Background In patients with Multiple Sclerosis (pwMS) disease-modifying therapies (DMTs) affects immune response to antigens. Therefore, post-vaccination serological assessments are needed to evaluate the effect of the vaccine on SARS-CoV-2 antibody response. Methods We designed a prospective multicenter cohort study enrolling pwMS who were scheduled for SARS-Cov-2 vaccination with mRNA vaccines (BNT162b2, Pfizer/BioNTech,Inc or mRNA-1273, Moderna Tx,Inc). A blood collection before the first vaccine dose and 4 weeks after the second dose was planned, with a centralized serological assessment (electrochemiluminescence immunoassay, ECLIA, Roche-Diagnostics). The log-transform of the antibody levels was analyzed by multivariable linear regression. Findings 780 pwMS (76% BNT162b2 and 24% mRNA-1273) had pre- and 4-week post-vaccination blood assessments. 87 (11·2%) were untreated, 154 (19·7%) on ocrelizumab, 25 (3·2%) on rituximab, 85 (10·9%) on fingolimod, 25 (3·2%) on cladribine and 404 (51·7%) on other DMTs. 677 patients (86·8%) had detectable post-vaccination SARS-CoV-2 antibodies. At multivariable analysis, the antibody levels of patients on ocrelizumab (201-fold decrease (95%CI=128–317), p < 0·001), fingolimod (26-fold decrease (95%CI=16–42), p < 0·001) and rituximab (20-fold decrease (95%CI=10–43), p < 0·001) were significantly reduced as compared to untreated patients. Vaccination with mRNA-1273 resulted in a systematically 3·25-fold higher antibody level (95%CI=2·46–4·27) than with the BNT162b2 vaccine ( p < 0·001). The antibody levels on anti-CD20 therapies correlated to the time since last infusion, and rituximab had longer intervals (mean=386 days) than ocrelizumab patients (mean=129 days). Interpretation In pwMS, anti-CD20 treatment and fingolimod led to a reduced humoral response to mRNA-based SARS-CoV-2 vaccines. As mRNA-1273 elicits 3·25-higher antibody levels than BNT162b2, this vaccine may be preferentially considered for patients under anti-CD20 treatment or fingolimod. Combining our data with those on the cellular immune response to vaccines, and including clinical follow-up, will contribute to better define the most appropriate SARS-CoV-2 vaccine strategies in the context of DMTs and MS. Funding FISM[2021/Special-Multi/001]; Italian Ministry of Health‘Progetto Z844A 5 × 1000′.

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The aim of this study was to evaluate the diagnostic performance of four new enzyme immunoassays (EIAs) for anti-double-stranded-DNA (anti-dsDNA) antibodies, in comparison with the Farr assay and the Crithidia luciliae immunofluorescence test (CLIFT). To this purpose, sera from four patient groups were collected: 52 sera from patients with systemic lupus erythematosus (SLE); 28 from patients with other connective tissue diseases (CTD); 36 from patients with hepatitis C virus (HCV) infection; and 24 from those with acute viral infection. All sera were tested for anti-dsDNA antibodies by four EIA methods using a different antigenic DNA source [synthetic oligonucleotide (Method A), circular plasmid (Method B), recombinant (Method C), and purified extracted (Method D)], and by CLIFT and Farr assays. The diagnostic sensitivity of the assays was as follows: 84.6% (Method A), 73% (B), 82.7% (C), 84.6% (D), 55.8% (CLIFT), and 78.8% (Farr). Specificity was 82.9% (A), 97.7% (B), 96.5% (C), 94.3% (D), 96.5% (CLIFT), and 90.9% (Farr). From these data, we can conclude that the new-generation EIA methods evaluated in this study have higher sensitivity than the CLIFT and Farr assays and, with the exception of Method A, have specificity similar to the CLIFT and slightly higher than the Farr assay. These findings suggest that EIA tests may replace CLIFT as a screening test and the Farr assay as a specific test, for anti-dsDNA antibody detection.

