Anti-viral citrullinated peptides antibodies in rheumatoid arthritis

Rheumatoid arthritis (RA) sera contain antibodies specific for deiminated peptide/proteins (ACPA). The deimination (or citrullination) is a post-translational modification catalysed by the calcium dependent enzyme peptidylarginine deiminase: an arginyl residue within protein is converted to a cytrullyl residue, with the consequent loss of a net positive charge. Citrullination is a physiologic event, occurring during cell differentiation, and particularly in inflammation and cell death; on the contrary, antibodies directed towards citrullinated proteins/peptides are highly specific for RA. A number of endogenous proteins have been found to be citrullinated and target of ACPA Recently, a viral peptide corresponding to the sequence 35-58 Epstein Barr Nuclear Antigen-1 containing citrulline in place of arginine (VCP – Viral Citrullinated Peptide) has been shown to be a specific probe for the detection of ACPA. To analyze the immune response to citrullinated Epstein-Barr antigens in RA, we tested peptides derived from different viral proteins and characterised by different rates of citrullination. Studying a large panel of peptides, we found high reactivity against another deiminated viral peptide (DVP). Thus, we measured anti-DVP antibodies of IgG, IgM and IgA isotype in 100 normal sera, 107 RA and 196 disease controls. Setting the threshold value at the 99° percentile of the normal population, IgG anti-DVP were found in 65%; IgM anti-DVP in 44%; and IgA anti-DVP in 48% of RA patients. In some cases IgM or IgA were present in the absence of IgG; moreover, anti-DVP and anti-VCP antibodies were found to be overlapping but distinct populations. Rheumatoid arthritis is a multifactorial disease. Among genes, the major contribution to RA susceptibility is given by HLA-DRB1 alleles encoding the “shared epitope” (SE) sequences. The presence of these alleles is strictly associated with the production of ACPAs. However, the genetic background underlying the production of single ACPA specificities has not been thoroughly investigated. Thus, we analysed the reactivity to two different viral citrullinated peptides (VCP and DVP) and studied their association with genetic variants within HLA-DRB1 SE alleles in a RA Caucasian population. One hundred and seventy-two French RA patients were characterized in terms of HLA-DRB1 genotype, anti VCP-A and anti-DVP antibodies. HLA-DRB1 SE alleles were classified into four SE+ groups (S1, S2, S3P, S3D) and one SE- group (X) according to a new classification of HLA-DRB1 alleles, “reshaping the shared epitope (SE) hypothesis”. Anti-viral citrullinated peptides antibodies were assessed by home made ELISA on VCP and DVP coated plates. We found that anti-VCP and anti-DVP antibodies showed a trend in association to HLA-SE alleles but not significant (odds ratio > 1, P>0.05). However, subgrouping the SE alleles according to the new classification, the presence of anti-VCP or anti-DVP antibodies is associated with S2 allele (OR>1, P1, P<0.05). We have tested the peptides for their capacity to bind various HLA-DR molecules. In no instance we found that any of the peptides bound any of the DR molecules at the highest dose tested. Therefore, these results challenge the role of HLA-SE in the pathogenesis of rheumatoid arthritis. More studies are needed to clarify whether HLA-SE may act as a classic immune response gene, thus presenting citrullinated peptide to T cells or whether they may play other roles, directly stimulating immune system cells

Immunoglobulin G subclass profile of anticitrullinated peptide antibodies specific for Epstein Barr virus-derived and histone-derived citrullinated peptides.

To the Editor: Studies have shown that the anticitrullinated peptide antibodies (ACPA) response is highly polyclonal, in terms of epitope specificity, V genes, and isotype usage1,2. Longitudinal studies of patients with rheumatoid arthritis (RA) have documented epitope spreading, and ACPA, specific for distinct citrullinated epitopes, have been described. By using different citrullinated antigens, ACPA from immunoglobulin (Ig)G, IgA, and IgM isotype have been detected3. ACPA are polyclonal in the usage of different IgG subclasses, but in this case the pattern is more heterogeneous. So far the studies conducted indicate the dominance of IgG1 and IgG4, while IgG3 have been detected with cyclic citrullinated peptide (CCP) and vimentin, but not with fibrinogen4,5. The production of specific IgG subclasses might help in deciphering the mechanisms eliciting B cell expansion in response to different antigens. Thus, it is of interest to explore the profile of IgG subclasses of antibodies reactive with novel citrullinated substrates, already known to be tools for ACPA detection. Ninety-three patients with RA, 25 with psoriatic arthritis, 15 with ankylosing spondylitis, and … Address correspondence to Professor P. Migliorini, Department of Clinical and Experimental Medicine, University of Pisa, Via Roma 67, 56126 – Pisa, Italy. E-mail: paola.migliorini{at}med.unipi.i

