Extracellular vesicles as contributors in the pathogenesis of multiple sclerosis.

Extracellular vesicles (EVs) are a heterogeneous family of extracellular structures bounded by a phospholipid bilayer, released by all cell types in various biological fluids, such as blood and cerebrospinal fluid (CSF), playing important roles in intercellular communication, both locally and systemically. EVs carry and deliver a variety of bioactive molecules (proteins, nucleic acids, lipids and metabolites), conferring epigenetic and phenotypic changes to the recipient cells and thus resulting as important mediators of both homeostasis and pathogenesis. In neurological diseases, such as multiple sclerosis (MS), the EV ability to cross Blood-Brain Barrier (BBB), moving from central nervous system (CNS) to the peripheral circulation and vice versa, has increased the interest in EV study in the neurological field. In the present review, we will provide an overview of the recent advances made in understanding the pathogenic role of EVs regarding the immune response, the BBB dysfunction and the CNS inflammatory processes.

Anti-GAPDH Autoantibodies as a Pathogenic Determinant and Potential Biomarker of Neuropsychiatric Diseases.

OBJECTIVE: To investigate the potential role of circulating autoantibodies specific to neuronal cell surface antigens in the pathophysiology of neuropsychiatric disorders. METHODS: Two different kinds of immunoscreening approaches were used to identify autoantigens associated with neuropsychiatric disorders in the serum of patients with schizophrenia. The presence of autoantibodies specific to the identified autoantigens was then tested in patients with various psychiatric disorders and in patients with systemic lupus erythematosus (SLE) and concomitant neuropsychiatric manifestations. Furthermore, the potential pathogenic role of these autoantibodies was assessed both in vitro and in vivo. RESULTS: GAPDH was identified as a novel autoantigen associated with neuropsychiatric disorders. Serum anti-GAPDH IgG was detected in the serum of 51% of patients with schizophrenia and 50% of patients with major depression. Moreover, SLE patients with comorbid psychiatric manifestations presented significantly higher serum levels of anti-GAPDH antibodies than did SLE patients without psychiatric manifestations (P = 0.004 by chi-square test). Of note, a significant positive correlation (R = 0.48, P = 0.0049, by Spearman's rank correlation test) was found between the levels of serum anti-GAPDH antibodies and cognitive dysfunction in patients with SLE. In vitro analysis of the effects of purified human anti-GAPDH autoantibodies on SH-SY5Y cells showed an immediate neurite retraction. Finally, in vivo administration of anti-GAPDH autoantibodies in the right cerebral ventricle of C57BL/6J mice resulted in specific behavioral changes associated with a detrimental cognitive and emotional profile. CONCLUSION: Overall, these data suggest that anti-GAPDH autoantibodies play a role in the pathogenesis of neuropsychiatric disorders, thus representing a potentially promising tool for the screening of individual vulnerability to these disabling conditions.

Peripheral blood biomarkers in multiple sclerosis.

Multiple sclerosis is the most common autoimmune disorder affecting the central nervous system. The heterogeneity of pathophysiological processes in MS contributes to the highly variable course of the disease and unpredictable response to therapies. The major focus of the research on MS is the identification of biomarkers in biological fluids, such as cerebrospinal fluid or blood, to guide patient management reliably. Because of the difficulties in obtaining spinal fluid samples and the necessity for lumbar puncture to make a diagnosis has reduced, the research of blood-based biomarkers may provide increasingly important tools for clinical practice. However, currently there are no clearly established MS blood-based biomarkers. The availability of reliable biomarkers could radically alter the management of MS at critical phases of the disease spectrum, allowing for intervention strategies that may prevent evolution to long-term neurological disability. This article provides an overview of this research field and focuses on recent advances in blood-based biomarker research.

Autoantibodies specific to a peptide of ß2-glycoprotein I cross-react with TLR4, inducing a proinflammatory phenotype in endothelial cells and monocytes.

