Ruggero Beri

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 [3H] 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.

In chronic idiopathic urticaria autoantibodies against FcεRII/CD23 induce histamine release via eosinophil activation

Background Chronic idiopathic urticaria is a common skin disorder characterized by recurrent, transitory, itchy weals for more than 6 weeks. An autoimmune origin has been suggested based on the findings of auto‐antibodies (Abs) directed against either the α subunit of the high‐affinity IgE receptor or the IgE molecule in nearly half of the patients.

Endothelial Cells' Activation and Apoptosis Induced by a Subset of Antibodies against Human Cytomegalovirus: Relevance to the Pathogenesis of Atherosclerosis

Background Human cytomegalovirus (hCMV) is involved in the pathogenesis of atherosclerosis. We have previously shown in patients with atherosclerosis that antibodies directed against the hCMV-derived proteins US28 and UL122 are able to induce endothelial cell damage and apoptosis of non-stressed endothelial cells through cross-rection with normally expressed surface molecules. Our aim was to dissect the molecular basis of such interaction and to investigate mechanisms linking innate immunity to atherosclerosis. Methodology/Principal Findings We analysed the gene expression profiles in endothelial cells stimulated with antibodies affinity-purified against either the UL122 or the US28 peptides using the microarray technology. Microarray results were validated by quantitative PCR and by detection of proteins in the medium. Supernatant of endothelial cells incubated with antibodies was analysed also for the presence of Heat Shock Protein (HSP)60 and was used to assess stimulation of Toll-Like Receptor-4 (TLR4). Antibodies against UL122 and US28 induced the expression of genes encoding for adhesion molecules, chemokines, growth factors and molecules involved in the apoptotis process together with other genes known to be involved in the initiation and progression of the atherosclerotic process. HSP60 was released in the medium of cells incubated with anti-US28 antibodies and was able to engage TLR4. Conclusions/Significance Antibodies directed against hCMV modulate the expression of genes coding for molecules involved in activation and apoptosis of endothelial cells, processes known to play a pivotal role in the pathogenesis of atherosclerosis. Moreover, endothelial cells exposed to such antibodies express HSP60 on the cell surface and release HSP60 in the medium able to activate TLR4. These data confirm that antibodies directed against hCMV-derived proteins US28 and UL122 purified from patients with coronary artery disease induce endothelial cell damage and support the hypothesis that hCMV infection may play a crucial role in mediating the atherosclerotic process.

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.

Endothelin‐1 serum levels correlate with MCP‐1 but not with homocysteine plasma concentration in patients with systemic sclerosis

Objectives: To determine whether homocysteine (Hcy) plasma levels are correlated with molecules indicative of endothelial cell and fibroblast activation, including endothelin‐1 (ET‐1) and monocyte chemoattractant protein‐1 and ‐3 (MCP‐1, MCP‐3), in patients with systemic sclerosis (SSc). Methods: Eighty‐two patients were enrolled in this study; the control group included 75 age‐ and sex‐matched subjects. Plasma Hcy was determined by high‐performance liquid chromatography; folic acid, and vitamin B12 plasma levels were determined by a chemiluminescence method. ET‐1, MCP‐1, and MCP‐3 were determined by enzyme‐linked immunosorbent assay (ELISA). Analysis of the 677C→T mutation in the methylenetetrahydrofolate reductase (MTHFR) gene was performed by polymerase chain reaction (PCR) and digestion with the enzyme HinfI. Results: Hcy levels were lower in patients whereas ET‐1 was significantly higher in patients and correlated with MCP‐1. Stratification of the patients on the basis of Hcy levels was not associated with any statistical difference in the concentration of ET‐1, MCP‐1, and MCP‐3. Patients with diffuse disease presented the highest levels of ET‐1 and MCP‐1. The distribution of the MTHFR genotypes was not different in patients and controls. Conclusions: In SSc, Hcy plasma concentration does not influence ET‐1, MCP‐1, or MCP‐3 levels. On the contrary, ET‐1, a marker of vascular activation, correlates with MCP‐1, a chemokine involved in the fibrotic process of SSc.

