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.

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.

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.

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.

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.

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.

Oxidized beta2-glycoprotein I induces human dendritic cell maturation and promotes a T helper type 1 response.

The human plasma protein beta2-glycoprotein I (beta2-GPI) is the most common target for antiphospholipid antibodies associated with thrombotic events in chronic disorders related to endothelial cell dysfunction. Crucial information is needed to clarify why this self-abundant protein is targeted by autoimmune responses. In this study, we investigated whether oxidative modification of beta2-GPI, either spontaneous in culture wells or induced by treatment with H2O2, renders this self-protein able to activate immature monocyte-derived dendritic cells (DCs) from healthy human donors. Oxidized beta2-GPI caused DCs to mature so that CD83 appeared and CD80, CD86, human leukocyte antigen-D region related (HLA-DR), and CD40 increased. The interaction between oxidized beta2-GPI and DCs specifically stimulated these cells to secrete interleukin 12 (IL-12), IL-1beta, IL-6, IL-8, tumor necrosis factor alpha (TNF-alpha), and IL-10. Oxidized beta2-GPI-stimulated DCs had increased allostimulatory ability and primed naive T lymphocytes, thus inducing T helper 1 (Th1) polarization. The interaction between oxidized beta2-GPI and DCs involved interleukin-1 receptor associated kinase (IRAK) phosphorylation and nuclear factor kappaB (NFkappaB) activation. Pretreatment of beta2-GPI with the antioxidant alpha-tocopherol prevented DC maturation. These findings show that human oxidized beta2-GPI, probably by interacting with a member of the Toll-like receptor (TLR) family, causes DCs to mature. Because this key beta2-GPI function requires oxidative modification, in several chronic disorders related to endothelial cell dysfunction oxidative stress might trigger the "autoimmune spiral."

Screening of an endothelial cDNA library identifies the C-terminal region of Nedd5 as a novel autoantigen in systemic lupus erythematosus with psychiatric manifestations.

Anti-endothelial-cell antibodies are associated with psychiatric manifestations in systemic lupus erythematosus (SLE). Our primary aim in this study was to seek and characterize molecules that behave as endothelial autoantigens in SLE patients with psychiatric manifestations. By screening a cDNA library from human umbilical artery endothelial cells with serum from an SLE patient with psychosis, we identified one positive strongly reactive clone encoding the C-terminal region (C-ter) of Nedd5, an intracytoplasmatic protein of the septin family. To evaluate anti-Nedd5 serum immunoreactivity, we analyzed by ELISA specific IgG responses in 17 patients with SLE and psychiatric manifestations (group A), 34 patients with SLE without psychiatric manifestations (group B), 20 patients with systemic sclerosis, 20 patients with infectious mononucleosis, and 35 healthy subjects. IgG specific to Nedd5 C-ter was present in 14 (27%) of the 51 SLE patients. The mean optical density value for IgG immunoreactivity to Nedd5 C-ter was significantly higher in patients of group A than in those of group B, those with infectious mononucleosis, or healthy subjects (0.17 +/- 0.14 vs, respectively, 0.11 +/- 0.07, P = 0.04; 0.11 +/- 0.06, P = 0.034; and 0.09 +/- 0.045, P = 0.003, on Student's t-test). Moreover, IgG immunoreactivity to Nedd5 C-ter was significantly higher in patients with systemic sclerosis than in patients of group B or healthy subjects (0.18 +/- 0.18 vs, respectively, 0.11 +/- 0.07, P = 0.046; and 0.09 +/- 0.045, P = 0.003). The percentage of patients with anti-Nedd5 C-ter serum IgG was higher in group A than in group B (8 (47%) of 17, vs 6 (17%) of 34, P = 0.045, on Fisher's exact test). In order to clarify a possible mechanism by which Nedd5 might be autoantigenic, we observed that Nedd5 relocated from cytoplasm to the plasma membrane of EAhy926 endothelial cells after apoptotic stimuli. In conclusion, Nedd5 is a novel autoantigen of potential clinical importance that could be successfully used for a more thorough investigation of the pathogenesis of psychiatric manifestations in SLE. Although anti-Nedd5 autoantibodies are not specific to SLE, they are significantly associated with neuropsychiatric SLE and may represent immunological markers of psychiatric manifestations in this pathology.

Identification and relevance of the CD95-binding domain in the N-terminal region of ezrin.

The CD95 (Fas/APO-1) linkage to the actin cytoskeleton through ezrin is an essential requirement for susceptibility to the CD95-mediated apoptosis in CD4+ T cells. We have previously shown that moesin was not involved in the binding to CD95. Here we further support the specificity of the ezrin/CD95 binding, showing that radixin did not bind CD95. The ezrin region specifically and directly involved in the binding to CD95 was located in the middle lobe of the ezrin FERM domain, between amino acids 149 and 168. In this region, ezrin, radixin, and moesin show 60-65% identity, as compared with the 86% identity in the whole FERM domain. Transfection of two different human cell lines with a green fluorescent protein-tagged ezrin mutated in the CD95-binding epitope, induced a marked inhibition of CD95-mediated apoptosis. In these cells, the mutated ezrin did not co-localize or co-immunoprecipitate with CD95. Further analysis showed that the mutated ezrin, while unable to bind CD95, was fully able to bind actin, thus preventing the actin linkage to CD95. Altogether, our results support the specificity of ezrin in the association to CD95 and the importance of the ezrin-to-CD95 linkage in CD95-mediated apoptosis. Moreover, this study suggests that a major role of ezrin is to connect CD95 to actin, thus allowing the CD95 polarization on the cells and the occurrence of the following multiple cascades of the CD95 pathway.

An update on immunodiagnosis of cystic echinococcosis.

Immunological parameters are increasingly investigated as possible markers for the development of cystic echinococcosis. Among the newer immunologic tests for assessing the host-parasite relationship, assay of immunoglobulin isotypes with the use of distinct parasite antigens and detection of Th1/Th2 cytokine expression are an interesting new approach. The findings upon which we have constructed our immunological hypothesis of the host-parasite relationship are: (1) immunoglobulin isotype profiles differ in patients with distinct clinical outcomes of the disease; in particular, antigen B is the antigen of choice to detect specific IgG4, which is the immunoglobulin isotype most clearly associated with the progression of the disease; (2) the isolation and characterisation of recombinant parasite proteins that behave as molecular markers of allergic reactions associated with cystic echinococcosis; (3) Th1/Th2 cell activation is involved in the clinical outcome of Echinococcus granulosus infection and, in particular Th2 response, is associated with susceptibility to the disease, whereas a Th1 response is associated with protective immunity.