Tracking the immune response profiles elicited by the BNT162b2 vaccine in COVID-19 unexperienced and experienced individuals.

Multiple vaccines have been approved to control COVID-19 pandemic, with Pfizer/BioNTech (BNT162b2) being widely used. We conducted a longitudinal analysis of the immune response elicited after three doses of the BNT162b2 vaccine in individuals who have previously experienced SARS-CoV-2 infection and in unexperienced ones. We conducted immunological analyses and single-cell transcriptomics of circulating T and B lymphocytes, combined to CITE-seq or LIBRA-seq, and VDJ-seq. We found that antibody levels against SARS-CoV-2 Spike, NTD and RBD from wild-type, delta and omicron VoCs show comparable dynamics in both vaccination groups, with a peak after the second dose, a decline after six months and a restoration after the booster dose. The antibody neutralization activity was maintained, with lower titers against the omicron variant. Spike-specific memory B cell response was sustained over the vaccination schedule. Clonal analysis revealed that Spike-specific B cells were polyclonal, with a partial clone conservation from natural infection to vaccination. Spike-specific T cell responses were oriented towards effector and effector memory phenotypes, with similar trends in unexperienced and experienced individuals. The CD8 T cell compartment showed a higher clonal expansion and persistence than CD4 T cells. The first two vaccinations doses tended to induce new clones rather than promoting expansion of pre-existing clones. However, we identified a fraction of Spike-specific CD8 T cell clones persisting from natural infection that were boosted by vaccination and clones specifically induced by vaccination. Collectively, our observations revealed a moderate effect of the second dose in enhancing the immune responses elicited after the first vaccination. Differently, we found that a third dose was necessary to restore comparable levels of neutralizing antibodies and Spike-specific T and B cell responses in individuals who experienced a natural SARS-CoV-2 infection.

Corrigendum: TMEM123 a key player in immune surveillance of colorectal cancer.

[This corrects the article DOI: 10.3389/fimmu.2023.1194087.].

TMEM123 a key player in immune surveillance of colorectal cancer.

Colorectal cancer (CRC) is a leading cause of cancer-associated death. In the tumor site, the interplay between effector immune cells and cancer cells determines the balance between tumor elimination or outgrowth. We discovered that the protein TMEM123 is over-expressed in tumour-infiltrating CD4 and CD8 T lymphocytes and it contributes to their effector phenotype. The presence of infiltrating TMEM123+ CD8+ T cells is associated with better overall and metastasis-free survival. TMEM123 localizes in the protrusions of infiltrating T cells, it contributes to lymphocyte migration and cytoskeleton organization. TMEM123 silencing modulates the underlying signaling pathways dependent on the cytoskeletal regulator WASP and the Arp2/3 actin nucleation complex, which are required for synaptic force exertion. Using tumoroid-lymphocyte co-culture assays, we found that lymphocytes form clusters through TMEM123, anchoring to cancer cells and contributing to their killing. We propose an active role for TMEM123 in the anti-cancer activity of T cells within tumour microenvironment.

SARS-COV-2 specific t-cells in patients with thyroid disorders related to COVID-19 are enriched in the thyroid and acquire a tissue-resident memory phenotype.

