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Introduction: The role of the complement inhibitory proteins CD46 and CD59 in the immune response to an acute coronary syndrome (ACS) is unknown. We investigated the relationships between the shedding of CD46 and CD59 into the circulation, reflected by plasma levels of soluble CD46 and CD59, and the risk for post-ACS complications., Methods: We measured plasma sCD46 and sCD59 in a cohort of 546 ACS patients within 24 h after hospital admission, and after 6-weeks in a subgroup of 114 patients. Study outcomes were incident heart failure (HF), major adverse cardiovascular events (MACE) and mortality during a median follow-up period of up to 3.3 years. Echocardiography at 1-year was performed in the follow-up subgroup., Results: Elevated sCD46 and sCD59 were correlated with increased levels of inflammatory mediators and metalloproteinases in plasma, and were associated with increased risk for MACE in Cox proportional hazard models adjusted for cardiovascular risk factors and revascularization [HR 95% CI 1.24 (1.02-1.52), p = 0.034 for sCD46 and 1.18 (1.00-1.38), p = 0.049 for sCD59]. Elevated sCD59 was also associated with higher incidence of HF [HR 95% CI 1.41 (1.15-1.74), p = 0.001], and with lower left ventricular ejection fraction at 1-year post-ACS (Spearman r = - 0.234, p = 0.020). We found no associations between plasma levels of the proteins at 6 weeks and outcomes., Conclusions: Shedding of the complement regulators CD46 and CD59 in plasma in the acute phase of ACS is associated with a negative prognosis. Plasma sCD46 and sCD59 could reflect the degree of local immune activation and serve as prognostic biomarkers in ACS patients., Competing Interests: Declarations Ethics approval and consent to participate All patients signed an informed consent form before inclusion in the study. The study was approved by the Regional Ethics Committee for human studies in Lund, Sweden (reference number 211/2007). Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests., (© 2024. The Author(s).)

Background: Cartilage oligomeric matrix protein (COMP), an extracellular matrix glycoprotein, is vital in preserving cartilage integrity. Further, its overexpression is associated with the aggressiveness of several types of solid cancers. This study investigated COMP's role in ovarian cancer, exploring clinicopathological links and mechanistic insights., Methods: To study the association of COMP expression in cancer cells and stroma with clinicopathological features of ovarian tumor patients, we analyzed an epithelial ovarian tumor cohort by immunohistochemical analysis. Subsequently, to study the functional mechanisms played by COMP, an in vivo xenograft mouse model and several molecular biology techniques such as transwell migration and invasion assay, tumorsphere formation assay, proximity ligation assay, and RT-qPCR array were performed., Results: Based on immunohistochemical analysis of epithelial ovarian tumor tissues, COMP expression in the stroma, but not in cancer cells, was linked to worse overall survival (OS) of ovarian cancer patients. A xenograft mouse model showed that carcinoma-associated fibroblasts (CAFs) expressing COMP stimulate the growth and metastasis of ovarian tumors through the secretion of COMP. The expression of COMP was upregulated in CAFs stimulated with TGF-β. Functionally, secreted COMP by CAFs enhanced the migratory capacity of ovarian cancer cells. Mechanistically, COMP activated the Notch3 receptor by enhancing the Notch3-Jagged1 interaction. The dependency of the COMP effect on Notch was confirmed when the migration and tumorsphere formation of COMP-treated ovarian cancer cells were inhibited upon incubation with Notch inhibitors. Moreover, COMP treatment induced epithelial-to-mesenchymal transition and upregulation of active β-catenin in ovarian cancer cells., Conclusion: This study suggests that COMP secretion by CAFs drives ovarian cancer progression through the induction of the Notch pathway and epithelial-to-mesenchymal transition., (© 2024. The Author(s).)

Background: The complement inhibitor CSMD1 acts as a tumor suppressor in various types of solid cancers. Despite its high level of expression in the brain, its function in gliomas, malignant brain tumors originating from glial cells, has not been investigated., Methods: Three cohorts of glioma patients comprising 1500 patients were analyzed in our study along with their clinical data. H4, U-118 and U-87 cell lines were used to investigate the tumor suppressor function of CSMD1 in gliomas. PDGFB-induced brain tumor model was utilized for the validation of in vitro data., Results: The downregulation of CSMD1 expression correlated with reduced overall and disease-free survival, elevated tumor grade, wild-type IDH genotype, and intact 1p/19q status. Moreover, enhanced activity was noted in the neuroinflammation pathway. Importantly, ectopic expression of CSMD1 in glioma cell lines led to decreased aggressiveness in vitro. Mechanically, CSMD1 obstructed the TNF-induced NF-kB and STAT3 signaling pathways, effectively suppressing the secretion of IL-6 and IL-8. There was also reduced survival in PDGFB-induced brain tumors in mice when Csmd1 was downregulated., Conclusions: Our study has identified CSMD1 as a tumor suppressor in gliomas and elucidated its role in TNF-induced neuroinflammation, contributing to a deeper understanding of glioma pathogenesis., (© 2024. The Author(s).)

