Elevated Concentrations of Serum Immunoglobulin Free Light Chains in Systemic Lupus Erythematosus Patients in Relation to Disease Activity, Inflammatory Status, B Cell Activity and Epstein-Barr Virus Antibodies

In this study, we examined the concentration of serum immunoglobulin free light chains (FLCs) in systemic lupus erythematosus (SLE) patients and investigated its association with various disease parameters in order to evaluate the role of FLCs as a potential biomarker in SLE. Furthermore, FLCs' association with Epstein-Barr virus (EBV) antibodies was examined.Using a nephelometric assay, κFLC and λFLC concentrations were quantified in sera from 45 SLE patients and 40 healthy controls. SLE patients with renal insufficiency were excluded in order to preclude high concentrations of serum FLCs due to decreased clearance.Serum FLC concentrations were significantly elevated in SLE patients compared to healthy controls (p<0.0001) also after adjusting for Ig levels (p<0.0001). The concentration of serum FLCs correlated with a global disease activity (SLE disease activity index (SLEDAI)) score of the SLE patients (r = 0.399, p = 0.007). Furthermore, concentrations of FLCs correlated with titers of dsDNA antibodies (r = 0.383, p = 0.009), and FLC levels and SLEDAI scores correlated in the anti-dsDNA-positive SLE patients, but not in anti-dsDNA-negative SLE patients. Total immunoglobulin (IgG and IgA) concentrations correlated with FLC concentrations and elevated FLC levels were additionally shown to associate with the inflammatory marker C-reactive protein and also with complement consumption determined by low C4 in SLE patients. Collectively, results indicated that elevated serum FLCs reflects increased B cell activity in relation to inflammation. SLE patients had an increased seropositivity of EBV-directed antibodies that did not associate with elevated FLC concentrations. An explanation for this could be that serum FLC concentrations reflect the current EBV activity (reactivation) whereas EBV-directed antibodies reflect the extent of previous infection/reactivations.SLE patients have elevated concentrations of serum FLCs that correlate with global disease activity scores and especially serologic markers for active disease. These findings are suggestive of circulating FLCs having potential as a new supplementary serologic biomarker in SLE

Efficient evaluation of humoral immune responses by the use of serum pools

Background: Collection and testing of individual serum samples are often used in research to gain knowledge about e.g. the humoral response against bacteria or virus. This is a valid but time-consuming method and might be a waste of valuable serum samples for inefficient research. So far, no study has considered using serum pools as a quick and efficient screening method to confirm or deny hypotheses. Methods: We created serum pools from four different patient groups (systemic lupus erythematosus n = 85, rheumatoid arthritis n = 77, Sjögren's syndrome n = 91, systemic sclerosis n = 66) and one healthy control group (n = 67). Each serum pool was analyzed using three well-known immunoassays: enzyme-linked immunosorbent assay (ELISA), line blot, and immunofluorescence microscopy (anti-nuclear antibody (ANA) screening). The presence of Epstein-Barr virus (EBV) EA/D-, EBNA-1-, VCA p23-, and gp350-directed antibodies was used to validate serum pools as an efficient tool for further investigations by comparison to previous findings in this area. Results: The presence of EBV EA/D-, EBNA-1-, VCA p23-, and gp350-directed antibodies in each pool was consistent within the obtained ELISA and line blot results, as increased titers of IgG against the four antigens were found in all patient serum pools and also in individual sera regarding gp350. These results correspond to previous findings on individual samples from patients with these diseases. The presence of ANAs was observed in all four patient serum pools and not in the HC pool by both line blots and immunofluorescence microscopy, which corresponds with the expectations and further corroborate the application of serum pools for screenings. Conclusion: We developed and validated the use of serum pools that reliably and rapidly can confirm or deny hypotheses, which enables a more efficient research concentrating on the most evident factors

Increased antibody levels to stage-specific Epstein–Barr virus antigens in systemic autoimmune diseases reveal a common pathology

The immune responses to antigens from different stages of the Epstein–Barr virus (EBV) life cycle were investigated in systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), Sjögren’s syndrome (SS), and systemic sclerosis (SSc) to gain knowledge of EBV’s involvement in the etiology of systemic autoimmune diseases (SADs) and for an overview of the humoral immune responses against EBV. Investigations were performed by the use of ELISA. IgM, IgA, and IgG antibody binding to 11 EBV antigens: EBNA1, EBNA2, BALF5, EAD, BALF2, EA/R, VCA p18, VCA p23, gB, gp350, and gp42 were examined in serum pools from SAD patients and healthy controls (HCs). Increased antibody levels against the 11 EBV antigens in the SAD pools were seen compared to the HC pool. Specifically, SLE was characterized by strongly increased IgA to EAD both compared to HCs and other SADs, and RA was characterized by increased IgM levels to several EBV antigens. The SADs may be partly distinguished by their differential immune responses to various antigens in the EBV life cycle. All together, these findings support an association between EBV infection and SADs

EBV antibodies in SLE patients and healthy controls (%).

<p>EBV—Epstein-Barr virus, SLE—systemic lupus erythematosus, EBNA-1 –EBV nuclear antigen 1, EBV-VCA—EBV viral capsid antigen, EBV-EA/D—EBV early antigen diffuse, NS—not significant.</p><p>EBV antibodies in SLE patients and healthy controls (%).</p

Concentration of serum FLCs in SLE patients and healthy controls.

<p>Total FLC (A), λFLC (B) and κFLC (C) levels in SLE patients (n = 45) and healthy controls (n = 40) measured by quantitative nephelometry. SLE patients suffering from renal insufficiency (eGFR<60 ml/min/1.73m²) were excluded. Middle horizontal bars represent median and statistical significant differences are indicated with *, ** or *** for p-values less than 0.05, 0.01 or 0.001. p-values for comparison of FLC levels in SLE patients and healthy controls are <0.0001. Maximum and minimum values of the normal ranges of λFLCs and κFLCs are indicated on the y-axis as dotted lines. FLCs—free light chains, SLE—systemic lupus erythematosus.</p

Correlation between serum FLC concentrations and SLEDAI scores of SLE patients.

<p>Correlation between SLEDAI scores and total FLC (A), λFLC (B) and κFLC (C) levels in SLE patients (n = 45). r-values are 0.399 (p = 0.007), 0.413 (p = 0.005) and 0.317 (p = 0.034) in A, B and C, respectively. FLCs—free light chains, SLE—systemic lupus erythematosus, SLEDAI—SLE disease activity index.</p

Correlation between serum FLC concentrations and SLEDAI scores in anti-dsDNA positive SLE patients.

<p>Correlation between SLEDAI scores and total FLC, λFLC and κFLC levels in anti-dsDNA positive SLE patients (A) (n = 21) and in anti-dsDNA negative SLE patients (B) (n = 24). <b>A: Anti-dsDNA positive SLE patients</b>. r-values are 0.498 (p = 0.022), 0.555 (p = 0.009) and 0.458 (p = 0.037) regarding total FLC, λFLC and κFLC levels, respectively. <b>B: Anti-dsDNA negative SLE patients</b>. r-values are 0.105 (p = 0.626), 0.159 (p = 0.458) and 0.030 (p = 0.888) regarding total FLC, λFLC and κFLC levels, respectively. FLCs—free light chains, SLE—systemic lupus erythematosus, SLEDAI—SLE disease activity index.</p