BACKGROUND: SARS-CoV-2 infections have been associated with the onset of thyroid disorders like classic subacute thyroiditis (SAT) or atypical SAT upon severe COVID disease (COV-A-SAT). Little is known about thyroid anti-viral immune responses. OBJECTIVES: To define the role of T-cells in COV-A-SAT. METHODS: T-cells from COV-A-SAT patients were analyzed by multi-dimensional flow cytometry, UMAP and DiffusionMap dimensionality reduction and FlowSOM clustering. T-cells from COVID-naïve healthy donors, patients with autoimmune thyroiditis (ATD) and with SAT following COVID vaccination were analyzed as controls. T-cells were analyzed four and eight months post-infection in peripheral blood and in thyroid specimen obtained by ultrasound-guided fine needle aspiration. SARS-COV2-specific T-cells were identified by cytokine production induced by SARS-COV2-derived peptides and with COVID peptide-loaded HLA multimers after HLA haplotyping. RESULTS: COV-A-SAT was associated with HLA-DRB1*13 and HLA-B*57. COV-A-SAT patients contained activated Th1- and cytotoxic CD4+ and CD8+ effector cells four months post-infection, which acquired a quiescent memory phenotype after eight months. Anti-SARS-CoV-2-specific T-cell responses were readily detectable in peripheral blood four months post-infection, but were reduced after eight months. CD4+ and CD8+ tissue-resident memory cells (TRM) were present in the thyroid, and circulating CXCR3+T-cells identified as their putative precursors. SARS-CoV-2-specific T-cells were enriched in the thyroid, and acquired a TRM phenotype eight months post-infection. CONCLUSIONS: The association of COV-A-SAT with specific HLA haplotypes suggests a genetic predisposition and a key role for T-cells. COV-A-SAT is characterized by a prolonged systemic anti-viral effector T-cell response and the late generation of COVID-specific TRM in the thyroid target tissue.

Chronic cholesterol administration to the brain supports complete and long-lasting cognitive and motor amelioration in Huntington's disease.

Evidence that Huntington's disease (HD) is characterized by impaired cholesterol biosynthesis in the brain has led to strategies to increase its level in the brain of the rapidly progressing R6/2 mouse model, with a positive therapeutic outcome. Here we tested the long-term efficacy of chronic administration of cholesterol to the brain of the slowly progressing zQ175DN knock-in HD mice in preventing ("early treatment") or reversing ("late treatment") HD symptoms. To do this we used the most advanced formulation of cholesterol loaded brain-permeable nanoparticles (NPs), termed hybrid-g7-NPs-chol, which were injected intraperitoneally. We show that one cycle of treatment with hybrid-g7-NPs-chol, administered in the presymptomatic ("early treatment") or symptomatic ("late treatment") stages is sufficient to normalize cognitive defects up to 5 months, as well as to improve other behavioral and neuropathological parameters. A multiple cycle treatment combining both early and late treatments ("2 cycle treatment") lasting 6 months generates therapeutic effects for more than 11 months, without severe adverse reactions. Sustained cholesterol delivery to the brain of zQ175DN mice also reduces mutant Huntingtin aggregates in both the striatum and cortex and completely normalizes synaptic communication in the striatal medium spiny neurons compared to saline-treated HD mice. Furthermore, through a meta-analysis of published and current data, we demonstrated the power of hybrid-g7-NPs-chol and other strategies able to increase brain cholesterol biosynthesis, to reverse cognitive decline and counteract the formation of mutant Huntingtin aggregates. These results demonstrate that cholesterol delivery via brain-permeable NPs is a therapeutic option to sustainably reverse HD-related behavioral decline and neuropathological signs over time, highlighting the therapeutic potential of cholesterol-based strategies in HD patients. DATA AVAILABILITY: This study does not include data deposited in public repositories. Data are available on request to the corresponding authors.

Mapping of functional SARS-CoV-2 receptors in human lungs establishes differences in variant binding and SLC1A5 as a viral entry modulator of hACE2.