Background: Sushi domain-containing protein 4 (SUSD4) is a recently discovered protein with unknown cellular functions. We previously revealed that SUSD4 can act as complement inhibitor and as a potential tumor suppressor., Methods: In a syngeneic mouse model of breast cancer, tumors expressing SUSD4 had a smaller volume compared with the corresponding mock control tumors. Additionally, data from three different expression databases and online analysis tools confirm that for breast cancer patients, high mRNA expression of SUSD4 in the tumor tissue correlates with a better prognosis. In vitro experiments utilized triple-negative breast cancer cell lines (BT-20 and MDA-MB-468) stably expressing SUSD4. Moreover, we established a cell line based on BT-20 in which the gene for EGFR was knocked out with the CRISPR-Cas9 method., Results: We discovered that the Epithelial Growth Factor Receptor (EGFR) interacts with SUSD4. Furthermore, triple-negative breast cancer cell lines stably expressing SUSD4 had higher autophagic flux. The initiation of autophagy required the expression of EGFR but not phosphorylation of the receptor. Expression of SUSD4 in the breast cancer cells led to activation of the tumor suppressor LKB1 and consequently to the activation of AMPKα1. Finally, autophagy was initiated after stimulation of the ULK1, Atg14 and Beclin-1 axis in SUSD4 expressing cells., Conclusions: In this study we provide novel insight into the molecular mechanism of action whereby SUSD4 acts as an EGFR inhibitor without affecting the phosphorylation of the receptor and may potentially influence the recycling of EGFR to the plasma membrane., (© 2022. The Author(s).)

Background: Human CUB and Sushi multiple domains 1 (CSMD1) is a large membrane-bound tumor suppressor in breast cancer. The current study aimed to elucidate the molecular mechanism underlying the effect of CSMD1 in highly invasive triple negative breast cancer (TNBC)., Methods: We examined the antitumor action of CSMD1 in three TNBC cell lines overexpressing CSMD1, MDA-MB-231, BT-20 and MDA-MB-486, in vitro using scanning electron microscopy, proteome array, qRT-PCR, immunoblotting, proximity ligation assay, ELISA, co-immunoprecipitation, immunofluorescence, tumorsphere formation assays and flow cytometric analysis. The mRNA expression pattern and clinical relevance of CSMD1 were evaluated in 3520 breast cancers from a modern population-based cohort., Results: CSMD1-expressing cells had distinct morphology, with reduced deposition of extracellular matrix components. We found altered expression of several cancer-related molecules, as well as diminished expression of signaling receptors including Epidermal Growth Factor Receptor (EGFR), in CSMD1-expressing cells compared to control cells. A direct interaction of CSMD1 and EGFR was identified, with the EGF-EGFR induced signaling cascade impeded in the presence of CSMD1. Accordingly, we detected increased  ubiquitination levels of EGFR upon activation in CSMD1-expressing cells, as well as increased degradation kinetics and chemosensitivity. Accordingly, CSMD1 expression rendered tumorspheres pretreated with gefitinib more sensitive to chemotherapy. In addition, higher mRNA levels of CSMD1 tend to be associated with better outcome of triple negative breast cancer patients treated with chemotherapy., Conclusions: Our results indicate that CSMD1 cross-talks with the EGFR endosomal trafficking cascade in a way that renders highly invasive breast cancer cells sensitive to chemotherapy. Our study unravels one possible underlying molecular mechanism of CSMD1 tumor suppressor function and may provide novel avenues for design of better treatment., (© 2021. The Author(s).)

