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OBJECTIVE: To investigate the potential role of circulating autoantibodies specific to neuronal cell surface antigens in the pathophysiology of neuropsychiatric disorders. METHODS: We performed two different kinds of immunoscreening approaches using sera from patients with schizophrenia to identify autoantigens associated with neuropsychiatric disorders. Then, we tested the presence of autoantibodies specific to the identified autoantigen in patients with various psychiatric disorders and systemic lupus erythematosus (SLE) with neuropsychiatric manifestations. Furthermore, we evaluated the potential pathogenic role of these autoantibodies both in vitro and in vivo. RESULTS: We identified glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a novel autoantigen associated with neuropsychiatric disorders. We detected serum anti-GAPDH IgG in 51% patients with schizophrenia and 50% patients with major depression. Moreover, patients suffering by SLE with comorbid psychiatric manifestations presented significantly higher serum level of anti-GAPDH antibodies than SLE patients without psychiatric manifestations (P= 0.004 by square Chi test). To note, a significant positive correlation (R = 0.48, P= 0.0049 by Spearman rank correlation test) was found between levels of serum anti-GAPDH antibodies and cognitive dysfunctions in patients with SLE. In vitro analysis of purified human anti-GAPDH autoantibodies effects on SH-SY5Y cells showed an immediate neurite retraction. Finally, in vivo administration of the anti-GAPDH autoantibodies in the right cerebral ventricle of C57BL6/J mice resulted in specific behavioral changes associated to a detrimental cognitive and emotional profile. CONCLUSION: Overall, these data suggest that anti-GAPDH autoantibodies play a role in the etiopathogenesis of neuropsychiatric disorders possibly representing a promising tool for the screening of individual vulnerability. This article is protected by copyright. All rights reserved.

SUMMARY Seeking biomarkers reflecting disease development in cystic echinococcosis (CE), we used a proteomic approach linked to immunological characterisation for the identification of respective antigens. Two-dimensional gel electrophoresis (2-DE) of sheep hydatid fluid, followed by immunoblot analysis (IB) with sera from patients with distinct phases of disease, enabled us to identify by mass spectrometry heat shock protein 20 (HSP20) as a potential marker of active CE. Using IB, antibodies specific to the 34 kDa band of HSP20 were detected in sera from 61 ⁄ 95 (64%) patients with CE, but not in sera from healthy subjects. IB revealed anti-HSP20 antibodies in a higher percentage of sera from patients with active disease than in sera from patients with inactive disease (81 vs. 24%; P = 10 )4 ). These primary results were confirmed in a long-term follow-up study after pharmacological and surgical treatment. Herewith antiHSP20 antibody levels significantly decreased over the course of treatment in sera from patients with cured disease, relative to sera from patients with progressive disease (P = 0AE017). Thus, during CE, a comprehensive strategy of proteomic identification combined with immunological validation represents a promising approach for the identification of biomarkers useful for the prognostic assessment of treatment of CE patients.

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.

To investigate the role of T lymphocytes in the immune response to Echinococcus granulosus, using sheep hydatid fluid (SHF) and antigen B (AgB), we generated T-cell lines from patients with active, transitional and inactive hydatid cysts. We established 16 T-cell lines, eight specific to SHF and eight specific to AgB. At surface phenotyping 88-98% of cells displayed the helper/inducer CD4 antigen. In all patients, at all clinical stages of hydatid cyst disease, T-cell stimulation with SHF and AgB invariably amplified a large number of almost identical Vbeta subfamily fragments. Irrespective of antigen-specificity, the two cell lines from the patient with an inactive cyst had a Th1 profile, because they exclusively expressed and produced IFN-gamma. Conversely, the T-cell lines derived from the seven patients with active and transitional hydatid cysts had mixed Th1/Th2 and Th0 clones. The functional characteristics of the 16 T-cell lines differed markedly in the various clinical stages of cystic echinococcosis, thus providing new in vitro evidence that Th1 lymphocytes contribute decisively to the inactive stage of hydatid disease, Th2 lymphocytes in the active and transitional stages. The parasite-specific T-cell lines, especially the two Th1 lines from the patient with an inactive cyst, may help identify Th1 protective epitopes on SHF and AgB.

By screening an expression library of Echinococcus granulosus with IgE from sera of patients with cystic echinococcosis (CE) and allergic reactions, we isolated the C-terminal region of a new heat shock protein (HSP)70 of E. granulosus. The protein, named Eg2HSP70, has close homology with the C-terminal region of Dermatophagoides farinae and human HSP70. We investigated the humoral and cell-mediated immune responses to this antigen in patients with CE grouped according to the presence of allergic reactions. Immunoblotting detected total IgG, IgG4 and IgE specific to Eg2HSP70 (83% of sera contained IgG, 31% IgG4 and 57% IgE). No significant difference was found in immunoglobulin percentages according to the presence of allergic reactions. Immunoblotting inhibition showed that no IgG or IgE specific to Eg2HSP70 cross-reacted with D. farinae or human HSP70. Eg2HSP70-stimulated PBMC from 26 patients produced significantly greater amounts of TNF-alpha, IFN-gamma, and IL-10 than unstimulated cultures in all patients, irrespective of the presence of allergic reactions (P < 0.05). They also produced significantly greater amounts of IL-4 than unstimulated cultures exclusively in patients with allergic reactions (P < 0.05). These findings show that Eg2HSP70 is a new antigenic molecule inducing both B and T cell responses.

A cDNA clone (Eg EF-1beta/delta) of Echinococcus granulosus has been isolated by an expression library screened with immunoglobulin (Ig)E of sera from patients with cystic echinococcosis (CE). The Eg EF-1beta/delta was identified on the basis of sequence homology to the subunits beta or delta of the elongation factor-1. The amino acid sequence deduced from this open reading frame is 244 residues long with a predicted molecular mass of 31 kDa. In Southern blot under high stringent condition, Eg EF-1beta/delta hybridized to genomic DNA of E.granulosus at two bands of 4 and 2.5 Kb. In immunoblotting analysis, the Eg EF-1beta/delta protein shows immunological reactivity with sera from CE patients: 51.7% of sera contained IgE, 41.7% IgG and 18.3% IgG4 specific to the recombinant protein. We identify the Eg EF-1beta/delta by immunoblotting with specific monoclonal antibody both in protoscoleces and in sheep hydatid fluid. The higher percentage of humoral immune response against Eg EF-1beta/delta observed in CE patients with calcified cysts than in patients with active cysts indicates the possible release of the protein in the hydatid fluid after protoscoleces degeneration suggesting the possible use of this antigen in the immunosurveillance of CE. Overall, these findings seem to assign to Eg EF-1beta/delta a key role in the allergic disorders and in the complex host-parasite relationship in CE.

Oropharyngeal washings (Ophs) from 27 HIV infected patients (18 with P. carinii pneumonia, PCP, and 9 without PCP) were examined for P. carinii using morphological staining and DNA amplification with PCR-SHELA and nested PCR methods. The comparison of these techniques shows that 1. the amplification of P. carinii DNA is more sensitive than (and as specific as) morphological staining; 2. PCR-SHELA is less sensitive than (and as specific as) nested PCR.

To understand the way of reinfection of Pneumocystis carinii we have analyzed the genetic variation at the internal transcribed spacer (ITS) in DNA samples from bronchoalveolar lavage fluid of Italian HIV patients who had multiple episodes of P.carinii pneumonia. The presence of the same and/or a new type in both episodes suggest the possible occurrence of both reactivation of a previously acquired infection and reinfection from an exogenous source. Furthermore the occurrence of two different types in the same episode indicate that a mixed infection is common.