Two of Them Do It Better: Novel Serum Biomarkers Improve Autoimmune Hepatitis Diagnosis.

BACKGROUND: Autoimmune hepatitis (AIH) is a chronic liver disease of unknown aetiology and characterized by continuing hepatocellular inflammation and necrosis. Autoantibodies represent accessible markers to measure the adaptive immune responses in the clinical investigation. Protein microarrays have become an important tool to discriminate the disease state from control groups, even though there is no agreed-upon standard to analyze the results. RESULTS: In the present study 15 sera of patients with AIH and 78 healthy donors (HD) have been tested against 1626 proteins by an in house-developed array. Using a Partial Least Squares Discriminant Analysis (PLS-DA) the resulting data interpretation led to the identification of both new and previously identified proteins. Two new proteins AHPA9419 and Chondroadherin precursor (UNQ9419 and CHAD, respectively), and previously identified candidates as well, have been confirmed in a validation phase by DELFIA assay using a new cohort of AIH patients. A receiver operating characteristic analysis was used for the evaluation of biomarker candidates. The sensitivity of each autoantigen in AIH ranged from 65 to 88%; moreover, when the combination of the two new autoantigens was analyzed, the sensitivity increased to 95%. CONCLUSIONS: Our findings demonstrate that the detection of autoantibodies against the two autoantigens could improve the performance in discriminating AIH patients from control classes and in combination with previously identified autoantigens and they could be used in diagnostic/prognostic markers.

Identification of new autoantigens by protein array indicates a role for IL4 neutralization in autoimmune hepatitis.

Autoimmune hepatitis (AIH) is an unresolving inflammation of the liver of unknown cause. Diagnosis requires the exclusion of other conditions and the presence of characteristic features such as specific autoantibodies. Presently, these autoantibodies have relatively low sensitivity and specificity and are identified via immunostaining of cells or tissues; therefore, there is a diagnostic need for better and easy-to-assess markers. To identify new AIH-specific autoantigens, we developed a protein microarray comprising 1626 human recombinant proteins, selected in silico for being secreted or membrane associated. We screened sera from AIH patients on this microarray and compared the reactivity with that of sera from healthy donors and patients with chronic viral hepatitis C. We identified six human proteins that are specifically recognized by AIH sera. Serum reactivity to a combination of four of these autoantigens allows identification of AIH patients with high sensitivity (82%) and specificity (92%). Of the six autoantigens, the interleukin-4 (IL4) receptor fibronectin type III domain of the IL4 receptor (CD124), which is expressed on the surface of both lymphocytes and hepatocytes, showed the highest individual sensitivity and specificity for AIH. Remarkably, patients' sera inhibited STAT6 phosphorylation induced by IL4 binding to CD124, demonstrating that these autoantibodies are functional and suggesting that IL4 neutralization has a pathogenetic role in AIH.

Protective activity of Streptococcus pneumoniae Spr1875 protein fragments identified using a phage displayed genomic library.

There is considerable interest in pneumococcal protein antigens capable of inducing serotype-independent immunoprotection and of improving, thereby, existing vaccines. We report here on the immunogenic properties of a novel surface antigen encoded by ORF spr1875 in the R6 strain genome. An antigenic fragment encoded by spr1875, designated R4, was identified using a Streptococcus pneumoniae phage displayed genomic library after selection with a human convalescent serum. Immunofluorescence analysis with anti-R4 antisera showed that Spr1875 was expressed on the surface of strains belonging to different serotypes. Moreover, the gene was present with little sequence variability in 27 different pneumococcal strains isolated worldwide. A mutant lacking Spr1875 was considerably less virulent than the wild type D39 strain in an intravenous mouse model of infection. Moreover, immunization with the R4 recombinant fragment, but not with the whole Spr1875 protein, induced significant protection against sepsis in mice. Lack of protection after immunization with the whole protein was related to the presence of immunodominant, non-protective epitopes located outside of the R4 fragment. In conclusion, our data indicate that Spr1875 has a role in pneumococcal virulence and is immunogenic. As the R4 fragment conferred immunoprotection from experimental sepsis, selected antigenic fragments of Spr1875 may be useful for the development of a pneumococcal protein-based vaccine.

Immunoprotective activities of a Streptococcus suis pilus subunit in murine models of infection.

Pili of gram-positive bacteria are key virulence factors and their subunits are considered excellent vaccine candidates. Streptococcus suis is an emerging zoonotic agent that can cause epidemics of life-threatening infections in humans, but the functional role or immunoprotective potential of its pilus components have not been studied yet. Using a selective proteomics approach, we have identified a surface protein of serotype 2 S. suis showing features of an ancillary pilus subunit, as evidenced by bioinformatics analysis, immunoblot and immunoelectron microscopy. Immunization with recombinant fragments of this protein, designated herein as PAPI-2b, markedly protected mice from systemic S. suis infection.