A novel DNA/histone H4 peptide complex detects autoantibodies in systemic lupus erythematosus sera

Background: The detection of anti-dsDNA antibodies is critical for the diagnosis and follow-up of systemic lupus erythematosus (SLE) patients. The presently available assays are characterized by a non-optimal specificity (solid phase assays) or sensitivity (Crithidia Luciliae immunofluorescence test (CLIFT)). To overcome the limits of CLIFT and solid phase chromatin assays, we explored the diagnostic potential of an assay based on plasmid DNA containing a highly bent fragment of 211 bp from Crithidia Luciliae minicircles, complexed with histone peptides. Methods: Electrically neutral complexes of PK201/CAT plasmid (PK) DNA and histone 4 (H4) peptides were evaluated by electromobility shift assay. Complexes of H4 peptides and PK were absorbed to the solid phase to detect specific immunoglobulin G (IgG) in sera. Sera from 109 SLE patients, 100 normal healthy subjects, and 169 disease controls were tested. Results: H4(14-34) containing the consensus sequence for DNA binding interacts with PK, retarding its migration. H4(14-34)/PK complexes were used to test sera by ELISA. Anti-H4-PK antibodies were detected in 56 % of SLE sera (more frequently in patients with skin or joint involvement) versus 5.9 % in disease controls; inhibition assays show that sera react with epitopes present on DNA or on the complex, not on the peptide. Antibody titer is correlated with European Consensus Lupus Activity Measurement (ECLAM) score and anti-complement component 1q (C1q) antibodies, negatively with C3 levels. Anti-H4-PK antibodies compared with CLIFT and solid phase dsDNA assays display moderate concordance. Conclusions: The H4/PK assay is a simple and reliable test which is useful for the differential diagnosis and evaluation of disease activity in SLE patients

Induction of neutralizing antibodies in CLL patients after SARS-CoV-2 mRNA vaccination: a monocentric experience

Vaccination represents the best strategy to fight COVID-19 pandemics, especially in immune compromised subjects. In chronic lymphatic leukemia patients, a marked impairment of the immune response to mRNA SARS-CoV-2 vaccine was observed. In this report, we analyzed anti-RBD and neutralizing antibodies in CLL patients after two doses of mRNA SARS CoV 2 vaccine and evaluated the impact of Bruton kinase inhibitory agents. Twenty-seven CLL patients vaccinated with mRNA vaccines against SARS CoV-2 were recruited. Serum IgG, IgM and IgA anti-RBD antibodies and neutralizing antibodies were detected, and antibody avidity was measured. Peripheral blood leukocytes subsets were evaluated by flow cytometry. After two vaccine doses anti-RBD IgG were produced in 11/27 (40.5%) of patients and levels of IgG and IgA anti RBD in CLL patients were sensibly lower than in controls. Neutralizing antibodies were detectable in 12/27 (44.5%) of the patients and their level was lower than that observed in controls. Disease burden and treatment with Bruton kinases inhibitors markedly impaired vaccine induced antibody response. However, in responder patients, antibody avidity was comparable to normal subjects, indicating that the process of clonal selection and affinity maturation takes place as expected. Taken together, these data confirm the impact of disease burden and therapy on production of anti-RBD and neutralizing antibodies and support the current policy of vaccinating CLL patients