Serum anti-mitochondrial antibodies (AMA) are the serological hallmark of primary biliary cirrhosis (PBC), yet up to 15% of PBC sera are AMA negative at routine indirect immunofluorescence (IIF) while being referred to as "probable" cases. The diagnostic role of PBC-specific antinuclear antibodies (ANA) remains to be determined. We will report herein data on the accuracy of new laboratory tools for AMA and PBC-specific ANA in a large series of PBC sera that were AMA-negative at IIF. We will also provide a discussion of the history and current status of AMA detection methods. We included IIF AMA-negative PBC sera (n=100) and sera from patients with other chronic liver diseases (n=104) that had been independently tested for IIF AMA and ANA; sera were blindly tested with an ELISA PBC screening test including two ANA (gp210, sp100) and a triple (pMIT3) AMA recombinant antigens. Among IIF AMA-negative sera, 43/100 (43%) manifested reactivity using the PBC screening test. The same test was positive for 6/104 (5.8%) control sera. IIF AMA-negative/PBC screen-positive sera reacted against pMIT3 (11/43), gp210 (8/43), Sp100 (17/43), both pMIT3 and gp210 (1/43), or both pMIT3 and Sp100 (6/43). Concordance rates between the ANA pattern on HEp-2 cells and specific Sp100 and gp210 ELISA results in AMA-negative subjects were 92% for nuclear dots and Sp100 and 99% for nuclear rim and gp210. Our data confirm the hypothesis that a substantial part of IIF AMA-negative (formerly coined "probable") PBC cases manifest disease-specific autoantibodies when tested using newly available tools and thus overcome the previously suggested diagnostic classification. As suggested by the recent literature, we are convinced that the proportion of AMA-negative PBC cases will be significantly minimized by the use of new laboratory methods and recombinant antigens.

Autoantibodies against DFS70 (dense fine speckles 70) antigen have recently been identified among antinuclear antibodies (ANA) in patients with various inflammatory diseases and in patients with different types of cancer. These antibodies are recognized using indirect immunofluorescence (IIF) on HEp-2 cells, by a fine speckled nuclear staining in interphase HEp-2 cells and a positive reaction in the chromosome region of mitotic cells. Given that the DFS70 protein is also known as the lens epithelium-derived growth factor, this study was performed with two objectives: (a) to assess the prevalence of these antibodies in patients sent for ANA testing and in 334 patients with different types of neoplasia and (b) to determine whether the lens tissue was a suitable substrate for the detection of antibodies specific to lens proteins. During routine workup for ANA detection by the IIF method, we found 172 DFS70-positive sera among 21,516 consecutive samples (prevalence, 0.8%). In the group of patients with neoplastic disease, 6 of 334 (1.8%) were anti-DFS70-positive. DFS70-positive sera were then assayed by the IIF method on cryostatic sections of mouse eye at a dilution of 1:40 with an anti-human IgG conjugate. Among the 172 DFS70-positive samples detected by the ANA screening, 32 (19%) were strongly reactive against the reticular fibers of the lens; 8 (5%) were positive only to the corneal epithelium (nuclear negative); 5 (3%) were positive both for the cornea and the lens fibers; 13 (7%) stained only the nuclei of lens and cornea cells, and 4 (2%) were positive against the ciliary muscle. Among the patients with neoplastic diseases, only one with lung cancer reacted weakly with the reticular fibers of the lens. Sera from 20 healthy blood donors were negative. In this preliminary study, we have shown that the prevalence of anti-DFS70 antibodies is much lower than previously reported, both in patients screened for ANA and in patients with cancer. We have also seen that some DFS70-positive sera have antibodies that recognize antigens of the lens. Further studies are needed to investigate the fine specificity and the possible significance of these new autoantibodies.

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After the success of genome-wide association studies to uncover complex trait loci, attempts to explain the remaining genetic heritability (h ) are mainly focused on unraveling rare variant associations and gene-gene or gene-environment interactions. Little attention is paid to the possibility that h estimates are inflated as a consequence of the epidemiological study design. We studied the time series of 54 biochemical traits in 4373 individuals from the Cooperative Health Research In South Tyrol (CHRIS) study, a pedigree-based study enrolling ten participants/day over several years, with close relatives preferentially invited within the same day. We observed distributional changes of measured traits over time. We hypothesized that the combination of such changes with the pedigree structure might generate a shared-environment component with consequent h inflation. We performed variance components (VC) h estimation for all traits after accounting for the enrollment period in a linear mixed model (two-stage approach). Accounting for the enrollment period caused a median h reduction of 4%. For 9 traits, the reduction was of>20%. Results were confirmed by a Bayesian Markov chain Monte Carlo analysis with all VCs included at the same time (one-stage approach). The electrolytes were the traits most affected by the enrollment period. The h inflation was independent of the h magnitude, laboratory protocol changes, and length of the enrollment period. The enrollment process may induce shared-environment effects even under very stringent and standardized operating procedures, causing h inflation. Including the day of participation as a random effect is a sensitive way to avoid overestimation.