BACKGROUND: The COVID-19 pandemic is an infectious disease caused by SARS-CoV-2. The first step of SARS-CoV-2 infection is the recognition of angiotensin-converting enzyme 2 (ACE2) receptors by the receptor-binding domain (RBD) of the viral Spike (S) glycoprotein. Although the molecular and structural bases of the SARS-CoV-2-RBD/hACE2 interaction have been thoroughly investigated in vitro, the relationship between hACE2 expression and in vivo infection is less understood. METHODS: Here, we developed an efficient SARS-CoV-2-RBD binding assay suitable for super resolution microscopy and simultaneous hACE2 immunodetection and mapped the correlation between hACE2 receptor abundance and SARS-CoV-2-RBD binding, both in vitro and in human lung biopsies. Next, we explored the specific proteome of SARS-CoV-2-RBD/hACE2 through a comparative mass spectrometry approach. FINDINGS: We found that only a minority of hACE2 positive spots are actually SARS-CoV-2-RBD binding sites, and that the relationship between SARS-CoV-2-RBD binding and hACE2 presence is variable, suggesting the existence of additional factors. Indeed, we found several interactors that are involved in receptor localization and viral entry and characterized one of them: SLC1A5, an amino acid transporter. High-resolution receptor-binding studies showed that co-expression of membrane-bound SLC1A5 with hACE2 predicted SARS-CoV-2 binding and entry better than hACE2 expression alone. SLC1A5 depletion reduces SARS-CoV-2 binding and entry. Notably, the Omicron variant is more efficient in binding hACE2 sites, but equally sensitive to SLC1A5 downregulation. INTERPRETATION: We propose a method for mapping functional SARS-CoV-2 receptors in vivo. We confirm the existence of hACE2 co-factors that may contribute to differential sensitivity of cells to infection. FUNDING: This work was supported by an unrestricted grant from "Fondazione Romeo ed Enrica Invernizzi" to Stefano Biffo and by AIRC under MFAG 2021 - ID. 26178 project - P.I. Manfrini Nicola.

Anti-Phospholipid Antibodies and Coronavirus Disease 2019: Vaccination Does Not Trigger Early Autoantibody Production in Healthcare Workers.

A molecular mimicry between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and human proteins supports the possibility that autoimmunity takes place during coronavirus disease 2019 (COVID-19) contributing to tissue damage. For example, anti-phospholipid antibodies (aPL) have been reported in COVID-19 as a result of such mimicry and thought to contribute to the immunothrombosis characteristic of the disease. Consistently, active immunization with the virus spike protein may elicit the production of cross-reactive autoantibodies, including aPL. We prospectively looked at the aPL production in healthcare workers vaccinated with RNA- (BNT162b2, n. 100) or adenovirus-based vaccines (ChAdOx1, n. 50). Anti-cardiolipin, anti-beta2 glycoprotein I, anti-phosphatidylserine/prothrombin immunoglobulin G (IgG), IgA, and IgM before and after vaccination were investigated. Anti-platelet factor 4 immunoglobulins were also investigated as autoantibodies associated with COVID-19 vaccination. Additional organ (anti-thyroid) and non-organ (anti-nuclear) autoantibodies and IgG against human proteome were tested as further post-vaccination autoimmunity markers. The antibodies were tested one or three months after the first injection of ChAdOx1 and BNT162b2, respectively; a 12-month clinical follow-up was also performed. Vaccination occasionally induced low titers of aPL and other autoantibodies but did not affect the titer of pre-existing autoantibodies. No significant reactivities against a microarray of approximately 20,000 human proteins were found in a subgroup of ChAdOx1-vaccinees. Consistently, we did not record any clinical manifestation theoretically associated with an underlying autoimmune disorder. The data obtained after the vaccination (two doses for the RNA-based and one dose for the adenovirus-based vaccines), and the clinical follow-up are not supporting the occurrence of an early autoimmune response in this cohort of healthcare workers.

Immunosuppressant Treatment in Rheumatic Musculoskeletal Diseases Does Not Inhibit Elicitation of Humoral Response to SARS-CoV-2 Infection and Preserves Effector Immune Cell Populations.