Analysis of CIITA encoding AIR-1 gene promoters in insulin-dependent diabetes mellitus and rheumatoid arthritis patients from the Northeast of Italy: Absence of sequence variability

Qualitative and/or quantitative alterations in the expression of the MHC class II molecules affect the onset and maintenance of the immune response and may be the basis of a wide variety of disease states, such as autoimmunity and immunodeficiency.CIITA is a major physiological regulator of the expression of MHC class II genes. The availability of CIITA ap- pears generally essential for MHC class II gene expression, and hence its own transcriptional regulatory mechanisms result of fundamental importance for a correct homeostasis of the immune response. Therefore, it is possible to hypothesize that variability at the CIITA-encoding locus, AIR-1, could constitute an additional source of susceptible traits to autoimmune diseases. Mutations at AIR-1/CIITA promoters could modulate expression of CIITA. Variations in CIITA expression could influence the qualitative and quantitative expression of MHC class II molecules at cell surface. We have analyzed sequence variation at AIR-1/CIITA promoters by PCR-SSCP in 23 IDDM and 30 RA patients compared to a sample of 19 unaffected normal controls and 16 unaffected IDDM family members, for a total of 88 Caucasian subjects from the Northeast of Italy. No sequence difference was found at the four AIR-1/CIITA promoters between autoimmune patients and normal controls. Moreover, the promoters resulted invariant within the entire group of 88 subjects analyzed, comprising patients and controls. This finding suggests a possible selective advantage in maintaining CIITA upstream regulatory sequences invariant.

Gene expression profiling in circulating endothelial cells from systemic sclerosis patients shows an altered control of apoptosis and angiogenesis that is modified by iloprost infusion

IntroductionCirculating endothelial cells are increased in patients affected by systemic sclerosis (SSc) and their number strongly correlates with vascular damage. The effects of iloprost in systemic sclerosis are only partially known. We aimed at studying the gene expression profile of circulating endothelial cells and the effects of iloprost infusion and gene expression in patients with systemic sclerosis.MethodsWe enrolled 50 patients affected by systemic sclerosis, 37 patients without and 13 patients with digital ulcers. Blood samples were collected from all patients before and 72 hours after either a single day or five days eight hours iloprost infusion. Blood samples were also collected from 50 sex- and age-matched healthy controls. Circulating endothelial cells and endothelial progenitors cells were detected in the peripheral blood of patients with systemic sclerosis by flow cytometry with a four-colour panel of antibodies. Statistical analysis was performed with the SPSS 16 statistical package.Circulating endothelial cells were then isolated from peripheral blood by immunomagnetic CD45 negative selection for the gene array study.ResultsThe number of both circulating endothelial cells and progenitors was significantly higher in patients affected by systemic sclerosis than in controls and among patients in those with digital ulcers than in patients without them. Circulating endothelial cells and progenitors number increased after iloprost infusion. Gene array analysis of endothelial cells showed a different transcriptional profile in patients compared to controls. Indeed, patients displayed an altered expression of genes involved in the control of apoptosis and angiogenesis. Iloprost infusion had a profound impact on endothelial cells gene expression since the treatment was able to modulate a very high number of transcripts.ConclusionsWe report here that circulating endothelial cells in patients with systemic sclerosis show an altered expression of genes involved in the control of apoptosis and angiogenesis. Moreover we describe that iloprost infusion has a strong effect on endothelial cells and progenitors since it is able to modulate both their number and their gene expression profile.

Gene Expression Profiling in Peripheral Blood Mononuclear Cells of Patients with Common Variable Immunodeficiency: Modulation of Adaptive Immune Response following Intravenous Immunoglobulin Therapy