Background: In the present study, we sought to evaluate the complement activation product C4d as a marker for lupus nephritis in systemic lupus erythematosus (SLE)., Methods: C4d levels were determined by enzyme-linked immunosorbent assay in plasma samples of patients with established SLE using a novel approach based on detection of a short linear cleavage neoepitope. Cross-sectional associations were studied in 98 patients with SLE with samples taken at lower or higher respective disease activity. Temporal associations were investigated in 69 patients with SLE who were followed longitudinally for up to 5 years. Plasma samples from 77 healthy donors were included as controls., Results: C4d levels were negligible in healthy control subjects and significantly increased in patients with SLE in the cross-sectional study (p < 0.0001). C4d levels discriminated between higher and lower disease activity according to ROC curve analysis (p < 0.001), exhibiting a positive predictive value of 68%. At higher disease activity, C4d levels correlated with the modified Systemic Lupus Erythematosus Disease Activity Index (p = 0.011) and predominantly with lupus nephritis (p = 0.003), exhibiting a sensitivity of 79% to identify patients with nephritis. High C4d levels together with the presence of anti-dsDNA autoantibodies preceded and thus predicted future lupus nephritis in the longitudinal study (OR 5.4, 95% CI 1.4-21.3). When we considered only patients with renal involvement (19 of 69) during the longitudinal study, we found that high C4d levels alone could forecast recurrence of future lupus nephritis (OR 3.3, 95% CI 1.2-9.6)., Conclusions: C4d appears to be a valuable marker for use in monitoring of patients with SLE, particularly for lupus nephritis. Importantly, C4d levels can predict impending flares of lupus nephritis and may thus be useful for informing treatment.

Background: 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., Methods: 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., Results: 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., Conclusions: The data indicate that complement activation is an important feature of SRC.

Background: The complement system is suggested to be involved in the pathogenesis of osteoarthritis (OA), and proinflammatory cytokines may play a role in OA development by inducing proteases. The association between complement factors, cytokines and OA has not been investigated. The aim of the present study was to explore the involvement of the complement system after knee trauma and in OA., Methods: C4d, C3bBbP and soluble terminal complement complex (sTCC) resulting from complement activation were immunoassayed in synovial fluid from subjects with healthy knees (reference), OA, rheumatoid arthritis (RA; positive control), pyrophosphate arthritis (PPA; positive control) and knee injury; other biomarkers were previously assessed. Magnetic resonance imaging was used to assess joint injuries., Results: Compared with levels in the reference group, the median concentrations of C4d, C3bBbP and sTCC in the OA, RA, PPA and knee injury groups were 2- to 34-fold increased (p < 0.001 to p = 0.044). For the knee injury group, the median concentrations of C4d, C3bBbP and sTCC were 5- to 12-fold increased (p < 0.001) at the day of injury; after 3-12 weeks, C3bBbP and sTCC concentrations were similar to reference levels; and C4d was still increased several years after injury. In the 0-12 weeks period after injury, the concentrations of C4d, C3bBbP and sTCC correlated positively with levels of interleukin (IL)-1β, IL-6 and tumour necrosis factor α (r s range 0.232-0.547); none of the measured complement factors correlated with proteolytic fragments of aggrecan or cartilage oligomeric matrix protein. Knees with osteochondral fracture, with or without disrupted cortical bone, had higher concentrations of C4d (p = 0.014, p = 0.004) and sTCC (p = 0.004, p < 0.001) compared with knees without fractures., Conclusions: The complement system is activated in OA and after knee injury. Following knee injury, this activation is instant and associated with inflammation as well as with the presence of osteochondral fractures.

Background: The human Sushi Domain-Containing Protein 4 (SUSD4) was recently shown to function as a novel inhibitor of the complement system, but its role in tumor progression is unknown., Methods: Using immunohistochemistry and quantitative PCR, we investigated SUSD4 expression in breast cancer tissue samples from two cohorts. The effect of SUSD4 expression on cell migration and invasion was studied in vitro using two human breast cancer cell lines overexpressing SUSD4., Results: Tissue stainings revealed that both tumor cells and tumor-infiltrating cells expressed SUSD4. The highest SUSD4 expression was detected in differentiated tumors with decreased rate of metastasis, and SUSD4 expression was associated with improved survival of the patients. Moreover, forced SUSD4 expression in human breast cancer cells attenuated their migratory and invasive traits in culture. SUSD4 expression also inhibited colony formation of human breast cancer cells cultured on carcinoma-associated fibroblasts. Furthermore, large numbers of SUSD4-expressing T cells in the tumor stroma associated with better overall survival of the breast cancer patients., Conclusion: Our findings indicate that SUSD4 expression in both breast cancer cells and T cells infiltrating the tumor-associated stroma is useful to predict better prognosis of breast cancer patients.