COVID-19 has proven to be particularly serious and life-threatening for patients presenting with pre-existing pathologies. Patients affected by rheumatic musculoskeletal disease (RMD) are likely to have impaired immune responses against SARS-CoV-2 infection due to their compromised immune system and the prolonged use of disease-modifying anti-rheumatic drugs (DMARDs), which include conventional synthetic (cs) DMARDs or biologic and targeted synthetic (b/ts) DMARDs. To provide an integrated analysis of the immune response following SARS-CoV-2 infection in RMD patients treated with different classes of DMARDs we carried out an immunological analysis of the antibody responses toward SARS-CoV-2 nucleocapsid and RBD proteins and an extensive immunophenotypic analysis of the major immune cell populations. We showed that RMD individuals under most DMARD treatments mount a sustained antibody response to the virus, with neutralizing activity. In addition, they displayed a sizable percentage of effector T and B lymphocytes. Among b-DMARDs, we found that anti-TNFα treatments are more favorable drugs to elicit humoral and cellular immune responses as compared to CTLA4-Ig and anti-IL6R inhibitors. This study provides a whole picture of the humoral and cellular immune responses in RMD patients by reassuring the use of DMARD treatments during COVID-19. The study points to TNF-α inhibitors as those DMARDs permitting elicitation of functional antibodies to SARS-CoV-2 and adaptive effector populations available to counteract possible re-infections.

Corrigendum: The Impact of Anti-rheumatic Drugs on the Seroprevalence of Anti-SARS-CoV-2 Antibodies in a Cohort of Patients With Inflammatory Arthritis: The MAINSTREAM Study.

[This corrects the article DOI: 10.3389/fmed.2022.850858.].

Synthetic carbohydrate-binding agents neutralize SARS-CoV-2 by inhibiting binding of the spike protein to ACE2.

Developing strategies against the SARS-CoV-2 is currently a main research subject. SARS-CoV-2 infects host cells by binding to human ACE2 receptors. Both, virus and ACE2, are highly glycosylated, and exploiting glycans of the SARS-CoV-2 envelope as binding sites for ACE2 represents a virus strategy for attacking the human host. We report here that a family of mannose-binding synthetic carbohydrate-binding agents (CBAs) inhibits SARS-CoV-2 infection, showing broad neutralizing activity vs. several variants of the spike protein. Preliminary tests indicated that the investigated CBAs interact with the spike protein rather than with ACE2. For a lead compound (IDS060), which has been selected among others for its lack of cytotoxicity, evidence of binding to the RBD of the spike protein has been found by NMR experiments, while competitive binding assays in the presence of IDS060 showed inhibition of binding of RBD to hACE2, although neutralizing activity was also observed with variants showing reduced or depleted binding.

The Impact of Anti-rheumatic Drugs on the Seroprevalence of Anti-SARS-CoV-2 Antibodies in a Cohort of Patients With Inflammatory Arthritis: The MAINSTREAM Study.

Objectives: Given the high occurrence of asymptomatic subsets, the true prevalence of SARS-CoV-2 infection in rheumatic patients is still underestimated. This study aims to evaluate the seroprevalence of SARS-CoV-2 antibodies in rheumatic musculoskeletal diseases (RMD) patients receiving immunomodulatory drugs. Methods: All consecutive patients with rheumatoid arthritis or spondyloarthritis receiving disease-modifying antirheumatic drugs (DMARDs) evaluated between 4th May and 16th June 2020 were included. All participants were tested for anti-SARS-CoV-2 antibodies (IgG, IgM, IgA) by ELISA and were questioned about previous COVID-19 symptoms and clinical course. Results were compared with healthy population from the same region and with a control group of healthy subjects diagnosed with confirmed COVID-19. Results: The study population includes 358 patients. The overall prevalence of anti-SARS-CoV-2 antibodies (18.4%) was higher than prevalence rate based on swab-positivity (1.12%) or clinically suspected cases (10.6%), but consistent with seroprevalence observed in the healthy population. Among seropositive patients 58% were asymptomatic. Mean anti-SARS-CoV-2 titer was comparable with the control group. No differences in seroprevalence were observed according to age, sex, rheumatic disease and treatment with conventional, biologic or targeted synthetic DMARDs, whereas glucocorticoids and comorbidities resulted in higher seroprevalence rate. Conclusions: The results of this study are reassuring about the low impact of RMDs and immunomodulatory therapies on the risk and clinical course of COVID-19 and on humoral immune response to SARS-CoV-2 infection.

Elucidating the 3D Structure of a Surface Membrane Antigen from Trypanosoma cruzi as a Serodiagnostic Biomarker of Chagas Disease.

