Identification of a novel antibody associated with autoimmune pancreatitis.

BACKGROUND: Autoimmune pancreatitis is characterized by an inflammatory process that leads to organ dysfunction. The cause of the disease is unknown. Its autoimmune origin has been suggested but never proved, and little is known about the pathogenesis of this condition. METHODS: To identify pathogenetically relevant autoantigen targets, we screened a random peptide library with pooled IgG obtained from 20 patients with autoimmune pancreatitis. Peptide-specific antibodies were detected in serum specimens obtained from the patients. RESULTS: Among the detected peptides, peptide AIP(1-7) was recognized by the serum specimens from 18 of 20 patients with autoimmune pancreatitis and by serum specimens from 4 of 40 patients with pancreatic cancer, but not by serum specimens from healthy controls. The peptide showed homology with an amino acid sequence of plasminogen-binding protein (PBP) of Helicobacter pylori and with ubiquitin-protein ligase E3 component n-recognin 2 (UBR2), an enzyme highly expressed in acinar cells of the pancreas. Antibodies against the PBP peptide were detected in 19 of 20 patients with autoimmune pancreatitis (95%) and in 4 of 40 patients with pancreatic cancer (10%). Such reactivity was not detected in patients with alcohol-induced chronic pancreatitis or intraductal papillary mucinous neoplasm. The results were validated in another series of patients with autoimmune pancreatitis or pancreatic cancer: 14 of 15 patients with autoimmune pancreatitis (93%) and 1 of 70 patients with pancreatic cancer (1%) had a positive test for anti-PBP peptide antibodies. When the training and validation groups were combined, the test was positive in 33 of 35 patients with autoimmune pancreatitis (94%) and in 5 of 110 patients with pancreatic cancer (5%). CONCLUSIONS: The antibody that we identified was detected in most patients with autoimmune pancreatitis but also in some patients with pancreatic cancer, making it an imperfect test to distinguish between these two conditions.

Generation of anti-NAG-2 mAb from patients' memory B cells: implications for a novel therapeutic strategy in systemic sclerosis.

We have previously reported that antibodies directed against the cytomegalovirus-derived protein UL94 cross react with the cell surface tetraspanin transmembrane 4 superfamily member 7 (TM4SF7 or NAG-2) molecule inducing apoptosis of endothelial cells and activation of fibroblasts in patients with systemic sclerosis (SSc). We aimed at generating a non-functional mAb directed against NAG-2 from patients' memory B cells. Direct and competitive ELISA methods have been used to evaluate the binding of antibodies from scleroderma patients' and controls' sera to the NAG-2 peptide. IgG memory B cells were sorted, EBV transformed and cloned to obtain NAG-2-specific mAbs. Endothelial cells and fibroblasts were cultured under standard conditions and used for functional assays. Anti-NAG-2-purified antibodies obtained from patients' Ig induce endothelial cell apoptosis and fibroblast proliferation. Patients' Igs depleted of the anti-NAG-2 fraction do not exert such functional activity. Therefore, the NAG-2 molecule represents a potential novel candidate for therapeutic intervention in SSc. Here, we describe the generation of a human mAb directed against the NAG-2 molecule. Such mAb does not retain any functional property and is able to block the effect of serum pathogenetic anti-NAG-2 antibodies. The majority of SSc patients present antibodies directed against tetraspanin NAG-2 and mediate both endothelial cell apoptosis and fibroblast proliferation, features of the disease. The anti-NAG-2 human mAb we have obtained blocks signal transduction and therefore may be a potential candidate for a new treatment in SSc, a disease where the current biological therapies have little or no efficacy.

Serum DNase I, soluble Fas/FasL levels and cell surface Fas expression in patients with SLE: a possible explanation for the lack of efficacy of hrDNase I treatment.