ß(2)-glycoprotein I (ß(2)GPI) is the major antigenic target for antiphospholipid Abs. Anti-ß(2)GPI Abs are a heterogeneous population of Igs targeting all domains of the molecule. Abs specific to ß(2)GPI domain I are strongly associated with thrombosis and obstetric complications. In the present study, we sought to understand the possible pathogenic mechanism for this subset of anti-ß(2)GPI Abs, investigating their potential cross-reactivity with other self-proteins involved in inflammatory or coagulant events. We compared the amino acid sequence of the ß(2)GPI domain I with human proteins in a protein databank and identified a peptide sharing 88% identity with an epitope of human TLR4. A high percentage of patients with antiphospholipid syndrome (41%) and systemic lupus erythematosus (50%) presented serum IgG specific to this peptide. Anti-ß(2)GPI peptide Abs binding the TLR4 were able to induce NF-κB activation in HEK293 cells that were stably transfected with the TLR4 gene. Anti-ß(2)GPI peptide Abs induced activation of TLR4 and triggered interleukin-1 receptor-associated kinase phosphorylation and NF-κB translocation, promoting VCAM expression on endothelial cells and TNF-α release by monocytes. In conclusion, our observations suggest a novel pathogenic mechanism in the TLR4 stimulation by anti-ß(2)GPI peptide Abs that links adaptive immune responses with innate immunity in antiphospholipid syndrome and systemic lupus erythematosus.

Gender disparity in susceptibility to oxidative stress and apoptosis induced by autoantibodies specific to RLIP76 in vascular cells.

AIM: Ral-binding protein 1 (RLIP76) is a cell surface protein that catalyzes the extrusion from the cell of reduced glutathione (GSH) conjugates. We recently demonstrated the presence of serum antibodies to RLIP76 (aaRLIP76) in patients with immune-mediated diseases characterized by vascular dysfunction. The aim of this work was to analyze the possible implication of gender in this issue, investigating the effects of aaRLIP76 in rat vascular smooth muscle cells and human endothelial cells from males and females. RESULTS: We observed that, after aaRLIP76 treatment, vascular cells from females showed a significantly higher susceptibility to the disturbance of intracellular redox balance, in terms of H(2)O(2) and O(2)(*) production, 4-hydroxy-t-2,3-nonenal and GSH levels, C-Jun NH2 kinase signaling activation, and apoptosis in comparison with cells from males. Interestingly, under mild oxidative stress (H(2)O(2) 30 µm for 30 min), these sex-associated differences became significantly more pronounced. Experiments carried out in the presence of sex hormones in the culture medium clearly suggested that estrogens could significantly increase the susceptibility of cells from females to the effects of aaRLIP76, whereas cells from males appeared unaffected. INNOVATION: These results open a new perspective in the gender-dependent pathogenic mechanisms of autoimmune diseases characterized by vascular dysfunction. CONCLUSIONS: Altogether these results suggest that the impairment of RLIP76 by aaRLIP76 can play a role in the damage of vascular cells from females, contributing to the gender-associated pathogenesis of immune-mediated vascular diseases.

Vimentin/cardiolipin complex as a new antigenic target of the antiphospholipid syndrome.

Antiphospholipid syndrome (APS) is an autoimmune disease characterized by arterial and venous thrombosis, recurrent abortions, and antiphospholipid antibodies (aPL). However, it is possible to find patients with clinical signs of APS who persistently test negative for aPL (seronegative APS, or SN-APS). The aim of this study was to identify new antigenic target(s) of autoantibodies in APS patients, which may also be recognized in SN-APS. We tested sera from patients with SN-APS with a proteomic approach by analyzing endothelial cell-surface membrane proteins. Sera from SN-APS patients revealed 2 reactive spots corresponding to vimentin, a protein that is shown to bind cardiolipin in vitro. Antivimentin/cardiolipin antibodies were tested in 29 SN-APS patients, 40 APS patients, 30 patients with systemic lupus erythematosus, 30 with rheumatoid arthritis, 30 with venous or arterial thrombosis, and 32 healthy control patients. We observed that not only a large proportion of SN-APS patients but also almost all the APS patients displayed the presence of antivimentin/cardiolipin antibodies. To verify the possible pathogenic role of these autoantibodies, we demonstrated that affinity-purified antivimentin/cardiolipin antibodies induced interleukin receptor-associated kinase phosphorylation and nuclear factor-κB activation in endothelial cells. Our results prompt to identify vimentin as a "new" cofactor for aPL, which may represent a useful tool mainly in SN-APS patients.