Chagas disease (CD) is a vector-borne parasitosis, caused by the protozoan parasite Trypanosoma cruzi, that affects millions of people worldwide. Although endemic in South America, CD is emerging throughout the world due to climate change and increased immigratory flux of infected people to non-endemic regions. Containing of the diffusion of CD is challenged by the asymptomatic nature of the disease in early infection stages and by the lack of a rapid and effective diagnostic test. With the aim of designing new serodiagnostic molecules to be implemented in a microarray-based diagnostic set-up for early screening of CD, herein, we report the recombinant production of the extracellular domain of a surface membrane antigen from T. cruzi (TcSMP) and confirm its ability to detect plasma antibodies from infected patients. Moreover, we describe its high-resolution (1.62 Å) crystal structure, to which in silico epitope predictions were applied in order to locate the most immunoreactive regions of TcSMP in order to guide the design of epitopes that may be used as an alternative to the full-length antigen for CD diagnosis. Two putative, linear epitopes, belonging to the same immunogenic region, were synthesized as free peptides, and their immunological properties were tested in vitro. Although both peptides were shown to adopt a structural conformation that allowed their recognition by polyclonal antibodies raised against the recombinant protein, they were not serodiagnostic for T. cruzi infections. Nevertheless, they represent good starting points for further iterative structure-based (re)design cycles.

Integrated longitudinal immunophenotypic, transcriptional and repertoire analyses delineate immune responses in COVID-19 patients.

To understand how a protective immune response against SARS-CoV-2 develops over time, we integrated phenotypic, transcriptional and repertoire analyses on PBMCs from mild and severe COVID-19 patients during and after infection, and compared them to healthy donors (HD). A type I IFN-response signature marked all the immune populations from severe patients during the infection. Humoral immunity was dominated by IgG production primarily against the RBD and N proteins, with neutralizing antibody titers increasing post infection and with disease severity. Memory B cells, including an atypical FCRL5+ T-BET+ memory subset, increased during the infection, especially in patients with mild disease. A significant reduction of effector memory, CD8+ T cells frequency characterized patients with severe disease. Despite such impairment, we observed robust clonal expansion of CD8+ T lymphocytes, while CD4+ T cells were less expanded and skewed toward TCM and TH2-like phenotypes. MAIT cells were also expanded, but only in patients with mild disease. Terminally differentiated CD8+ GZMB+ effector cells were clonally expanded both during the infection and post-infection, while CD8+ GZMK+ lymphocytes were more expanded post-infection and represented bona fide memory precursor effector cells. TCR repertoire analysis revealed that only highly proliferating T cell clonotypes, which included SARS-CoV-2-specific cells, were maintained post-infection and shared between the CD8+ GZMB+ and GZMK+ subsets. Overall, this study describes the development of immunity against SARS-CoV-2 and identifies an effector CD8+ T cell population with memory precursor-like features.

Structure, Immunoreactivity, and In Silico Epitope Determination of SmSPI S. mansoni Serpin for Immunodiagnostic Application.

The human parasitic disease Schistosomiasis is caused by the Schistosoma trematode flatworm that infects freshwaters in tropical regions of the world, particularly in Sub-Saharan Africa, South America, and the Far-East. It has also been observed as an emerging disease in Europe, due to increased immigration. In addition to improved therapeutic strategies, it is imperative to develop novel, rapid, and sensitive diagnostic tests that can detect the Schistosoma parasite, allowing timely treatment. Present diagnosis is difficult and involves microscopy-based detection of Schistosoma eggs in the feces. In this context, we present the 3.22 Å resolution crystal structure of the circulating antigen Serine protease inhibitor from S. mansoni (SmSPI), and we describe it as a potential serodiagnostic marker. Moreover, we identify three potential immunoreactive epitopes using in silico-based epitope mapping methods. Here, we confirm effective immune sera reactivity of the recombinant antigen, suggesting the further investigation of the protein and/or its predicted epitopes as serodiagnostic Schistosomiasis biomarkers.