The 75-Gram Glucose Load in Pregnancy

OBJECTIVE—To investigate, in pregnant women without gestational diabetes mellitus (GDM), the relation among obstetric/demographic characteristics; fasting, 1-h, and 2-h plasma glucose values resulting from a 75-g glucose load; and the risk of abnormal neonatal anthropometric features and then to verify the presence of a threshold glucose value for a 75-g glucose load above which there is an increased risk for abnormal neonatal anthropometric characteristics. RESEARCH DESIGN AND METHODS—The study group consisted of 829 Caucasian pregnant women with singleton pregnancy who had no history of pregestational diabetes or GDM, who were tested for GDM with a 75-g, 2-h glucose load, used as a glucose challenge test, in two periods of pregnancy (early, 16–20 weeks; late, 26–30 weeks), and who did not meet the criteria for a GDM diagnosis. In the newborns, the following abnormal anthropometric characteristics were considered as outcome measures: cranial/thoracic circumference (CC/TC) ratio ≤10th percentile for gestational age (GA), ponderal index (birth weight/length3 × 100) ≥90th percentile for GA, and macrosomia (birth weight ≥90th percentile for GA), on the basis of growth standard development for our population. For the first part of the objective, logistic regression models were used to identify 75-g glucose load values as well as obstetric and demographic variables as markers for abnormal neonatal anthropometric characteristics. For the second part, the receiver operating characteristic (ROC) curve was performed for the 75-g glucose load values to determine the plasma glucose threshold value that yielded the highest combined sensitivity and specificity for the prediction of abnormal neonatal anthropometric characteristics. RESULTS—In both early and late periods, maternal age &gt;35 years was a predictor of neonatal CC/TC ratio ≤10th percentile and macrosomia, with fasting 75-g glucose load values being independent predictors of neonatal CC/TC ratio ≤10th percentile. In both periods, 1-h values gave a strong association with all abnormal neonatal anthropometric characteristics chosen as outcome measures, with maternal age &gt;35 years being an independent predictor for macrosomia. The 2-h, 75-g glucose load values were significantly associated in both periods with neonatal CC/TC ratio ≤10th percentile and ponderal index ≥90th percentile, whereas maternal age &gt;35 years was an independent predictor of both neonatal CC/TC ratio ≤10th percentile and macrosomia. In the ROC curves for the prediction of neonatal CC/TC ratio ≤10th percentile for GA in both early and late periods of pregnancy, inflection points were identified for a 1-h, 75-g glucose load threshold value of 150 mg/dl in the early period and 160 mg/dl in the late period. CONCLUSIONS—This study documented a significant association, seen even in the early period of pregnancy, between 1-h, 75-g glucose load values and abnormal neonatal anthropometric features, and provided evidence of a threshold relation between 75-g glucose load results and clinical outcome. Our results would therefore suggest the possibility of using a 75-g, 1-h oral glucose load as a single test for the diagnosis of GDM, adopting a threshold value of 150 mg/dl at 16–20 weeks and 160 mg/dl at 26–30 weeks

Efectos psicosociales y sociopolíticos de la Guerra de Malvinas en las provincias de Chaco y Corrientes, 1982 →. 16H241

El proyecto plantea un estudio psicosocial y antropológico del impacto que tuvo la Guerra de Malvinas sobre la política y la identidad de la población de las provincias de Chaco y Corrientes, región desde la que se movilizó un nutrido contingente de soldados hacia el frente de guerra. El periodo a investigar abarca desde el acontecimiento en 1982 hasta nuestros días y pone la mirada sobre los protagonistas del momento: soldados, gobernantes, dirigentes y activistas sociales, y sobre las generaciones posteriores al hecho que lo conocen a partir de la memoria transmitida. El tema es abordado a través de la investigación documental y de entrevistas en profundidad. Constituye una etapa en el desarrollo de la línea de investigación sobre la Guerra de Malvinas en la región del nordeste argentino que se inició en el año 2004, por lo cual contamos con resultados que nos permiten reorientar la investigación hacia distintos aspectos de los efectos del acontecimiento en la sociedad. Este avance nos permite un mayor acercamiento con las organizaciones de ex combatientes que desarrollan su actividad en ambas provincias

Repeated SARS-CoV-2 vaccination in cancer patients treated with immune checkpoint inhibitors: induction of high-avidity anti-RBD neutralizing antibodies

BackgroundCancer patients are more vulnerable to COVID-19 and are thus given high priority in vaccination campaigns. In solid cancer patients treated with checkpoint inhibitors, we evaluated the amount of anti-RBD and neutralizing antibodies and antibody avidity after two or three doses of the vaccine.MethodsThirty-eight solid cancer patients, 15 untreated hematological patients and 21 healthy subjects were enrolled in the study. Blood was collected before the first dose (T0), 21 days after the second (T2) and in 18 solid cancer patients also 15 days after the third dose of vaccine (T3). IgG, IgM and IgA anti-RBD antibodies were detected by ELISA. Neutralizing antibodies were measured testing the inhibition of RBD binding to ACE2. Antibody avidity was evaluated in 18 patients by a urea avidity ELISA.ResultsIgG anti-RBD antibodies were produced in 65.8% of the cancer patients at T2, and in 60% of hematological patients at levels lower than healthy controls. IgM and IgA anti-RBD antibodies were also produced in 5.3% and 21% cancer patients, respectively. At T3, a significant increase in anti-RBD IgG levels was observed. Neutralizing antibodies were produced in 68.4% of cancer patients as compared with 93% of untreated hematological patients and 100% of controls, at titers lower than in healthy subjects. At T3, neutralizing antibodies and avidity of IgG anti-RBD increased; 6/18 patients negative at T2 developed neutralizing antibodies at T3.ConclusionThe data indicate that in cancer patients mRNA vaccine induces high avidity anti-RBD antibodies and neutralizing antibodies that increase after the third dose. The process of induction and selection of high-affinity antibodies is apparently unaffected by the treatment with anti-PD-1 or anti-PD-L1 antibodies