Identification of a novel 19 kDa Echinococcus granulosus antigen.

By screening an Echinococcus granulosus cDNA library with IgG4 from patients with active cystic echinococcosis (CE), we identified a cDNA encoding a protein of 19.0 kDa (Eg19). Eg19, in 12% SDS-PAGE in reducing and non-reducing conditions, showed several bands between 19 and 100 kDa. Immunoblotting (IB) analysis detected total IgG, IgG1 and IgG4 specific to the 38/40 kDa band of Eg19 in the 10% of patients' sera. The percentage of total IgG, IgG1 and IgG4-positive sera were significantly higher in sera from patients with active disease and cyst in multiple sites than from patients with inactive disease and cyst in the liver (P<10(-4)). ELISA analysis disclosed that during the follow-up anti-Eg19 antibody concentration decreased over the course of treatment in sera from patients with cured disease. Even if Eg19 appear to have no benefit in the diagnosis of the disease, our data, confirming the presence of antigens inducing both IgG1 and IgG4 during active development of CE, suggest that Eg19 might be a marker of disease status.

Autoantibodies involved in neuropsychiatric manifestations associated with systemic lupus erythematosus.

In the course of Systemic Lupus Erythematosus (SLE), a variety of neuropsychiatric disturbances is reported with a prevalence ranging from 17% to 75%. The diagnosis of these syndromes is difficult and requires a careful psychiatric evaluation. Distinct autoantibodies detectable in serum or cerebrospinal fluid of patients with SLE are associated with the presence of neuropsychiatric disorders. These autoantibodies may have a pathogenic relevance in neuropsychiatric SLE or they may be merely an epiphenomenon. This review describes the various autoantibodies reported to be associated with neuropsychiatric manifestations in SLE and discusses their possible role.

Molecular cross-talk in host-parasite relationships: the intriguing immunomodulatory role of Echinococcus antigen B in cystic echinococcosis.

Cystic echinococcosis (CE), a zoonosis caused by the development of Echinococcus granulosus tapeworm larvae in the internal organs of ungulates and humans, continues to pose a major public health burden in underdeveloped and industrialised areas worldwide. Research designed to improve parasitic disease control and find out more about parasite biology has already identified a number of E. granulosus antigenic molecules. The major E. granulosus immunomodulant antigen isolated from hydatid fluid is antigen B, a 120 kDa polymeric lipoprotein consisting of various 8 kDa subunits. By inhibiting elastase activity and neutrophil chemotaxis and eliciting a non-protective Th2 cell response, antigen B helps the parasite evade the human response. In this review, we briefly discuss current information on the molecular characteristics and immunomodulatory properties of E. granulosus antigen B. Besides focusing on findings that provide intriguing insights into the complex interplay between host and parasite, we suggest how this information could extend the current therapeutic options in inflammatory diseases.

Redox state, cell death and autoimmune diseases: a gender perspective.

Autoimmune disorders, redox balance and gender differences are closely connected. In fact, activation, proliferation and death of cells of different histotype, including blood and vascular cells, are under control of oxidative balance and are key players in autoimmune disease pathogenesis and progression. However, cells from male and female appear characterized by a huge series of differences in terms of reactive oxygen species production and oxidative stress susceptibility. In this review, we briefly summarize the possible implications of the redox state in the onset and progression of autoimmune diseases in a gender perspective.

Immunomodulatory mechanisms during Echinococcus granulosus infection.