ADAM10 hyperactivation acts on piccolo to deplete synaptic vesicle stores in Huntington's disease.

Synaptic dysfunction and cognitive decline in Huntington's disease (HD) involve hyperactive A disintegrin and metalloproteinase domain-containing protein 10 (ADAM10). To identify the molecular mechanisms through which ADAM10 is associated with synaptic dysfunction in HD, we performed an immunoaffinity purification-mass spectrometry (IP-MS) study of endogenous ADAM10 in the brains of wild-type and HD mice. We found that proteins implicated in synapse organization, synaptic plasticity, and vesicle and organelles trafficking interact with ADAM10, suggesting that it may act as hub protein at the excitatory synapse. Importantly, the ADAM10 interactome is enriched in presynaptic proteins and ADAM10 co-immunoprecipitates with piccolo (PCLO), a key player in the recycling and maintenance of synaptic vesicles. In contrast, reduced ADAM10/PCLO immunoprecipitation occurs in the HD brain, with decreased density of synaptic vesicles in the reserve and docked pools at the HD presynaptic terminal. Conditional heterozygous deletion of ADAM10 in the forebrain of HD mice reduces active ADAM10 to wild-type level and normalizes ADAM10/PCLO complex formation and synaptic vesicle density and distribution. The results indicate that presynaptic ADAM10 and PCLO are a relevant component of HD pathogenesis.

Insights into kinetics, release, and behavioral effects of brain-targeted hybrid nanoparticles for cholesterol delivery in Huntington's disease.

Supplementing brain cholesterol is emerging as a potential treatment for Huntington's disease (HD), a genetic neurodegenerative disorder characterized, among other abnormalities, by inefficient brain cholesterol biosynthesis. However, delivering cholesterol to the brain is challenging due to the blood-brain barrier (BBB), which prevents it from reaching the striatum, especially, with therapeutically relevant doses. Here we describe the distribution, kinetics, release, and safety of novel hybrid polymeric nanoparticles made of PLGA and cholesterol which were modified with an heptapeptide (g7) for BBB transit (hybrid-g7-NPs-chol). We show that these NPs rapidly reach the brain and target neural cells. Moreover, deuterium-labeled cholesterol from hybrid-g7-NPs-chol is released in a controlled manner within the brain and accumulates over time, while being rapidly removed from peripheral tissues and plasma. We confirm that systemic and repeated injections of the new hybrid-g7-NPs-chol enhanced endogenous cholesterol biosynthesis, prevented cognitive decline, and ameliorated motor defects in HD animals, without any inflammatory reaction. In summary, this study provides insights about the benefits and safety of cholesterol delivery through advanced brain-permeable nanoparticles for HD treatment.

Exosomes Recovered From the Plasma of COVID-19 Patients Expose SARS-CoV-2 Spike-Derived Fragments and Contribute to the Adaptive Immune Response.

Coronavirus disease 2019 (COVID-19) is an infectious disease caused by beta-coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that has rapidly spread across the globe starting from February 2020. It is well established that during viral infection, extracellular vesicles become delivery/presenting vectors of viral material. However, studies regarding extracellular vesicle function in COVID-19 pathology are still scanty. Here, we performed a comparative study on exosomes recovered from the plasma of either MILD or SEVERE COVID-19 patients. We show that although both types of vesicles efficiently display SARS-CoV-2 spike-derived peptides and carry immunomodulatory molecules, only those of MILD patients are capable of efficiently regulating antigen-specific CD4+ T-cell responses. Accordingly, by mass spectrometry, we show that the proteome of exosomes of MILD patients correlates with a proper functioning of the immune system, while that of SEVERE patients is associated with increased and chronic inflammation. Overall, we show that exosomes recovered from the plasma of COVID-19 patients possess SARS-CoV-2-derived protein material, have an active role in enhancing the immune response, and possess a cargo that reflects the pathological state of patients in the acute phase of the disease.

