spreading in the diversification of autoimmune response and the anti-idiotypic antibodies in the regulation of autoantibodies (idiotypic) response are addressed.

Autoimmune response and target autoantigens in Sjogren's syndrome.

BACKGROUND: Primary Sjogren's syndrome (pSS) is characterized by the presence of autoantibodies targeting mainly the Ro/La ribonucleoprotein complex. It is now appreciated that the production of autoantibodies is an antigen-driven immune response. DESIGN: In this review, candidate mechanisms for autoantigen presentation and perpetuation of the autoimmune response within the autoimmune tissue lesion of pSS are discussed. RESULTS: Several studies have shown that the epithelial cell in labial salivary glands of patients with Sjogren's syndrome is activated, bearing characteristics of an antigen-presenting cell, as suggested by inappropriate expression of class II HLA and co-stimulatory molecules. Other studies have confirmed that in salivary glands, there is an increased autoantigen presentation via apoptotic blebs and bodies, exosomes and heat shock protein-mediated cross-priming. There is also an increased expression of interferon (IFN)-induced genes, such as the autoantigen Ro52, which provide negative feedback regulation in inflammation. Ro60 and La autoantigens also appear to play a major role in the local autoimmune response in Sjogren's syndrome. In this regard, La and Ro60 the messenger RNA (mRNA) expression is upregulated in the affected salivary glands with different isoforms of La autoantigen mRNA to be expressed in patients with pSS. At the protein level, La/SSB in pSS salivary glands is found to be post-translationally modified. CONCLUSIONS: Autoantigen alterations in a microenvironment of local inflammation with increased in situ apoptosis, Toll-like receptor (TLR) signalling and antigen presentation may drive the autoimmune response and local autoantibody production in pSS.

Development of a new spectrophotometric assay for rapid detection and differentiation of KPC, MBL and OXA-48 carbapenemase-producing Klebsiella pneumoniae clinical isolates.

The increased prevalence of carbapenemase-producing Enterobacteriaceae (CPE) has made essential the design of quicker tests for CPE detection. In the present study, a simple and rapid assay was developed based on measurement of the hydrolytic activity of imipenem at a final concentration of 65 µg/mL (100 µM) through ultraviolet-visible (UV-Vis) spectrophotometry. All measurements were conducted at 297 nm. A total of 83 carbapenem-non-susceptible CPE, consisting of Klebsiella pneumoniae clinical isolates and genotypically characterised as KPC-, VIM-, NDM- or OXA-48-producers, were tested. For comparison, 30 carbapenem-non-susceptible clinical isolates, consisting of Escherichia coli and K. pneumoniae and genotypically confirmed as non-CPE, were also examined. The spectrophotometric assay enabled efficient discrimination of CPE from non-CPE isolates even in 45 min (P < 0.0001). Moreover, the presence of phenylboronic acid (PBA) or ethylene diamine tetra-acetic acid (EDTA) in the reaction mixture was able to inhibit the hydrolytic capacity of KPC- or metallo-ß-lactamase (MBL)-producers, respectively, while the hydrolytic activity of OXA-48-producing strains was not affected by the presence of these inhibitors (P < 0.001). The newly developed assay presented 100% sensitivity and specificity to detect and differentiate KPC-, MBL- and OXA-48-producers compared with genotypic characterisation. Thus, the proposed spectrophotometric method can be considered as an easy, fast, accurate and cost-effective diagnostic tool for screening carbapenem-non-susceptible K. pneumoniae isolates in the clinical laboratory.

Functional expression of the alpha 2-macroglobulin receptor CD91 in salivary gland epithelial cells.

CD91 molecule is a multifunctional receptor of alpha 2-macroglobulin, heat-shock proteins and calreticulin. CD91 has been implicated in cross-presentation of peptides chaperoned by these proteins to MHC molecules, thus eliciting antigen-specific immune responses. Hence, CD91 is considered as a major regulator of innate and acquired immune responses. Herein, we show that CD91 molecules are expressed by human salivary gland epithelial cells (SGEC), as indicated by immunohistochemical studies in minor salivary gland biopsy tissues (n = 21) as well as by the analyses of human long-term cultured non-neoplastic SGEC lines (n = 11) and the neoplastic HSG cell line. In these cell lines CD91 expression was evaluated by RT-PCR, flow cytometry and confocal microscopy. Standard internalization assays revealed that HSG and SGECs are capable to bind and internalize the CD91 ligand alpha 2-macroglobulin. This internalization is specific, as attested by inhibition studies using unlabeled alpha 2-macroglobulin and a blocking antibody against human CD91 receptor. Conclusively, our findings indicate that SGEC functionally express CD91 receptor, suggesting that this pathway might be involved in the presentation of exogenous antigens in SGEC.

Sjögren's syndrome--study of autoantigens and autoantibodies.

The presence of autoantibodies is the hallmark of systemic autoimmune diseases. During the past 30 years, intense clinical and basic research have dissected the clinical value of autoantibodies in many autoimmune diseases and offered new insights into a better understanding of the molecular and functional properties of the targeted autoantigens. Unraveling the immunologic mechanisms underlying the autoimmune tissue injury, provided useful conclusions on the generation of autoantibodies and the perpetuation of the autoimmune response. Primary Sjögren's syndrome (pSS) is characterized by the presence of autoantibodies binding on a vast array of organ and non-organ specific autoantigens. The most common autoantibodies are those targeting the Ro/La RNP complex, and they serve as disease markers, as they are included in the European-American Diagnostic Criteria for pSS. Other autoantibodies are associated with particular disease manifestations, such as anti-centromere antibodies with Raynaud's phenomenon, anti-carbonic anhydrase II with distal renal tubular acidosis, anti-mitochondrial antibodies with liver pathology, and cryoglobulins with the evolution to non-Hodgkin's lymphoma. Finally, autoantibodies against autoantigens such as alpha- and beta-fodrin, islet cell autoantigen, poly(ADP)ribose polymerase (PARP), NuMA, Golgins, and NOR-90 are found in a subpopulation of SS patients without disease specificity, and their utility remains to be elucidated. In this review, the molecular and clinical characteristics (divided according to their clinical utility) of the autoantigens and autoantibodies associated with pSS are discussed.

The role of chaperone proteins in autoimmunity.

Heat-shock or stress proteins (HSPs) are intracellular molecules that are expressed under cellular stress and have housekeeping and cytoprotective functions. Many of them act also as molecular chaperones, assisting the correct folding, stabilization, and translocation of proteins. In pathological situations, such as necrotic cell death, they can be released into the extracellular environment complexed with intact or fragmented cellular proteins. Evidence is now accumulating to indicate that, under certain circumstances, these complexes can contribute to induction of autoimmunity by receptor-mediated activation of the innate immune response (signaling the "danger") and by participation in the presentation of autoantigens for the adaptive immune response (acting as natural adjuvants). In addition, the conservation of HSPs through prokaryotes and eukaryotes, together with the increased production of host and microbial HSPs at the site of infection, has led to the proposition that these proteins may provide a link between infection and autoimmunity. This review outlines the mechanisms for the potential involvement of chaperones in the induction of autoimmune disease.