The pathologic events that ensue after humans ingest the eggs of Echinococcus granulosus and continue while cystic echinococcosis develops, provide an excellent example illustrating the evasive strategies helminth parasites use to develop, progress and cause chronic disease. The hydatid cyst secretes and exposes numerous immunomodulatory molecules to the host's immune system. By characterizing these molecules we can understand the mechanisms that E. granulosus uses for increasing the efficiency and persistency of infection in the host. These molecules modulate both the innate and adaptive arms of the immune response and appear to target cellular and humoral responses. In this review, we discuss recent advances in the immunobiology of host-E. granulosus interactions that provide intriguing insights into the complex interplay between host and parasite that ultimately facilitates parasite survival.

Thioredoxin peroxidase from Echinococcus granulosus: a candidate to extend the antigenic panel for the immunodiagnosis of human cystic echinococcosis.

The currently available tests for the diagnosis of cystic echinococcosis (CE), enzyme-linked immunosorbent assay (ELISA), and immunoblotting (IB) lack sensitivity and specificity, and antigen panels need standardizing. By screening an Echinococcus granulosus cDNA library with IgG1 from patients with CE, we identified E. granulosus thioredoxin peroxidase (EgTPx). Although IB and ELISA achieved the same specificity (92%), ELISA showed higher sensitivity than IB (83% versus 42%) in determining total immunoglobulin G (IgG) specific to EgTPx in CE sera. The percentage of total IgG- and IgG1-positive sera in ELISA was equally distributed in patients with active, transitional, and inactive disease. Conversely, the percentages of IgG4-positive sera were significantly higher in sera from patients with active than inactive disease (P = 0.03). Our data suggest that adding this highly specific recombinant antigen to the standard diagnostic panel of antigens used in ELISA would increase diagnostic sensitivity. Antibodies specific to EgTPx are of potential interest in the host-parasite relationship.

Autoantibodies to the C-terminal subunit of RLIP76 induce oxidative stress and endothelial cell apoptosis in immune-mediated vascular diseases and atherosclerosis.

Although detection of autoantibodies in the peripheral blood from patients with immune-mediated endothelial dysfunctions has so far failed to provide tools of diagnostic or pathogenetic value, putative bioindicators include anti-endothelial cell antibodies, a heterogeneous family of antibodies that react with autoantigens expressed by endothelial cells. In this study, to identify endothelial autoantigens involved in the autoimmune processes causing endothelial damage, we screened a human microvascular endothelial cell cDNA library with sera from patients with Behçet's disease. We identified antibodies to the C-terminus of Ral binding protein1 (RLIP76), a protein that catalyzes the ATP-dependent transport of glutathione (GSH) conjugates including GSH-4-hydroxy-t-2,3-nonenal, in the serum of a significant percentage of patients with various diseases characterized by immune-mediated endothelial dysfunction, including Behçet disease, systemic sclerosis, systemic lupus erythematosus and carotid atherosclerosis. These autoantibodies increased intracellular levels of 4-hydroxy-t-2,3-nonenal, decreased levels of GSH and activated C-Jun NH2 Kinase signaling (JNK), thus inducing oxidative stress-mediated endothelial cell apoptosis. The dietary antioxidant alpha-tocopherol counteracted endothelial cell demise. These findings suggest that autoantibodies to RLIP76 play a pathogenetic role in immune-mediated vascular diseases and represent a valuable peripheral blood bioindicator of atherosclerosis and immune-mediated vascular diseases.

Screening of a microvascular endothelial cDNA library identifies rabaptin 5 as a novel autoantigen in Alzheimer's disease.

The pathogenesis of Alzheimer's disease (AD) includes the participation of the immune system. To identify antigenic targets in AD, we screened a human microvascular endothelial cell cDNA library with sera from patients with AD, and we identified rabaptin 5 (RABPT5). We detected serum IgG specific to RABPT5 in 65% of patients with AD and in 35% of patients with systemic lupus erythematosus, but in no healthy controls. Our results demonstrated a massive redistribution of this protein in the cytoplasm of endothelial and neuronal cells in apoptosis. In conclusion, we identified RABPT5 as a novel autoantigen in AD.

Screening of endothelial expression libraries for the identification of novel autoantigens involved in distinct autoimmune diseases characterized by endothelial dysfunction.