The objectives of the study are to evaluate DNase I serum levels and their correlation with soluble Fas (sFas) and soluble Fas ligand (sFasL) and with cell surface Fas expression in patients with systemic lupus erythematosus (SLE), thus contributing to the dysregulated apoptosis typical of the disease. The methods include the following: Serum DNase I levels in patients and in controls were detected using the dot blot method and quantified by densitometry; sFas and sFasL were quantified using an ELISA system. Cell surface Fas expression was evaluated by FACS analysis. Apoptosis was studied by means of internucleosomal DNA degradation using a commercially available kit. The results are as follows: We found a significant difference in DNase I, sFas and sFasL serum levels between patients and controls. Levels of DNase I <7.79 ng ml(-1) are more represented in patients with SLE. Active SLE is strongly associated with high sFas levels and detectable sFasL. DNase I does not correlate with sFas or sFasL, whereas it correlates with T cell surface Fas expression that is higher in patients with active SLE than in healthy controls. Finally, administration of exogenous human recombinant DNase (hrDNase) I to freshly isolated T cells up-regulates cell surface Fas expression and induces increased susceptibility to Fas-mediated apoptosis. In conclusion, our findings confirm that DNase I is low in SLE and suggest that it may play a role in apoptosis in SLE by regulating the surface expression of the cell death molecule Fas. This role may contribute to explain the inefficacy of hrDNase I in SLE, a treatment proposed for the ability of DNase I to remove DNA from auto-antigenic nucleoprotein complexes.

Gene Expression Profiling in Fibromyalgia Indicates an Autoimmune Origin of the Disease and Opens New Avenues for Targeted Therapy.

Fibromyalgia is a chronic disorder characterized by widespread pain and by several non-pain symptoms. Autoimmunity, small fiber neuropathy and neuroinflammation have been suggested to be involved in the pathogenesis of the disease. We have investigated the gene expression profile in peripheral blood mononuclear cells obtained from ten patients and ten healthy subjects. Of the 545,500 transcripts analyzed, 1673 resulted modulated in fibromyalgic patients. The majority of these genes are involved in biological processes and pathways linked to the clinical manifestations of the disease. Moreover, genes involved in immunological pathways connected to interleukin-17 and to Type I interferon signatures were also modulated, suggesting that autoimmunity plays a role in the disease. We then aimed at identifying differentially expressed Long non-coding RNAs (LncRNAs) functionally connected to modulated genes both directly and via microRNA targeting. Only two LncRNAs of the 298 found modulated in patients, were able to target the most highly connected genes in the fibromyalgia interactome, suggesting their involvement in crucial gene regulation. Our gene expression data were confirmed by real time PCR, by autoantibody testing, detection of soluble mediators and Th-17 polarization in a validation cohort of 50 patients. Our results indicate that genetic and epigenetic mechanisms as well as autoimmunity play a pivotal role in the pathogenesis of fibromyalgia.

Pathogenesis of immune thrombocytopenia in common variable immunodeficiency.

Immune Thrombocitopenic Purpura (ITP) is an autoimmune disease characterized by antibody-mediated platelet destruction and variable reduced platelet production. Besides antibody-mediated platelet destruction, new pathogenic mechanisms have been reported to be involved in reducing platelet count. Among these, desialylation is one of the most recent and innovative mechanisms that has been found to be implied, at least in part, in non-antibody mediated platelet clearance. Common Variable Immunodeficiency (CVID) is the most common Primary Immunodeficiency seen in clinical practice. About 25-30% of CVID patients are affected by autoimmune manifestation, among which ITP is the most common. Little is know about pathophysiological mechanisms that lead to ITP in CVID. Given the poor antibody production typical of CVID patients, we aimed at verifying whether platelet desialylation could be responsible for CVID associated thrombocytopenia. According to our results, we may suggest that in CVID patients, ITP is due to a decreased bone marrow platelets production, rather than an increased peripheral platelet destruction, which is more common in patients with primary ITP. An increased platelet desialylation does not appear to be implicated in the thrombocytopenia secondary to CVID, while it is implicated in the pathogenesis of primary ITP. Nevertheless an intriguing aspect has emerged from this study: regardless the presence of thrombocytopenia, the majority of CVID patients present a double platelet population as far as desialylation concerns, whilst no one of the healthy donors and of the patients with primary ITP shows a similar characteristic.