A Structurally Simple Vaccine Candidate Reduces Progression and Dissemination of Triple-Negative Breast Cancer.

The Tn antigen is a well-known tumor-associated carbohydrate determinant, often incorporated in glycopeptides to develop cancer vaccines. Herein, four copies of a conformationally constrained mimetic of the antigen TnThr (GalNAc-Thr) were conjugated to the adjuvant CRM197, a protein licensed for human use. The resulting vaccine candidate, mime[4]CRM elicited a robust immune response in a triple-negative breast cancer mouse model, correlated with high frequency of CD4+ T cells and low frequency of M2-type macrophages, which reduces tumor progression and lung metastasis growth. Mime[4]CRM-mediated activation of human dendritic cells is reported, and the proliferation of mime[4]CRM-specific T cells, in cancer tissue and peripheral blood of patients with breast cancer, is demonstrated. The locked conformation of the TnThr mimetic and a proper presentation on the surface of CRM197 may explain the binding of the conjugate to the anti-Tn antibody Tn218 and its efficacy to fight cancer cells in mice.

Novel biomarkers for primary biliary cholangitis to improve diagnosis and understand underlying regulatory mechanisms.

BACKGROUND AND AIMS: Primary biliary cholangitis is an autoimmune biliary disease characterized by injury of bile ducts, eventually leading to cirrhosis and death. In most cases, anti-mitochondrial antibodies and persistently elevated serum alkaline phosphatase are the basis for the serological diagnosis. Anti-nuclear antibodies are also useful and may indicate a more aggressive diseases course. In patients in which anti-mitochondrial antibodies are not detected, an accurate diagnosis requires liver histology. This study aims at identifying specific biomarkers for the serological diagnosis of primary biliary cholangitis. METHODS: Sera from patients affected by primary biliary cholangitis, primary sclerosing cholangitis, hepatitis C virus (with and without cryoglobulinemia), hepatocarcinoma and healthy donors were tested on a protein array representing 1658 human proteins. The most reactive autoantigens were confirmed by DELFIA analysis on expanded cohorts of the same mentioned serum classes, and on autoimmune hepatitis sera, using anti-PDC-E2 as reference biomarker. RESULTS: Two autoantigens, SPATA31A3 and GARP, showed high reactivity with primary biliary cholangitis sera, containing or not anti-mitochondrial antibodies. Their combination with PDC-E2 allowed to discriminate primary biliary cholangitis from all tested control classes with high sensitivity and specificity. We found that GARP expression is upregulated upon exposure to biliary salts in human cholangiocytes, an event involving EGFR and insulin pathways. GARP expression was also detected in biliary duct cells of PBC patients. CONCLUSIONS: This study highlighted SPATA31A3 and GARP as new biomarkers for primary biliary cholangitis and unravelled molecular stimuli underlying GARP expression in human cholangiocytes.

Computation and Selection of Optimal Biomarker Combinations by Integrative ROC Analysis Using CombiROC.

The diagnostic accuracy of biomarker-based approaches can be considerably improved by combining multiple markers. A biomarker's capacity to identify specific subjects is usually assessed using receiver operating characteristic (ROC) curves. Multimarker signatures are complicated to select as data signatures must be integrated using sophisticated statistical methods. CombiROC, developed as a user-friendly web tool, helps researchers to accurately determine optimal combinations of markers identified by a range of omics methods. With CombiROC, data of different types, such as proteomics and transcriptomics, can be analyzed using Sensitivity/Specificity filters: the number of candidate marker panels arising from combinatorial analysis is easily optimized bypassing limitations imposed by the nature of different experimental approaches. Users have full control over initial selection stringency, then CombiROC computes sensitivity and specificity for all marker combinations, determines performance for the best combinations, and produces ROC curves for automatic comparisons. All steps can be visualized in a graphic interface. CombiROC is designed without hard-coded thresholds, to allow customized fitting of each specific dataset: this approach dramatically reduces computational burden and false-negative rates compared to fixed thresholds. CombiROC can be accessed at www.combiroc.eu .