Background Regular intravenous immunoglobulin treatment is used to replace antibody deficiency in primary immunodeficiency diseases; however the therapeutic effect seems to be related not only to antibody replacement but also to an active role in the modulation of the immune response. Common variable immunodeficiency is the most frequent primary immunodeficiency seen in clinical practice. Methods We have studied the effect of intravenous immunoglobulin replacement in patients with common variable immunodeficiency by evaluating the gene-expression profiles from Affimetrix HG-U133A. Some of the gene array results were validated by real time RT-PCR and by the measurement of circulating cytokines and chemokines by ELISA. Moreover we performed FACS analysis of blood mononuclear cells from the patients enrolled in the study. Results A series of genes involved in innate and acquired immune responses were markedly up- or down-modulated before therapy. Such genes included CD14, CD36, LEPR, IRF-5, RGS-1, CD38, TNFRSF25, IL-4, CXCR4, CCR3, IL-8. Most of these modulated genes showed an expression similar to that of normal controls after immunoglobulin replacement. Real time RT-PCR of selected genes and serum levels of IL-4, CXCR4 before and after therapy changed accordingly to gene array results. Interestingly, serum levels of IL-8 remained unchanged, as the corresponding gene, before and after treatment. FACS analysis showed a marked decrease of CD8+T cells and an increase of CD4+T cells following treatment. Moreover we observed a marked increase of CD23−CD27−IgM−IgG− B cells (centrocytes). Conclusions Our results are in accordance with previous reports and provide further support to the hypothesis that the benefits of intravenous immunoglobulin therapy are not only related to antibody replacement but also to its ability to modulate the immune response in common variable immunodeficiency.

Gene Expression Profiling in Peripheral Blood Cells and Synovial Membranes of Patients with Psoriatic Arthritis.

Background Psoriatic arthritis (PsA) is an inflammatory arthritis whose pathogenesis is poorly understood; it is characterized by bone erosions and new bone formation. The diagnosis of PsA is mainly clinical and diagnostic biomarkers are not yet available. The aim of this work was to clarify some aspects of the disease pathogenesis and to identify specific gene signatures in paired peripheral blood cells (PBC) and synovial biopsies of patients with PsA. Moreover, we tried to identify biomarkers that can be used in clinical practice. Methods PBC and synovial biopsies of 10 patients with PsA were used to study gene expression using Affymetrix arrays. The expression values were validated by Q-PCR, FACS analysis and by the detection of soluble mediators. Results Synovial biopsies of patients showed a modulation of approximately 200 genes when compared to the biopsies of healthy donors. Among the differentially expressed genes we observed the upregulation of Th17 related genes and of type I interferon (IFN) inducible genes. FACS analysis confirmed the Th17 polarization. Moreover, the synovial trascriptome shows gene clusters (bone remodeling, angiogenesis and inflammation) involved in the pathogenesis of PsA. Interestingly 90 genes are modulated in both compartments (PBC and synovium) suggesting that signature pathways in PBC mirror those of the inflamed synovium. Finally the osteoactivin gene was upregulared in both PBC and synovial biopsies and this finding was confirmed by the detection of high levels of osteoactivin in PsA sera but not in other inflammatory arthritides. Conclusions We describe the first analysis of the trancriptome in paired synovial tissue and PBC of patients with PsA. This study strengthens the hypothesis that PsA is of autoimmune origin since the coactivity of IFN and Th17 pathways is typical of autoimmunity. Finally these findings have allowed the identification of a possible disease biomarker, osteoactivin, easily detectable in PsA serum.

Plant-Derived Chimeric Virus Particles for the Diagnosis of Primary Sjögren Syndrome

Plants are ideal for the production of protein-based nanomaterials because they synthesize and assemble complex multimeric proteins that cannot be expressed efficiently using other platforms. Plant viruses can be thought of as self-replicating proteinaceous nanomaterials generally stable and easily produced in high titers. We used Potato virus X (PVX), chimeric virus particles, and Cowpea mosaic virus, empty virus-like particles to display a linear peptide (lipo) derived from human lipocalin, which is immunodominant in Sjögren’s syndrome (SjS) and is thus recognized by autoantibodies in SjS patient serum. These virus-derived nanoparticles were thus used to develop a diagnostic assay for SjS based on a direct enzyme linked immunosorbent assay format. We found that PVX-lipo formulations were more sensitive than the chemically synthesized immunodominant peptide and equally specific when used to distinguish between healthy individuals and SjS patients. Our novel assay therefore allows the diagnosis of SjS using a simple, low-invasive serum test, contrasting with the invasive labial biopsy required for current tests. Our results demonstrate that nanomaterials based on plant viruses can be used as diagnostic reagents for SjS, and could also be developed for the diagnosis of other diseases.