In Systemic Sclerosis, a Unique Long Non Coding RNA Regulates Genes and Pathways Involved in the Three Main Features of the Disease (Vasculopathy, Fibrosis and Autoimmunity) and in Carcinogenesis.

Systemic sclerosis (SSc) is an autoimmune disease characterized by three main features: vasculopathy, immune system dysregulation and fibrosis. Long non-coding RNAs (lncRNAs) may play a role in the pathogenesis of autoimmune diseases and a comprehensive analysis of lncRNAs expression in SSc is still lacking. We profiled 542,500 transcripts in peripheral blood mononuclear cells (PBMCs) from 20 SSc patients and 20 healthy donors using Clariom D arrays, confirming the results by Reverse Transcription Polymerase-chain reaction (RT-PCR). A total of 837 coding-genes were modulated in SSc patients, whereas only one lncRNA, heterogeneous nuclear ribonucleoprotein U processed transcript (ncRNA00201), was significantly downregulated. This transcript regulates tumor proliferation and its gene target hnRNPC (Heterogeneous nuclear ribonucleoproteins C) encodes for a SSc-associated auto-antigen. NcRNA00201 targeted micro RNAs (miRNAs) regulating the most highly connected genes in the Protein-Protein interaction (PPI) network of the SSc transcriptome. A total of 26 of these miRNAs targeted genes involved in pathways connected to the three main features of SSc and to cancer development including Epidermal growth factor (EGF) receptor, ErbB1 downstream, Sphingosine 1 phosphate receptor 1 (S1P1), Activin receptor-like kinase 1 (ALK1), Endothelins, Ras homolog family member A (RhoA), Class I Phosphoinositide 3-kinase (PI3K), mammalian target of rapamycin (mTOR), p38 mitogen-activated protein kinase (MAPK), Ras-related C3 botulinum toxin substrate 1 (RAC1), Transforming growth factor (TGF)-beta receptor, Myeloid differentiation primary response 88 (MyD88) and Toll-like receptors (TLRs) pathways. In SSc, the identification of a unique deregulated lncRNA that regulates genes involved in the three main features of the disease and in tumor-associated pathways, provides insight in disease pathogenesis and opens avenues for the design of novel therapeutic strategies.

Long Non-Coding RNAs Target Pathogenetically Relevant Genes and Pathways in Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease driven by genetic, environmental and epigenetic factors. Long non-coding RNAs (LncRNAs) are a key component of the epigenetic mechanisms and are known to be involved in the development of autoimmune diseases. In this work we aimed to identify significantly differentially expressed LncRNAs (DE-LncRNAs) that are functionally connected to modulated genes strictly associated with RA. In total, 542,500 transcripts have been profiled in peripheral blood mononuclear cells (PBMCs) from four patients with early onset RA prior any treatment and four healthy donors using Clariom D arrays. Results were confirmed by real-time PCR in 20 patients and 20 controls. Six DE-LncRNAs target experimentally validated miRNAs able to regulate differentially expressed genes (DEGs) in RA; among them, only FTX, HNRNPU-AS1 and RP11-498C9.15 targeted a large number of DEGs. Most importantly, RP11-498C9.15 targeted the largest number of signalling pathways that were found to be enriched by the global amount of RA-DEGs and that have already been associated with RA and RA-synoviocytes. Moreover, RP11-498C9.15 targeted the most highly connected genes in the RA interactome, thus suggesting its involvement in crucial gene regulation. These results indicate that, by modulating both microRNAs and gene expression, RP11-498C9.15 may play a pivotal role in RA pathogenesis.

