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.

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.

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.

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.

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.

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.

Synthetic peptides for efficient discrimination of anti-enterovirus antibodies at the serotype level.

Enteroviruses are important human pathogens, causing a broad spectrum of diseases from minor common colds to fatal myocarditis. However, certain disease syndromes are caused by one or few serotypes. Serotype identification is difficult due to the laborious neutralization tests that lack of sensitivity, while in commercial ELISAs homotypic antibodies' activities are largely masked by the recognition of genera-specific epitopes by heterotypic antibodies. In the present study homotypic assays were developed with the ability to discriminate different enterovirus serotypes. Seventy-three children sera, positive for IgM antibodies against enterovirus genus and 49 healthy children were examined for the presence of antibodies against 14 synthetic peptides derived from a non-conserved region of the VP1 protein of coxsackieviruses B2, B3, B4, B5, A9, A16, A24, echoviruses 6, 7, 9, 11, 30, enterovirus 71 and parechovirus 1. 50% of the anti-enterovirus IgM positive sera (>150 BU) reacted with the peptides with the majority of them to preferentially recognize one of them, supporting the homotypic nature of our assay. Inhibition studies yielded homologous inhibition rates 67-95% suggesting that specific peptide recognition actually occurred. The diagnostic value of our assay was tested in blood samples drawn over a 1.5-year period from a 5-year old patient. The anti-enterovirus reactivity was clearly attributed to echovirus serotype 11. The IgM/IgG antibody ratio was reversed 4 months later and subsequently IgM antibodies dropped below the cutoff point. In this paper we demonstrate that our assay can be used to discriminate between antibodies targeting different enterovirus serotypes.

Malignant lymphoma in primary Sjögren's syndrome: an update on the pathogenesis and treatment.

OBJECTIVES: Sjögren's syndrome (SS), a chronic autoimmune disorder, particularly compromises the function of exocrine glands. Its association with lymphoma is well documented. Our aim was to systematically review the molecular, clinical, histopathologic, and therapeutic aspects of these SS-related malignant lymphoproliferations. METHODS: The literature was searched for original articles published between 1968 and 2012 focusing on the risk factors for lymphoma development in Sjögren's syndrome using MEDLINE and PubMed. The search terms we used were "Sjögren's syndrome," "lymphoma," and "risk factors." All papers identified were English-language, full-text papers. RESULTS: A low-grade marginal-zone lymphoma related to mucosa-associated lymphoid tissue is the commonest lymphoid neoplasia in SS. The majority of SS-associated lymphomas are characterized by localized stage, indolent clinical course, and recurrence in other extranodal sites. Although the transition from a chronic inflammatory condition to malignant lymphoma is a multistep process that is yet poorly understood, there is increasing evidence that chronic antigenic stimulation by an exoantigen or autoantigens plays an essential role in the development of SS-associated lymphoproliferation. CONCLUSIONS: This review discusses the pathogenetic aspects of lymphomagenesis in SS. Recent advances in the treatment of lymphoma in SS are also stated.

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.

Seroepidemiological study of pandemic influenza H1N1 following the 2009-2010 wave in Greece.

Knowledge of seroprevalence rates against 2009 pandemic H1N1 virus will assist vaccination recommendations and the preparation of the health-care system during subsequent years. This study was conducted in Greece during June-August 2010 to estimate the seroprevalence rate against pandemic H1N1 virus. Persons presenting in 29 health-care facilities across the country were studied. Seroprevalence was estimated employing a virus-free ELISA that specifically recognizes 2009 H1N1 virus antibodies in human sera. Sera collected from 2005 to April 2009 were also used to estimate pre-pandemic seroprevalence rates. A total of 954 persons were studied. The overall seroprevalence rate was 28.5% (95% confidence interval=25.6-31.3%). Age-specific rates were 34.2% in persons 0-4 years, 36.3% in persons 5-19 years, 25.0% in persons 20-39 years, 23.4% in persons 40-59 years, and 31.8% in persons ≥ 60 years. The highest rates were recorded in the Regions of Ionian Islands (67%) and Epirus (42.9%), while the lowest (8.4%) in the Region of Thessaly. Age-specific attack rates of infection during 2009-2010 were 28.8% in persons 0-4 years, 32.5% in persons 5-19 years, 14.3% in persons 20-39 years, 19.1% in persons 40-59 years, and 14.4% in persons ≥ 60 years. Multivariate analysis revealed that Region of residence and caring for children <5 years were associated with increased risk for seropositivity. Urbanity, personal and family characteristics, working in a health-care facility or in a school, history of pandemic H1N1 vaccination or history of influenza-like illness during 2009-2010 were not associated with increased risk for seropositivity.

