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Introduction/objectives<p>In 2012, hypocomplementemia was included in the classification criteria of systemic lupus erythematosus (SLE). The suggested measurement of C3 or C4 often reflect disease activity poorly. Our objective was to establish an assay measuring C3dg, which is generated following complement activation, and to evaluate the assay in a cross-sectional SLE cohort.</p>Method<p>We included SLE patients (n = 169) and controls (n = 170) and developed a modified C3dg assay where C3dg fragments were separated from the large plasma proteins by polyethylene glycol (PEG), and the supernatant containing the C3dg fragment was used for analysis in an antibody-based sandwich-type assay. Gel permeation chromatography and western blotting were used to establish the optimal conditions for PEG precipitation.</p>Results<p>16% PEG was optimal for separating C3dg from C3 and the larger protein fragments. The assay showed a high degree of stability when using EDTA plasma, and measurements correlated well with commercially available complement activation assays. SLE patients had higher concentrations in plasma of C3dg than controls (p < 0.05). ROC analysis showed that the C3dg activation fragment of C3 with an AUC of 0.96 (CI 0.94–0.98) was superior to C3 (AUC 0.52) in differentiating between patients and controls.</p>Conclusion<p>Our results present a modified assay for the measurement of C3dg. We demonstrate that C3dg was superior to conventional C3 measurements in discriminating SLE patients from controls. We suggest that C3dg should be considered as a complement activation measurement in the SLE classification criteria.</p

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Introduction/objectives<p>In 2012, hypocomplementemia was included in the classification criteria of systemic lupus erythematosus (SLE). The suggested measurement of C3 or C4 often reflect disease activity poorly. Our objective was to establish an assay measuring C3dg, which is generated following complement activation, and to evaluate the assay in a cross-sectional SLE cohort.</p>Method<p>We included SLE patients (n = 169) and controls (n = 170) and developed a modified C3dg assay where C3dg fragments were separated from the large plasma proteins by polyethylene glycol (PEG), and the supernatant containing the C3dg fragment was used for analysis in an antibody-based sandwich-type assay. Gel permeation chromatography and western blotting were used to establish the optimal conditions for PEG precipitation.</p>Results<p>16% PEG was optimal for separating C3dg from C3 and the larger protein fragments. The assay showed a high degree of stability when using EDTA plasma, and measurements correlated well with commercially available complement activation assays. SLE patients had higher concentrations in plasma of C3dg than controls (p < 0.05). ROC analysis showed that the C3dg activation fragment of C3 with an AUC of 0.96 (CI 0.94–0.98) was superior to C3 (AUC 0.52) in differentiating between patients and controls.</p>Conclusion<p>Our results present a modified assay for the measurement of C3dg. We demonstrate that C3dg was superior to conventional C3 measurements in discriminating SLE patients from controls. We suggest that C3dg should be considered as a complement activation measurement in the SLE classification criteria.</p

The Spearman rank correlation coefficient, r, for the correlation between levels of IgG1 and IgG3 against human HSP60 and BASDAI, BASMI and BASMI in the total group of SpA patients, the group of HLA-B27 positive patients (HLA-B27⁺) and the group of HLA-B27 negative patients (HLA-B27⁻).

<p>The Spearman rank correlation coefficient, r, for the correlation between levels of IgG1 and IgG3 against human HSP60 and BASDAI, BASMI and BASMI in the total group of SpA patients, the group of HLA-B27 positive patients (HLA-B27⁺) and the group of HLA-B27 negative patients (HLA-B27⁻).</p

Antibody levels in the SpA group and the control group.

<p>Serum levels (µg/mL) of IgG1 and IgG3 antibodies against <i>C. trachomatis</i> HSP60 (A), <i>C. jejuni</i> HSP60 (B), <i>S. enteritidis</i> HSP60 (C) and human HSP60 (D) in the SpA group and the control group. No differences in serum levels of antibodies against the bacterial HSP60 were found between the two groups (A–C). Levels of anti-human HSP60 IgG1 and IgG3 were elevated in the SpA group compared with the control group (D). The two groups are compared using the non-parametric Mann-Whitney rank sum test. Bars represent medians with interquartile ranges (IQR).</p

Medians and interquartile ranges (IQR) of IgG1 and IgG3 antibody levels (µg/mL) against human and bacterial HSP60 in the SpA group.

<p>Medians and interquartile ranges (IQR) of IgG1 and IgG3 antibody levels (µg/mL) against human and bacterial HSP60 in the SpA group.</p

The Spearman rank correlation coefficient, r, for the correlation between levels of IgG1 and IgG3 against bacterial and human HSP60.

<p>The Spearman rank correlation coefficient, r, for the correlation between levels of IgG1 and IgG3 against bacterial and human HSP60.</p

Clinical disease course and IL-22-producing T helper cells.

<p>The percentages of IL-22-producing CD4⁺CD45RO⁺ T cells in AH patients whose condition improved (<b>↓</b>GAHS ≥2) versus AH patients whose condition did not improve (<b>↓</b>GAHS <2) at days 0, 14 and 30 after diagnosis measured by flow cytometry. The frequency of these cells is elevated in the patient who experience disease improvement at baseline compared with the group without disease improvement (p<0.05). This elevation is persistent and augmented through the follow-up period (p<0.05). Values are presented as median (IQR). (GAHS: Glasgow alcoholic hepatitis score).</p

Patient baseline characteristics.

<p>Baseline characteristics are presented for healthy controls, stable alcoholic cirrhosis patients and alcoholic hepatitis patients. The alcoholic hepatitis patients are divided into two groups based on whether or not they experience a decline in GAHS≥2 during the 30 days of follow-up. Values are reported as median (IQR).</p><p>ALT = Alanine Amino Transferase.</p><p>MELD = Model of End Stage Liver Disease.</p><p>GAHS = Glasgow Alcoholic Hepatitis Score.</p>*<p>a vs. b.</p

Flow plot of cytokine production.

<p>Peripheral blood mononuclear cells were stimulated with phorbol-12-myristate-13-acetate, ionomycin and brefeldin A for 4 hours followed by intracellular staining for IL-17A and IL-22. The flow plots depict the population of CD4⁺CD45RO⁺ T cells from a representative AH patient. The values in the gate represent the percentage of IL-17A⁺, IL-22⁺ and double positive cells within the population of CD4⁺CD45RO⁺ T cells (right). The gate is set based on the control staining (left).</p

Baseline IL-22-producing T helper cells.

<p>Flow cytometric measurement of the percentages of IL-22-producing CD4⁺CD45RO⁺ T cells in AH patients on day 0, alcoholic cirrhosis patients and healthy controls in peripheral blood. The frequency of IL-22-producing CD4⁺CD45RO⁺ T cells are increased in AH patients compared with both of the controls groups. The bars represent median values.</p