Long Non-Coding RNAs Modulate Sjögren's Syndrome Associated Gene Expression and Are Involved in the Pathogenesis of the Disease.

Primary Sjögren's syndrome (pSjS) is a chronic systemic autoimmune disorder, primarily affecting exocrine glands; its pathogenesis is still unclear. Long non-coding RNAs (lncRNAs) are thought to play a role in the pathogenesis of autoimmune diseases and a comprehensive analysis of lncRNAs expression in pSjS is still lacking. To this aim, the expression of more than 540,000 human transcripts, including those ascribed to more than 50,000 lncRNAs is profiled at the same time, in a cohort of 16 peripheral blood mononuclear cells PBMCs samples (eight pSjS and eight healthy subjects). A complex network analysis is carried out on the global set of molecular interactions among modulated genes and lncRNAs, leading to the identification of reliable lncRNA-miRNA-gene functional interactions. Taking this approach, a few lncRNAs are identified as targeting highly connected genes in the pSjS transcriptome, since they have a major impact on gene modulation in the disease. Such genes are involved in biological processes and molecular pathways crucial in the pathogenesis of pSjS, including immune response, B cell development and function, inflammation, apoptosis, type I and gamma interferon, epithelial cell adhesion and polarization. The identification of deregulated lncRNAs that modulate genes involved in the typical features of the disease provides insight in disease pathogenesis and opens avenues for the design of novel therapeutic strategies.

Gene Expression Profiles in Primary Sjögren's Syndrome With and Without Systemic Manifestations.

OBJECTIVE: To investigate the gene expression profile in patients with Sjögren's syndrome that is characterized by different clinical phenotypes. METHODS: RNA from peripheral blood mononuclear cells was purified in 8 patients with glandular features (GFs) and widespread pain (WP) and 11 with extraglandular manifestations (EGMs) and then was analyzed by hybridization on a human gene chip exploring more than 40,000 human genes. Differentially expressed genes (DEGs) in the two subgroups (ie, those with false discovery rate-corrected P values ≤ 0.01) with respect to 20 healthy controls have been submitted to functional classification using a Gene Ontology database and were mapped to define the networks of protein to protein interactions (PPIs). RESULTS: The enriched pathway analyses of DEGs and of the highly interconnected modules identified in the PPI networks showed that the pathological processes characterizing the two subgroups were substantially different. The predominant pathways in patients with EGMs are related to T- and B-cell activation, Toll-like receptor, interferon signaling, and apoptosis. Conversely, pathological processes related to pain transmission and modulation are preferentially operative in patients with GFs and WP. These data suggest that a neuroinflammatory pathway driven by cytokines and chemokines may play a central role in triggering WP features in this phenotype of patients. CONCLUSION: The present study supports the hypothesis that different biological pathways are operative in patients with primary Sjögren's syndrome with different clinical phenotypes. A better knowledge of these specific processes might help in tailoring more effective target therapies.

MicroRNA Expression Profiling in Behçet's Disease.

BACKGROUND: Behçet's disease (BD) is a chronic inflammatory multisystem disease characterized by oral and genital ulcers, uveitis, and skin lesions. MicroRNAs (miRNAs) are key regulators of immune responses. Differential expression of miRNAs has been reported in several inflammatory autoimmune diseases; however, their role in BD is not fully elucidated. We aimed to identify miRNA expression signatures associated with BD and to investigate their potential implication in the disease pathogenesis. METHODS: miRNA microarray analysis was performed in blood cells of BD patients and healthy controls. miRNA expression profiles were analyzed using Affymetrix arrays with a comprehensive coverage of miRNA sequences. Pathway analyses were performed, and the global miRNA profiling was combined with transcriptoma data in BD. Deregulation of selected miRNAs was validated by real-time PCR. RESULTS: We identified specific miRNA signatures associated with BD patients with active disease. These miRNAs target pathways relevant in BD, such as TNF, IFN gamma, and VEGF-VEGFR signaling cascades. Network analysis revealed several miRNAs regulating highly connected genes within the BD transcriptoma. CONCLUSIONS: The combined analysis of deregulated miRNAs and BD transcriptome sheds light on some epigenetic aspects of BD identifying specific miRNAs, which may represent promising candidates as biomarkers and/or for the design of novel therapeutic strategies in BD.

