Complement factors C3a and C5a mimick a proinflammatory microenvironment and increase HBV IGRA sensitivity

Abstract Background Hepatitis B virus (HBV) infections represent a global health problem and chronic hepatitis B (CHB) leads to liver cirrhosis and hepatocellular carcinoma. Thus, timely diagnosis of hepatitis B is crucial to ensure adequate treatment. We developed a powerful and rapid whole blood-based cytokine release assay assessing cellular immune responses to HBV antigens. IL-2 and IFNγ release in this assay depicts hepatitis B vaccination status. Of note, CHB goes along with elevated C5a concentrations in plasma. We aim at mimicking the proinflammatory microenvironment associated with HBV infection to enhance the diagnostic quality of our HBV specific cytokine release assay. We specifically investigated the potential of the complement factors C3a and C5a as costimulators and analyzed their potential effects on activation marker expression on T cells and antigen presenting cells. Results Whole blood from 87 healthy individuals (n = 59 hepatitis B vaccinated, n = 28 unvaccinated) was stimulated with HBV surface antigen (HBsAg) in presence or absence of C3a or C5a, respectively. Further, C3a and C5a were used in combination to investigate potential synergistic effects. IL-2 and IFNγ levels in plasma were quantified using ELISA. Complement factor C5a specifically enhances HBsAg-mediated IL-2 (690.3 ± 195.4 pg/ml vs. 789.4 ± 216.5 pg/ml) and IFNγ (146.0 ± 43.1 pg/ml vs. 336.7 ± 67.9 pg/ml) responses in whole blood. Similar cytokine levels were measured when both C3a and C5a were used. With a diagnostic specificity of 90% the IFNγ release assay reached a diagnostic sensitivity of 49.2% upon whole blood stimulation with HBsAg alone, but of 78.9% when HBsAg was combined with C3a and C5a. Conclusions Innate signals mediated via complement pathways contribute to HBV-specific cellular immune responses. The massively improved diagnostic sensitivity of the IFNγ release assay after addition of C3a and C5a demonstrates that these effects render whole blood-based cytokine release assays even more potent as screening tools in HBV immunology and HBV vaccination studies

Collapsing Polynomial-Time Degrees

. For reducibilities r and r 0 such that r is weaker than r 0 , we say that the r-degree of A, i.e., the class of sets which are r-equivalent to A, collapses to the r 0 -degree of A if both degrees coincide. We investigate for the polynomial-time bounded many-one, bounded truth-table, truthtable, and Turing reducibilities whether and under which conditions such collapses can occur. While we show that such collapses do not occur for sets which are hard for exponential time, we have been able to construct a recursive set such that its bounded truth-table degree collapses to its many-one degree. The question whether there is a set such that its Turing degree collapses to its many-one degree is still open; however, we show that such a set -- if it exists -- must be recursive. 1 Introduction and Notation 1.1 Introduction Ladner, Lynch and Selman [12] first compared the strength of the polynomialtime reducibilities. For the most common reducibilities -- namely Turing (p-T)..

Gremlin-1 expression on aortic tissue of LDS patients.

<p>Paraffin-embedded aortic tissue specimen of LDS patients (n = 3) were double stained with anti-Gremlin-1 (brown staining) and anti-CD34 (red staining; C) or anti-smooth muscle actin (red staining; B, D–F). Endothelial cells throughout the vessel wall showed expression for Gremlin-1 including ECs of the intima (B) and of vessels within the media (C) and the adventitia (D and in more detail in E). Gremlin-1 positive staining was also observed on smooth muscle cells of the media (B, C) as well as on vessel surrounding smooth muscle cells in the adventitial layer (D, E). In aortic tissue specimen of healthy controls (n = 3), a similar staining pattern without gross differences of staining intensity was observed as shown for a small vessel within the adventitia (F). In A, an isotype control instead of primary antibody was used revealing the specificity of the staining (EC = endothelial cell, SMC = smooth muscle cell; magnification A–D, F: 400×; E: 1000×; scale bar represents 20 µm in A–D and F and 8 µm in E).</p

Members of the TGF-β superfamily with altered mRNA expression levels in LDS-OECs compared to healthy controls.

<p>* signal log ratio of LDS-OEC compared to healthy control, determined in microarray analysis; ^† expression fold change of LDS-OEC compared to healthy control, converted from microarray data; ^‡ relative gene expression in LDS-OEC compared to healthy control, determined in quantitative PCR analysis and normalized to <i>GAPDH</i> expression.</p