Pharmacokinetics of intravitreal bevacizumab (Avastin®) in rabbits.

PURPOSE: To describe the pharmacokinetics of intravitreal bevacizumab (Avastin®) in rabbits. METHODS: The right eye of 20 rabbits was injected intravitreally with 1.25 mg/0.05 mL bevacizumab. Both eyes of four rabbits each time were enucleated at days 1, 3, 8, 15, and 29. Bevacizumab concentrations were measured in serum, aqueous humor, and vitreous. RESULTS: Maximum vitreous (406.25 µg/mL) and aqueous humor (5.83 µg/mL) concentrations of bevacizumab in the right eye were measured at day 1. Serum bevacizumab concentration peaked at day 8 (0.413 µg/mL) and declined to 0.032 µg/mL at 4 weeks. Half-life values in right vitreous, right aqueous humor, and serum were 6.61, 6.51, and 5.87 days, respectively. Concentration of bevacizumab in the vitreous of the noninjected eye peaked at day 8 (0.335 ng/mL) and declined to 0.218 ng/mL at 4 weeks. In the aqueous humor of the noninjected eye, maximum concentration of bevacizumab was achieved at day 8 (1.6125 ng/mL) and declined (to 0.11 ng/mL) at 4 weeks. CONCLUSION: The vitreous half-life of 1.25 mg/0.05 mL intravitreal bevacizumab was 6.61 days in this rabbit model. Maximum concentrations of bevacizumab were reached at day 1 in both vitreous and aqueous humor of the right eye and at day 8 in the serum. Very low concentrations of bevacizumab were measured in the fellow noninjected eye.

Estimation of seroprevalence of the pandemic H1N1 2009 influenza virus using a novel virus-free ELISA assay for the detection of specific antibodies.

The pandemic H1N1 2009 influenza A emerged in April 2009 and spread rapidly all over the world. In Greece, the first case of the pandemic H1N1 was reported on May 18, 2009, while a considerable increase in the number of cases was noticed at the beginning of July 2009. The need for surveillance of the immune status of the Greek population led us to develop a virus-free ELISA that specifically recognizes pandemic H1N1 2009 influenza virus antibodies in human sera. The method is based on the use of synthetic peptides (H1-pep and N1-pep) that are derived from the hemagglutinin and neuraminidase of the 2009 pandemic strain, respectively, and differentiate the swine-origin influenza A/California/14/2009 (H1N1) from the seasonal influenza A viruses. Serum samples were obtained from 271 healthy blood donors during May, November, and December 2009. Among sera collected during May, November, and December, IgG antibodies against the peptide H1-pep were detected in 7.4, 13.8, and 19.3% of the donors, respectively, while IgG antibodies against the peptide N1-pep were detected in 5.3, 9.6, and 16.9% of the donors, respectively. The application of the immunoassay indicated a time-dependent increase of the prevalence of anti-H1-pep and anti-N1-pep IgG antibodies during the pandemic H1N1 outbreak in Greece. The method could be also indicative for the discrimination of immune persons from those susceptible to infection with the pandemic H1N1 strain, as well as for the establishment of effective vaccination programs.

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.

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.

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.

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.

In vitro bactericidal activity of human beta-defensin 2 against nosocomial strains.

Human beta-defensin 2 (hBD-2) is a 41-amino acid cationic peptide of the innate immune system that serves as antimicrobial molecule. We determined the bactericidal activity of synthetic hBD-2 against nosocomial strains belonging to eight different bacterial species and exhibiting various antimicrobial resistance phenotypes. The native disulfide connectivity was found essential for the bactericidal activity of hBD-2, while sodium chloride concentration was reversely associated with its potency. hBD-2 exhibited high bactericidal activity against Acinetobacter baumannii, Pseudomonas aeruginosa, Enterococcus faecalis, Enterococcus faecium and Staphylococcus aureus clinical strains. Characteristically, A. baumannii strains that exhibited multi-drug resistant (MDR) phenotypes were susceptible to lower concentrations of hBD-2 (vLD(90)=3.25-4.5 microg/ml) in comparison with non-MDR (wild-type) A. baumannii strains (vLD(90)=3.90-9.35 microg/ml). Bactericidal activity of hBD-2 was less pronounced against Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis strains but was significantly enhanced against strains of these species that exhibited resistance to several beta-lactam antibiotics. These observations give indications that the natural hBD-2 has a potential therapeutic role against bacterial pathogens and particularly against those exhibiting MDR phenotypes.