Long Non-Coding RNAs Play a Role in the Pathogenesis of Psoriatic Arthritis by Regulating MicroRNAs and Genes Involved in Inflammation and Metabolic Syndrome.

Psoriatic arthritis (PsA) is an inflammatory arthritis, characterized by inflammation of entheses and synovium, leading to joint erosions and new bone formation. It affects 10-30% of patients with psoriasis, and has an estimated prevalence of approximately 1%. PsA is considered to be primarily an autoimmune disease, driven by autoreactive T cells directed against autoantigens present in the skin and in the joints. However, an autoinflammatory origin has recently been proposed. Long noncoding RNAs (lncRNAs) are RNAs more than 200 nucleotides in length that do not encode proteins. LncRNAs play important roles in several biological processes, including chromatin remodeling, transcription control, and post-transcriptional processing. Several studies have shown that lncRNAs are expressed in a stage-specific or lineage-specific manner in immune cells that have a role in the development, activation, and effector functions of immune cells. LncRNAs are thought to play a role in several diseases, including autoimmune disorders. Indeed, a few lncRNAs have been identified in systemic lupus erythematosus, rheumatoid arthritis, and psoriasis. Although several high-throughput studies have been performed to identify lncRNAs, their biological and pathological relevance are still unknown, and most transcriptome studies in autoimmune diseases have only assessed protein-coding transcripts. No data are currently available on lncRNAs in PsA. Therefore, by microarray analysis, we have investigated the expression profiles of more than 50,000 human lncRNAs in blood samples from PsA patients and healthy controls using Human Clariom D Affymetrix chips, suitable to detect rare and low-expressing transcripts otherwise unnoticed by common sequencing methodologies. Network analysis identified lncRNAs targeting highly connected genes in the PsA transcriptome. Such genes are involved in molecular pathways crucial for PsA pathogenesis, including immune response, glycolipid metabolism, bone remodeling, type 1 interferon, wingless related integration site, and tumor necrosis factor signaling. Selected lncRNAs were validated by RT-PCR in an expanded cohort of patients. Moreover, modulated genes belonging to meaningful pathways were validated by RT-PCR in PsA PBMCs and/or by ELISA in PsA sera. The findings indicate that lncRNAs are involved in PsA pathogenesis by regulating both microRNAs and genes and open new avenues for the identification of new biomarkers and therapeutical targets.

MicroRNA Expression Profiling in Psoriatic Arthritis.

BACKGROUND: Psoriatic arthritis (PsA) is an inflammatory arthritis, characterized by bone erosions and new bone formation. MicroRNAs (miRNAs) are key regulators of the immune responses. Differential expression of miRNAs has been reported in several inflammatory autoimmune diseases; however, their role in PsA is not fully elucidated. We aimed to identify miRNA expression signatures associated with PsA and to investigate their potential implication in the disease pathogenesis. METHODS: miRNA microarray was performed in blood cells of PsA patients and healthy controls. miRNA pathway analyses were performed and the global miRNA profiling was combined with transcriptome data in PsA. Deregulation of selected miRNAs was validated by real-time PCR. RESULTS: We identified specific miRNA signatures associated with PsA patients with active disease. These miRNAs target pathways relevant in PsA, such as TNF, MAPK, and WNT signaling cascades. Network analysis revealed several miRNAs regulating highly connected genes within the PsA transcriptome. miR-126-3p was the most downregulated miRNA in active patients. Noteworthy, miR-126 overexpression induced a decreased expression of genes implicated in PsA. CONCLUSIONS: This study sheds light on some epigenetic aspects of PsA identifying specific miRNAs, which may represent promising candidates as biomarkers and/or for the design of novel therapeutic strategies in PsA.