Introduction: Patients with systemic lupus erythematosus (SLE) have a decreased ability to clear cell remnants and multiple deficiencies in the ability to degrade cellular chromatin have been linked to the disease. Since the discovery of neutrophil extracellular traps (NETs), a renewed interest has been sparked in this field of research with multiple studies reporting a decreased ability of patients with SLE to degrade NETs. In this study we extend these findings by investigating the ability of patients with SLE to degrade chromatin from multiple clinically relevant sources., Methods: We use flow cytometry in combination with NET degradation and DNA zymogram assays to investigate the ability of sera from SLE patients to degrade chromatin from three different sources of DNA such as NETs, apoptotic and necrotic cells. This ability was further associated with clinical manifestations., Results: We found that 61% of the patients had an affected degradation of at least one chromatin source. Further, degradation of NETs correlated with degradation of chromatin from secondary necrotic cells but not with degradation of chromatin from primary necrotic cells. Patients who fail to degrade several forms of DNA more often display anti-nuclear and nephritic involvement whereas this is not observed in patients with decreased ability to degrade chromatin from primary necrotic cells., Conclusions: The majority of patients with SLE has a decreased ability to degrade chromatin from clinically relevant sources. This decreased ability is further reflected in their clinical presentation.

Introduction: The ability to degrade neutrophil extracellular traps (NETs) is reduced in a subset of patients with systemic lupus erythematosus (SLE). NETs consist of chromatin covered with antimicrobial enzymes and are normally degraded by DNase-I, an enzyme which is known to have reduced activity in SLE. Decreased ability to degrade NETs is associated with disease activity. In the current study we investigated how the ability of serum from SLE patients to degrade NETs varies during the course of SLE as well as what impact this may have for the clinical phenotype of SLE., Methods: Serum from 69 patients with SLE, included in a prospective study, was taken every 60 days for a median of 784 days. The ability of serum to degrade NETs was determined and associated with clinical parameters occurring before and at the time of sampling, as well as after sampling by using conditional logistic regression., Results: As many as 41% of all patients in the study showed decreased ability to degrade NETs at least once, but with a median of 20% of all time points. Decreased degradation was associated with manifestations of glomerulonephritis as well as low complement levels and elevated levels of antibodies directed against histones and DNA. Furthermore, the odds ratio for the patient to develop alopecia and fever after an episode of decreased NETs degradation was increased by four to five times compared to normal., Conclusions: Decreased degradation of NETs is associated with clinical manifestations in SLE and may contribute to disease pathogenesis. Potential therapeutics restoring the ability to degrade NETs could be beneficial for certain patients with SLE.

Introduction: Complexes between cartilage oligomeric matrix protein (COMP) and the complement activation product C3b have been found in the circulation of patients with rheumatoid arthritis and systemic lupus erythematosus. In systemic sclerosis (SSc) COMP expression in the skin is upregulated both in lesional and non-lesional skin, which is also reflected in an increased amount of circulating COMP. We investigated the presence of COMP-C3b complexes in serum and skin biopsies of patients with SSc., Methods: The presence of COMP and COMP-C3b complexes in the serum of 80 patients with limited cutaneous SSc (lcSSc, n = 40) and diffuse cutaneous SSc (dcSSc, n = 40) and 97 healthy controls was measured by ELISA and correlated to different clinical parameters. Samples were collected both at baseline and after three to five years to assess longitudinal changes in COMP-C3b complex levels. Furthermore, skin biopsies from seven patients with dcSSc and three healthy controls were analyzed for expression of COMP and deposition of C3b and IgG., Results: Serum levels of COMP-C3b were found to be elevated in both dcSSc and lcSSc compared to healthy controls and decreased at the second measurement in patients on immunosuppressive therapy. No co-localization of COMP and C3b was found in the skin biopsies, indicating that the COMP-C3b complexes are formed upon release of COMP into the circulation., Conclusion: COMP-C3b complexes are found in the serum of patients with SSc. The lack of co-localization between COMP and C3b in the skin suggests that COMP does not drive complement activation in the skin in SSc.