Screening a cDNA expression library is a powerful technique that allows identification of previously uncharacterized antigens. Proteins recognized by antibodies from patients with autoimmune diseases have been intensively studied over the past two decades since cDNAs encoding autoantigens have become available. Identity of many of them has been defined, and specific structural motifs or post-translational modifications, which may be important to explain the generation of such antibodies during the autoimmune process, have been pointed out. The screening of human umbilical artery or microvascular endothelial cell expression libraries appears to be a useful tool for the characterization of endothelial autoantigens allowing us to identify several molecules recognized from serum anti-endothelial cell antibodies of patients with diseases characterized by immune-mediated endothelial dysfunctions.

Free hemoglobin: a dangerous signal for the immune system in patients with carotid atherosclerosis?

Atherosclerosis is a chronic inflammatory multifactorial disease in which immune responses are key pathogenetic factors. T cell-mediated immunity contributes to the initiation and progression of atherosclerotic disease, but the nature of antigens responsible for immune cell activation is still not completely elucidated. Convincing evidence supports a determinant role of autoimmune responses to self-structures in shaping the progression of the disease. Autoimmune responses may be directed against altered self-structures, such as oxidized low-density lipoproteins (LDL). Oxidative stress, increasingly reported in patients with atherosclerosis, is the major event causing protein structural modification, thus inducing the appearance of neo/cryptic epitopes on the molecule. Intraplaque hemorrhage, a common event in advanced lesions, causes the deposition of large amounts of hemoglobin (Hb). The pro-oxidative intraplaque microenvironment may induce structural changes in extra-erythrocytic free Hb, thus generating novel/cryptic autoantigenic epitopes. We demonstrated that an oxidized Hb preparation enriched in hemichromes expands IFN-gamma-secreting T lymphocytes in patients with advanced carotid atherosclerosis and enhances the phenotypical and functional maturation of human monocyte-derived dendritic cells induced by lipopolysaccharide (LPS). Overall, our findings suggest that oxidized forms of Hb could act as a dangerous signal for the immune system, thus contributing to the inflammatory process that takes place within the atherosclerotic plaque.

Echinococcus granulosus antigen B impairs human dendritic cell differentiation and polarizes immature dendritic cell maturation towards a Th2 cell response.

Despite inducing a strong host cellular and humoral immune response, the helminth Echinococcus granulosus is a highly successful parasite that develops, progresses, and ultimately causes chronic disease. Although surgery remains the preferred therapeutic option, pharmacological research now envisages antihelminthic strategies. To understand the mechanisms that E. granulosus uses to escape host immunosurveillance and promote chronic infection, we investigated how two hydatid cyst components, purified antigen B (AgB) and sheep hydatid fluid (SHF), act on host dendritic cell (DC) differentiation from monocyte precursors and how they influence maturation of DC that have already differentiated. We evaluated the immunomodulatory potential of these antigens by performing immunochemical and cytofluorimetric analyses of monocyte-derived DCs from healthy human donors. During monocyte differentiation, AgB and SHF downmodulated CD1a expression and upregulated CD86 expression. Compared with immature DCs differentiated in medium alone (iDCs), AgB- and SHF-differentiated cells stimulated with lipopolysaccharide included a significantly lower percentage of CD83(+) cells (P < 10(-4)) and had weaker costimulatory molecule expression. When stimulated with AgB and SHF, iDCs matured and primed lymphocytes towards the Th2 response typical of E. granulosus infection. SHF and particularly AgB reduced the production of interleukin-12p70 (IL-12p70) and tumor necrosis factor alpha in lipopolysaccharide-stimulated iDCs. Anti-IL-10 antibodies increased the levels of IL-12p70 secretion in AgB- and SHF-matured DCs. AgB and SHF induced interleukin-1 receptor-associated kinase phosphorylation and activated nuclear factor-kappaB, suggesting that Toll-like receptors could participate in E. granulosus-stimulated DC maturation. These results suggest that E. granulosus escapes host immunosurveillance in two ways: by interfering with monocyte differentiation and by modulating DC maturation.