Antibodies from patients with rheumatoid arthritis target citrullinated histone 4 contained in neutrophils extracellular traps.

Histone deimination regulates gene function and contributes to antimicrobial response, allowing the formation of neutrophil extracellular traps (NETs). Deiminated proteins are target of anti-citrullinated peptides antibodies (ACPA) in rheumatoid arthritis (RA). OBJECTIVE: The objective of this paper is to test the hypothesis that RA sera react with deiminated histones contained in NETs. METHODS: Neutrophils from peripheral blood were stimulated with A23187 and acid treated; NETosis was induced by phorbol myristate acetate, and NET proteins were isolated. Sera were tested by immunoblot on acid extracted proteins from neutrophils and from NETs, and by ELISA on deiminated histone H4 or H4-derived peptides. Bands reactive with RA sera were excised from gels, digested with trypsin and subjected to matrix-assisted laser desorption/ionisation time of flight (MALDI-TOF) analysis, before and after derivatisation to detect citrullinated peptides. RESULTS: RA sera reacted with a deiminated antigen of 11 KDa from activated neutrophils, recognised also by anti-H4 and antideiminated H4 antibodies. A similar reactivity was observed with NET proteins. The antigen from neutrophils or NETs was identified as citrullinated H4 by MALDI-TOF analysis. By ELISA, RA sera bound in vitro citrullinated H4. Citrullinated H4 14-34 and 31-50 peptides detected antibodies in 67% and 63% of RA sera and in less than 5% of controls; antibody titre was correlated with anti-CCP2. CONCLUSIONS: Citrullinated H4 from activated neutrophils and NETs is a target of antibodies in RA, and synthetic citrullinated H4-derived peptides are a new substrate for ACPA detection. As NETosis can generate antigens for ACPA, these data suggest a novel connection between innate and adaptive immunity in RA

A Straightforward Method to Produce Multi-Nanodrug Delivery Systems for Transdermal/Tympanic Patches Using Electrospinning and Electrospray

The delivery of drugs through the skin barrier at a predetermined rate is the aim of transdermal drug delivery systems (TDDSs). However, so far, TDDS has not fully attained its potential as an alternative to hypodermic injections and oral delivery. In this study, we presented a proof of concept of a dual drug-loaded patch made of nanoparticles (NPs) and ultrafine fibers fabricated by using one equipment, i.e., the electrospinning apparatus. Such NP/fiber systems can be useful to release drugs locally through the skin and the tympanic membrane. Briefly, dexamethasone (DEX)-loaded poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBHV) fiber meshes were decorated with rhodamine (RHO)-loaded poly(lactic-co-glycolic acid) (PLGA) NPs, with RHO representing as a second drug model. By properly tuning the working parameters of electrospinning, DEX-loaded PHBHV fibers (i.e., by electrospinning mode) and RHO-loaded PLGA NPs (i.e., by electrospray mode) were successfully prepared and straightforwardly assembled to form a TDDS patch, which was characterized via Fourier transform infrared spectroscopy and dynamometry. The patch was then tested in vitro using human dermal fibroblasts (HDFs). The incorporation of DEX significantly reduced the fiber mesh stiffness. In vitro tests with showed that HDFs were viable for 8 days in contact with drug-loaded samples, and significant signs of cytotoxicity were not highlighted. Finally, thanks to a beaded structure of the fibers, a controlled release of DEX from the electrospun patch was obtained over 4 weeks, which may accomplish the therapeutic objective of a local, sustained and prolonged anti-inflammatory action of a TDDS, as is requested in chronic inflammatory conditions, and other pathological conditions, such as in sudden sensorineural hearing loss treatment