Gene Expression Profiling in Behcet's Disease Indicates an Autoimmune Component in the Pathogenesis of the Disease and Opens New Avenues for Targeted Therapy.

Behçet disease (BD) is a chronic inflammatory multisystem disease characterized by oral and genital ulcers, uveitis, and skin lesions. Disease etiopathogenesis is still unclear. We aim to elucidate some aspects of BD pathogenesis and to identify specific gene signatures in peripheral blood cells (PBCs) of patients with active disease using novel gene expression and network analysis. 179 genes were modulated in 10 PBCs of BD patients when compared to 10 healthy donors. Among differentially expressed genes the top enriched gene function was immune response, characterized by upregulation of Th17-related genes and type I interferon- (IFN-) inducible genes. Th17 polarization was confirmed by FACS analysis. The transcriptome identified gene classes (vascular damage, blood coagulation, and inflammation) involved in the pathogenesis of the typical features of BD. Following network analysis, the resulting interactome showed 5 highly connected regions (clusters) enriched in T and B cell activation pathways and 2 clusters enriched in type I IFN, JAK/STAT, and TLR signaling pathways, all implicated in autoimmune diseases. We report here the first combined analysis of the transcriptome and interactome in PBCs of BD patients in the active stage of disease. This approach generates useful insights in disease pathogenesis and suggests an autoimmune component in the origin of BD.

Gene Profiling in Patients with Systemic Sclerosis Reveals the Presence of Oncogenic Gene Signatures.

Systemic sclerosis (SSc) is a rare connective tissue disease characterized by three pathogenetic hallmarks: vasculopathy, dysregulation of the immune system, and fibrosis. A particular feature of SSc is the increased frequency of some types of malignancies, namely breast, lung, and hematological malignancies. Moreover, SSc may also be a paraneoplastic disease, again indicating a strong link between cancer and scleroderma. The reason of this association is still unknown; therefore, we aimed at investigating whether particular genetic or epigenetic factors may play a role in promoting cancer development in patients with SSc and whether some features are shared by the two conditions. We therefore performed a gene expression profiling of peripheral blood mononuclear cells (PBMCs) derived from patients with limited and diffuse SSc, showing that the various classes of genes potentially linked to the pathogenesis of SSc (such as apoptosis, endothelial cell activation, extracellular matrix remodeling, immune response, and inflammation) include genes that directly participate in the development of malignancies or that are involved in pathways known to be associated with carcinogenesis. The transcriptional analysis was then complemented by a complex network analysis of modulated genes which further confirmed the presence of signaling pathways associated with carcinogenesis. Since epigenetic mechanisms, such as microRNAs (miRNAs), are believed to play a central role in the pathogenesis of SSc, we also evaluated whether specific cancer-related miRNAs could be deregulated in the serum of SSc patients. We focused our attention on miRNAs already found upregulated in SSc such as miR-21-5p, miR-92a-3p, and on miR-155-5p, miR 126-3p and miR-16-5p known to be deregulated in malignancies associated to SSc, i.e., breast, lung, and hematological malignancies. miR-21-5p, miR-92a-3p, miR-155-5p, and miR-16-5p expression was significantly higher in SSc sera compared to healthy controls. Our findings indicate the presence of modulated genes and miRNAs that can play a predisposing role in the development of malignancies in SSc and are important for a better risk stratification of patients and for the identification of a better individualized precision medicine strategy.

Immune Response to Rotavirus and Gluten Sensitivity.