Intracellular expression of cytokines in peripheral blood from patients with atherosclerosis before and after carotid endarterectomy.

OBJECTIVE: We investigated the possible association of intracellular cytokine profiles in peripheral mononuclear cells in whole blood from patients with carotid atherosclerosis in whom follow-up after carotid endarterectomy (CEA) showed the onset or progression of contralateral disease. METHODS AND RESULTS: Intracellular expression of tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma, interleukin (IL)-1beta, IL-6, IL-8, IL-4 and IL-10 was determined in 43 patients with carotid atherosclerosis, at baseline and at 1, 3, 6 and 12 months after CEA, and in 16 healthy subjects. When follow-up ended patients were divided into two groups, those with cured or stable disease (no onset of disease or unchanged stenosis in the contralateral vessel) and those with progressive disease (onset of disease or increased stenosis in the contralateral vessel). In patients with cured or stable disease, cytokine-positive cells significantly decreased or remained unchanged (IL-8 and IL-10) during follow-up, whereas in patients with progressive disease they increased or remained unchanged (TNF-alpha). Multivariate ANOVA confirmed that the trend of cytokine expression between baseline and 12 months differed significantly in the two groups. CONCLUSIONS: Intracellular expression of TNF-alpha, IFN-gamma, IL-1beta, IL-6, IL-8, IL-4 and IL-10 in peripheral mononuclear cells is associated with the outcome of contralateral disease after CEA.

Autoantibodies associated with psychiatric disorders.

Growing evidence suggests that autoantibodies to neuronal or endothelial targets in psychiatric disorders exist and may be pathogenic. This review describes and discusses the possible role of autoantibodies related to the psychiatric manifestations in autoimmune diseases, autoantibodies related to the psychiatric disorders present in post-streptococcal diseases, celiac disease, chronic fatigue syndrome and substance abuse, and autoantibodies related to schizophrenia and autism, disorders now considered of autoimmune origin.

Identification and characterization of the carboxy-terminal region of Sip-1, a novel autoantigen in Behçet's disease.

Given the lack of a serological test specific for Behçet's disease, its diagnosis rests upon clinical criteria. The clinical diagnosis is nevertheless difficult because the disease manifestations vary widely, especially at the onset of disease. The aim of this study was to identify molecules specifically recognized by serum autoantibodies in patients with Behçet's disease and to evaluate their diagnostic value. We screened a cDNA library from human microvascular endothelial cells with serum IgG from two patients with Behçet's disease and isolated a reactive clone specific to the carboxy-terminal subunit of Sip1 (Sip1 C-ter). Using ELISA, we measured IgG, IgM and IgA specific to Sip1 C-ter in patients with various autoimmune diseases characterized by the presence of serum anti-endothelial cell antibodies, such as Behçet's disease, systemic lupus erythematosus, systemic sclerosis and various forms of primary vasculitis, as well as in patients with diseases that share clinical features with Behçet's disease, such as inflammatory bowel disease and uveitis. IgM immunoreactivity to Sip1 C-ter was significantly higher in patients with Behçet's disease and in patients with primary vasculitis than in the other groups of patients and healthy subjects tested (P < 10-4 by Mann-Whitney test). ELISA detected IgG specific to Sip1 C-ter in sera from 11/56 (20%) patients with Behçet's disease, IgM in 23/56 (41%) and IgA in 9/54 (17%). No sera from patients with systemic lupus erythematosus, systemic sclerosis, inflammatory bowel disease, uveitis or healthy subjects but 45% of sera from patients with primary vasculitis contained IgM specific to Sip1 C-ter. Serum levels of soluble E-selectin, a marker of endothelial activation and inflammation, correlated with levels of serum IgM anti Sip-1 C-ter in patients with Behçet's disease (r = 0.36, P = 0.023). In conclusion, Sip1 C-ter is a novel autoantigen in Behçet's disease. IgM specific to Sip1 C-ter might be useful in clinical practice as an immunological marker of endothelial dysfunction in vasculitis.