Introduction: Complement activation is involved in rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) and atypical hemolytic uremic syndrome (aHUS). Autoantibodies to complement inhibitor factor H (FH), particularly in association with deletions of the gene coding for FH-related protein 1 (CFHR1), are associated with aHUS., Methods: Autoantibodies against FH, factor I (FI) and C4b-binding protein (C4BP) were measured by ELISA, while CFHR1 homozygous deletion was determined with Western blotting of sera. Epitopes for FH autoantibodies were mapped using recombinant fragments of FH., Results: FH autoantibodies were detected in SLE (6.7%, n = 60, RA patients (16.5%, n = 97 in the Swedish cohort and 9.2%, n = 217 in the Dutch cohort) and thrombosis patients positive for the lupus anticoagulants (LA+) test (9.4%, n = 64) compared with aHUS patients (11.7%, n = 103). In the control groups (n = 354), an average of 4% of individuals were positive for FH autoantibodies. The frequencies observed in both RA cohorts and LA+ patients were statistically significantly higher than in controls. We also found that an average of 15.2% of the FH-autoantibody positive individuals in all studied disease groups had homozygous deficiency of CFHR1 compared with 3.8% of the FH autoantibody negative patients. The levels of FH autoantibodies varied in individual patients over time. FH autoantibodies found in LA+, SLE and RA were directed against several epitopes across FH in contrast to those found in aHUS, which bound mainly to the C-terminus. Autoantibodies against FI and C4BP were detected in some patients and controls but they were not associated with any of the diseases analyzed in this study., Conclusions: Autoantibodies against FH are not specific for aHUS but are present at a significant frequency in rheumatic diseases where they could be involved in pathophysiological mechanisms.

Introduction: Cartilage oligomeric matrix protein (COMP) is found at elevated concentrations in sera of patients with joint diseases such as rheumatoid arthritis (RA) and osteoarthritis (OA). We recently showed that COMP activates complement via the alternative pathway and that COMP-C3b complexes are present in sera of RA patients, but not in healthy controls. We now set out to elaborate on the information provided by this marker in a variety of diseases and larger patient cohorts., Methods: COMP-C3b levels in sera were measured by using an enzyme-linked immunosorbent assay (ELISA) capturing COMP and detecting C3b. Serum COMP was measured by using ELISA., Results: COMP-C3b levels were significantly elevated in patients with RA as well as in systemic lupus erythematosus (SLE), compared with healthy controls. SLE patients with arthritis had significantly higher COMP-C3b levels than did those without. COMP-C3b was furthermore elevated in patients with ankylosing spondylitis (AS), psoriatic arthritis (PsA), reactive arthritis, systemic sclerosis, and OA. COMP-C3b did not correlate with COMP in any of the patient groups. COMP-C3b correlated with disease activity in RA, but not in other diseases. COMP-C3b levels in RA patients decreased on treatment with tumor necrosis factor (TNF)-α inhibitors, whereas the levels increased in patients with AS or PsA. The changes of COMP-C3b did not parallel the changes of C-reactive protein (CRP)., Conclusions: COMP-C3b levels are elevated in several rheumatologic diseases and correlate with inflammatory measures in RA. COMP-C3b levels in RA decrease during TNF-α inhibition differently from those of CRP, suggesting that formation of COMP-C3b relates to disease features not reflected by general inflammation measures.

Introduction: Inherited deficiencies of several complement components strongly predispose to systemic lupus erythematosus (SLE) while deficiencies of complement inhibitors are found in kidney diseases such as atypical hemolytic uremic syndrome (aHUS)., Methods: The exons of complement inhibitor genes CD46 and CFH (factor H) were fully sequenced using the Sanger method in SLE patients with nephritis originating from two cohorts from southern and mid Sweden (n = 196). All identified mutations and polymorphisms were then analyzed in SLE patients without nephritis (n = 326) and in healthy controls (n = 523)., Results: We found nonsynonymous, heterozygous mutations in CFH in 6.1% patients with nephritis, in comparison with 4.0% and 5.4% in patients without nephritis and controls, respectively. No associations of SLE or nephritis with common variants in CFH (V62I/Y402H/E936D) were found. Furthermore, we found two nonsynonymous heterozygous mutations in CD46 in SLE patients but not in controls. The A353V polymorphism, known to affect function of CD46, was found in 6.6% of nephritis patients versus 4.9% and 6.1% of the non-nephritis SLE patients and controls. The presence of mutations in CD46 and CFH did not predispose to SLE or nephritis but was associated with earlier onset of nephritis. Furthermore, we found weak indications that there is one protective and one risk haplotype predisposing to nephritis composed of several polymorphisms in noncoding regions of CD46, which were previously implicated in aHUS., Conclusions: SLE nephritis is not associated with frequent mutations in CFH and CD46 as found in aHUS but these may be modifying factors causing earlier onset of nephritis.