The complement system has been implicated in pathogenesis of systemic sclerosis (SSc). The goal of the present study was to evaluate improved complement biomarkers in SSc. The presence of C4d, reflecting activation of the classical/lectin pathways, C3bBbP corresponding to activation of the alternative pathway, and soluble terminal complement complexes (all complement pathways), was measured in plasma samples by enzyme-linked immunosorbent assay and correlated to clinical parameters. The study included 81 patients with limited cutaneous SSc and 41 with diffuse cutaneous SSc, as well as 47 matched healthy controls and 81 patients with rheumatoid arthritis, 22 with psoriatic arthritis and 20 with ankylosing spondylitis. Skin and kidney biopsies of selected patients were stained to detect deposited C3b as a marker of local complement activation. Biomarkers of activation of all complement pathways were increased in SSc compared with healthy controls and were similar to those in other rheumatic diseases. When patients with SSc were divided into subgroups, a distinct pattern of complement markers was observed in individuals with scleroderma renal crisis (SRC). By functional assay, we confirmed a significant decrease in complement haemolytic activity in SRC vs. non-SRC patients, indicating complement consumption. Further, we detected glomerular deposits of C3b in some patients with SRC. The data indicate that complement activation is an important feature of SRC.

Complement C4d-specific antibodies for the diagnosis of lung cancer Complement C4d-specific antibodies for the diagnosis of lung cancer

Overactivation of the alternative pathway of the complement system is associated with the renal diseases atypical hemolytic uremic syndrome (aHUS) and C3 glomerulopathy (C3G). C3 nephritic factors (C3NeF) play an important role in C3G pathogenesis by stabilizing the key enzymatic complex of complement, the C3 convertase. However, the reliability of assays detecting these autoantibodies is limited. Therefore, in this study we validated and optimized a prototype hemolytic method for robust detection and characterization of factors causing convertase overactivity in large patient cohorts. The assay assesses convertase activity directly in the physiological milieu of serum and therefore is not restricted to detection of stabilizing autoantibodies such as C3NeF, but may also reveal genetic variants resulting in prolonged convertase activity. We first defined clear cut-off values based on convertase activity in healthy controls. Next, we evaluated 27 C3G patient samples and found 16 positive for prolonged convertase activity, indicating the presence of factors influencing convertase stability. In three patients, the overactive convertase profile was persistent over disease course while in another patient the increased stability normalized in remission. In all these four patients, the convertase-stabilizing activity resided in the purified immunoglobulin fraction, demonstrating the autoantibody nature. In contrast, the immunoglobulins of a familial aHUS patient carrying the factor B mutation p.Lys323Glu did not reveal convertase stabilization. However, in serum prolonged convertase activity was observed and segregated with the mutation in both affected and unaffected family members. In conclusion, we present a robust and reliable method for the detection, characterization, and evaluation over time of factors prolonging convertase activity (C3NeF or certain mutations) in patient cohorts. This assay may provide new insights in disease pathogenesis and may contribute to the development of more personalized treatment strategies.

Autoantibodies termed C3-nephritic factor (C3NeF), which stabilize convertases of the alternative complement pathway, often stimulate autoinflammatory diseases. However, knowledge about analogous autoantibodies acting on the classical pathway (C4NeF) is limited to a few reports, which indicate association with kidney dysfunction, systemic lupus erythematous, and infections. C4NeF may appear independently from C3NeF, but the lack of a routine diagnostic method predisposes C4NeF for being an underestimated player in autoinflammatory episodes. We tested the activity of classical convertases directly in serum/plasma to screen samples from 13 patients with C3 glomerulopathies and identified one patient showing significantly prolonged half-life of these enzymes. Observed effect was reproduced by immunoglobulins purified from patient’s plasma and additionally confirmed on classical convertase built from purified components. Isolated immunoglobulins protected classical convertases from both spontaneous and inhibitor-driven decay but not from C4b proteolysis. The patient had a decreased serum level of C3, elevated sC5b-9, and normal concentrations of factor B and C4. Neither C3NeF nor other autoantibodies directed against alternative pathway proteins (factor H, factor B, factor I, C3, and properdin) were found. Genetic analysis showed no mutations in C3, CFB, CFH, CFI, MCP, THBD, and DGKE genes. Renal biopsy revealed a membranoproliferative pattern with intense C3 deposits. Our results underline the importance of C4NeF as an independent pathogenic factor and a need for the implementation of routine examination of classical convertase activity. Proposed method may enable robust inspection of such atypical cases.

As the COVID-19 outbreak is developing the two most frequently reported statistics seem to be the raw confirmed case and case fatalities counts. Focusing on Italy, one of the hardest hit countries, we look at how these two values could be put in perspective to reflect the dynamics of the virus spread. In particular, we find that merely considering the confirmed case counts would be very misleading. The number of daily tests grows, while the daily fraction of confirmed cases to total tests has a change point. It (depending on region) generally increases with strong fluctuations till (around, depending on region) 15–22 March and then decreases linearly after. Combined with the increasing trend of daily performed tests, the raw confirmed case counts are not representative of the situation and are confounded with the sampling effort. This we observe when regressing on time the logged fraction of positive tests and for comparison the logged raw confirmed count. Hence, calibrating model parameters for this virus’s dynamics should not be done based only on confirmed case counts (without rescaling by the number of tests), but take also fatalities and hospitalization count under consideration as variables not prone to be distorted by testing efforts. Furthermore, reporting statistics on the national level does not say much about the dynamics of the disease, which are taking place at the regional level. These findings are based on the official data of total death counts up to 15 April 2020 released by ISTAT and up to 10 May 2020 for the number of cases. In this work, we do not fit models but we rather investigate whether this task is possible at all. This work also informs about a new tool to collect and harmonize official statistics coming from different sources in the form of a package for the statistical environment and presents the “.”

Rituximab is a pioneering anti-CD20 monoclonal antibody that became the first-line drug used in immunotherapy of B-cell malignancies over the last twenty years. Rituximab activates the complement system in vitro, but there is an ongoing debate on the exact role of this effector mechanism in therapeutic effect. Results of both in vitro and in vivo studies are model-dependent and preclude clear clinical conclusions. Additional confounding factors like complement inhibition by tumor cells, loss of target antigen and complement depletion due to excessively applied immunotherapeutics, intrapersonal variability in the concentration of main complement components and differences in tumor burden all suggest that a personalized approach is the best strategy for optimization of rituximab dosage and therapeutic schedule. Herein we critically review the existing knowledge in support of such concept and present original data on markers of complement activation, complement consumption, and rituximab accumulation in plasma of patients with chronic lymphocytic leukemia (CLL) and non-Hodgkin’s lymphomas (NHL). The increase of markers such as C4d and terminal complement complex (TCC) suggest the strongest complement activation after the first administration of rituximab, but not indicative of clinical outcome in patients receiving rituximab in combination with chemotherapy. Both ELISA and complement-dependent cytotoxicity (CDC) functional assay showed that a substantial number of patients accumulate rituximab to the extent that consecutive infusions do not improve the cytotoxic capacity of their sera. Our data suggest that individual assessment of CDC activity and rituximab concentration in plasma may support clinicians’ decisions on further drug infusions, or instead prescribing a therapy with anti-CD20 antibodies like obinutuzumab that more efficiently activate effector mechanisms other than complement.