Rotavirus is a double-stranded RNA virus belonging to the family of Reoviridae. The virus is transmitted by the faecal-oral route and infects intestinal cells causing gastroenteritis. Rotaviruses are the main cause of severe acute diarrhoea in children less than 5 years of age worldwide. In our previous work we have shown a link between rotavirus infection and celiac disease. Nonceliac gluten sensitivity (NCGS) is emerging as new clinical entity lacking specific diagnostic biomarkers which has been reported to occur in 6-10% of the population. Clinical manifestations include gastrointestinal and/or extraintestinal symptoms which recede with gluten withdrawal. The pathogenesis of the disease is still unknown. Aim of this work is to clarify some aspects of its pathogenesis using a gene array approach. Our results suggest that NCGS may have an autoimmune origin. This is based both on gene expression data (i.e., TH17-interferon signatures) and on the presence of TH17 cells and of serological markers of autoimmunity in NCGS. Our results also indicate a possible involvement of rotavirus infection in the pathogenesis of nonceliac gluten sensitivity similarly to what we have previously shown in celiac disease.

Antibodies Directed against a Peptide Epitope of a Klebsiella pneumoniae-Derived Protein Are Present in Ankylosing Spondylitis.

Ankylosing spondylitis (AS) is a chronic inflammatory arthritis of unknown origin. Its autoimmune origin has been suggested but never proven. Several reports have implicated Klebsiella pneumoniae as a triggering or perpetuating factor in AS; however, its role in the disease pathogenesis remains debated. Moreover, despite extensive investigations, a biomarker for AS has not yet been identified. To clarify these issues, we screened a random peptide library with pooled IgGs obtained from 40 patients with AS. A peptide (AS peptide) selected from the library was recognized by serum IgGs from 170 of 200 (85%) patients with AS but not by serum specimens from 100 healthy controls. Interestingly, the AS peptide shows a sequence similarity with several molecules expressed at the fibrocartilaginous sites that are primarily involved in the AS inflammatory process. Moreover, the peptide is highly homologous to a Klebsiella pneumoniae dipeptidase (DPP) protein. The antibody affinity purified against the AS peptide recognizes the autoantigens and the DPP protein. Furthermore, serum IgG antibodies against the Klebsiella DPP121-145 peptide epitope were detected in 190 of 200 patients with AS (95%), 3 of 200 patients with rheumatoid arthritis (1.5%) and only 1 of 100 (1%) patients with psoriatic arthritis. Such reactivity was not detected in healthy control donors. Our results show that antibodies directed against an epitope of a Klebsiella pneumoniae-derived protein are present in nearly all patients with AS. In the absence of serological biomarkers for AS, such antibodies may represent a useful tool in the diagnosis of the disease.

Gene Expression Analysis before and after Treatment with Adalimumab in Patients with Ankylosing Spondylitis Identifies Molecular Pathways Associated with Response to Therapy.

The etiology of Ankylosing spondylitis (AS) is still unknown and the identification of the involved molecular pathogenetic pathways is a current challenge in the study of the disease. Adalimumab (ADA), an anti-tumor necrosis factor (TNF)-alpha agent, is used in the treatment of AS. We aimed at identifying pathogenetic pathways modified by ADA in patients with a good response to the treatment. Gene expression analysis of Peripheral Blood Cells (PBC) from six responders and four not responder patients was performed before and after treatment. Differentially expressed genes (DEGs) were submitted to functional enrichment analysis and network analysis, followed by modules selection. Most of the DEGs were involved in signaling pathways and in immune response. We identified three modules that were mostly impacted by ADA therapy and included genes involved in mitogen activated protein (MAP) kinase, wingless related integration site (Wnt), fibroblast growth factor (FGF) receptor, and Toll-like receptor (TCR) signaling. A separate analysis showed that a higher percentage of DEGs was modified by ADA in responders (44%) compared to non-responders (12%). Moreover, only in the responder group, TNF, Wnt, TLRs and type I interferon signaling were corrected by the treatment. We hypothesize that these pathways are strongly associated to AS pathogenesis and that they might be considered as possible targets of new drugs in the treatment of AS.

Antibodies against human cytomegalovirus in the pathogenesis of systemic sclerosis: a gene array approach.

BACKGROUND: Systemic sclerosis is an autoimmune disease characterized by immunological abnormalities, vascular damage, and fibroblast proliferation. We have previously shown that a molecular mimicry mechanism links antibodies against the human-cytomegalovirus-derived protein UL94 to the pathogenesis of systemic sclerosis. The UL94 epitope shows homology with NAG-2, a surface molecule highly expressed on endothelial cells. Anti-UL94 peptide antibodies purified from patients' sera induce apoptosis of endothelial cells upon engagement of the NAG-2-integrin complex. METHODS AND FINDINGS: We show here that NAG-2 is expressed on dermal fibroblasts and that anti-UL94 antibodies bind to fibroblasts. We have used the gene array strategy (Affimetrix oligonucleotide microarrays) to analyze the transcriptional profile in response to a 4-h and an 8-h treatment with antibodies against the UL94 peptide in endothelial cells and dermal fibroblasts. Exposure of endothelial cells to anti-UL94 antibodies had a profound impact on gene expression, resulting in the upregulation of 1,645 transcripts. Several gene clusters were upregulated including genes encoding adhesion molecules, chemokines, colony-stimulating factors (CSFs), growth factors, and molecules involved in apoptosis. Following antibody stimulation, dermal fibroblasts showed an upregulation of 989 transcripts and acquired a "scleroderma-like" phenotype. Indeed, genes involved in extracellular matrix deposition, growth factors, chemokines, and cytokines were upregulated. We confirmed the microarray results by real-time quantitative polymerase chain reaction and by measuring some of the corresponding proteins with ELISA and Western blotting. CONCLUSION: Our results show that anti-human-cytomegalovirus antibodies may be linked to the pathogenesis of systemic sclerosis not only by inducing endothelial cell activation and apoptosis but also by causing activation of fibroblasts, one of the hallmarks of the disease.

In celiac disease, a subset of autoantibodies against transglutaminase binds toll-like receptor 4 and induces activation of monocytes.

BACKGROUND: Celiac disease is a small intestine inflammatory disorder with multiple organ involvement, sustained by an inappropriate immune response to dietary gluten. Anti-transglutaminase antibodies are a typical serological marker in patients with active disease, and may disappear during a gluten-free diet treatment. Involvement of infectious agents and innate immunity has been suggested but never proven. Molecular mimicry is one of the mechanisms that links infection and autoimmunity. METHODS AND FINDINGS: In our attempt to clarify the pathogenesis of celiac disease, we screened a random peptide library with pooled sera of patients affected by active disease after a pre-screening with the sera of the same patients on a gluten-free diet. We identified a peptide recognized by serum immunoglobulins of patients with active disease, but not by those of patients on a gluten-free diet. This peptide shares homology with the rotavirus major neutralizing protein VP-7 and with the self-antigens tissue transglutaminase, human heat shock protein 60, desmoglein 1, and Toll-like receptor 4. We show that antibodies against the peptide affinity-purified from the sera of patients with active disease recognize the viral product and self-antigens in ELISA and Western blot. These antibodies were able to induce increased epithelial cell permeability evaluated by transepithelial flux of [(3)H] mannitol in the T84 human intestinal epithelial cell line. Finally, the purified antibodies induced monocyte activation upon binding Toll-like receptor 4, evaluated both by surface expression of activation markers and by production of pro-inflammatory cytokines. CONCLUSIONS: Our findings show that in active celiac disease, a subset of anti-transglutaminase IgA antibodies recognize the viral protein VP-7, suggesting a possible involvement of rotavirus infection in the pathogenesis of the disease, through a mechanism of molecular mimicry. Moreover, such antibodies recognize self-antigens and are functionally active, able to increase intestinal permeability and induce monocyte activation. We therefore provide evidence for the involvement of innate immunity in the pathogenesis of celiac disease through a previously unknown mechanism of engagement of Toll-like receptor 4.