Complement C1s Research Articles

Sutimlimab: A Complement C1s Inhibitor for the Management of Cold Agglutinin Disease-Associated Hemolysis.

To review the pharmacology, pharmacokinetics, efficacy, safety, dosing and administration, and place in therapy of sutimlimab for the management of cold agglutinin disease (CAD)-associated hemolysis. A literature search of PubMed (1966-October 2022) was conducted using the keywords sutimlimab, BIVV009, and cold agglutinin. Data were also obtained from prescribing information, meeting abstracts, and clinicaltrials.gov. All published prospective clinical trials, prescribing information, and meeting abstracts on sutimlimab for the treatment of CAD were reviewed. Sutimlimab is a first-in-class complement C1s inhibitor indicated for the treatment of CAD-associated hemolysis. This approval was based on the phase III CARDINAL trial, which evaluated sutimlimab in patients with CAD-associated hemolysis. The primary endpoint of achieving a hemoglobin of ≥12 g/dL or increase of ≥2 above baseline was achieved by 54% of patients with sutimlimab in the 26-week trial. The phase III CADENZA trial was a placebo-controlled trial in which sutimlimab has demonstrated a significant improvement in the composite endpoint of hemoglobin increase of ≥1.5 g/dL, avoidance of transfusion, and avoidance of additional CAD therapies (73% sutimlimab vs 15% placebo). Sutimlimab rapidly halts hemolysis, improves hemoglobin, and improves quality-of-life in patients with CAD. Safety issues with sutimlimab include infusion-related reactions and risk of serious infections with encapsulated bacteria. Sutimlimab provides an additional therapeutic option in the treatment of CAD-associated hemolysis that can lead to rapid improvement in hemoglobin and anemia-related symptoms.

Sustained Complement C1s Inhibition with Sutimlimab in Patients with Cold Agglutinin Disease Results in Continued Efficacy during Part B of the Randomized Placebo-Controlled Phase 3 Cadenza Study (NCT03347422)

Background: Cold agglutinin disease (CAD) is a rare chronic autoimmune hemolytic anemia characterized by classical complement pathway (CP)-mediated hemolysis. Sutimlimab (SUT) is a first-in-class humanized monoclonal antibody that prevents CP activation by selectively inhibiting C1s; both the alternative and lectin pathways remain intact. In the randomized, double-blind, placebo-controlled Part A (26 weeks) of Phase 3 CADENZA, SUT treatment resulted in rapid and sustained improvements in hemoglobin (Hb), hemolytic markers and quality of life (QoL). Aims: To report the long-term safety and efficacy of SUT treatment in patients with CAD from the open-label Part B extension of CADENZA. Methods: In Part A, eligible patients with CAD without a history of recent blood transfusion (≤1 during previous 12 months; 0 during last 6 months), received SUT or placebo (PBO) through intravenous infusions on Days 0 and 7, then biweekly. All patients completing Part A were eligible to enter Part B and receive biweekly SUT for ≥1 year after the last patient completed Part A. Efficacy data up to Week 79 as well as the last available patients' values are reported. Efficacy endpoints included change from baseline (BL) in Hb, hemolytic and pharmacodynamic markers, Functional Assessment of Chronic Illness Therapy (FACIT)-Fatigue, and blood transfusions. Safety data was recorded throughout the study; treatment emergent adverse event (TEAE) and serious TEAE (TESAE). Results: Of the 42 patients enrolled in Part A, 39 completed Part A and entered Part B, with 32 (82.1%) completing Part B; 7 (17.9%) patients discontinued the study prematurely, due to TESAE (n=1), lack of efficacy (n=3), withdrawal of consent (n=2) and withdrawal by sponsor (n=1). SUT treatment rapidly improved Hb levels; mean (SE) Hb at the end of Part A was 11.51 (0.40) g/dL and 9.43 (0.40) g/dL for SUT and PBO groups, respectively. In Part B, improvements in Hb were sustained in the SUT group and the ex-PBO group saw rapid and comparable increases in Hb upon initiation of SUT; at Week 79 mean (SE) Hb levels were 11.86 (0.54) g/dL and 11.76 (0.58) g/dL in the SUT only and ex-PBO groups, respectively (Panel A). Mean total bilirubin was normalized with SUT treatment in Part A and sustained in Part B; similar decreases were observed for patients in the ex-PBO group when they started receiving SUT in Part B (Panel B). Improvements in mean [SE] FACIT-Fatigue scores (BL: 32.96 [1.79]) observed in Part A were sustained in Part B in the SUT only group (44.31 [2.19] at Week 87); the mean FACIT-Fatigue score increased to comparable levels in the ex-PBO group (41.40 [2.71] at Week 87). Improvements in Hb, bilirubin, and FACIT-Fatigue correlated with normalization of C4 and near-complete inhibition of CP activity, that was maintained through the end of treatment. Reductions in mean absolute reticulocyte count and increases in haptoglobin levels observed with SUT treatment in Part A were maintained in Part B, and upon initiation of SUT in Part B, the ex-PBO group reached comparable levels. In Part B, 9 (23.1%) patients received a ≥1 transfusion. Thirty-six (92.3%) patients experienced ≥1 TEAE in Part B. Seven (17.9%) patients experienced a total of 11 TESAEs; one TESAE of hypertension was assessed as related to SUT by the investigator. Thromboembolic events (transient ischemic attack [n=1]; deep vein thrombosis [n=1]) were observed in 2 (5.1%) patients with underlying risk factors for thromboembolism. Both events were assessed as non-serious and unrelated to SUT by the Investigator. One TESAE of infection (urinary tract infection Grade ≥3) was reported. No meningococcal infections, serious events of hypersensitivity, anaphylaxis or systemic lupus erythematosus were reported. One patient with a history of tobacco use (received PBO in Part A) experienced a TESAE of squamous cell carcinoma of the lung with fatal outcome. SUT was withdrawn due to this TESAE prior to the patient's death. Conclusion: Long-term treatment with SUT, an anti-C1s CP inhibitor, maintained mean Hb levels >11 g/dL, achieved sustained normalization of bilirubin and led to clinically meaningful improvements of FACIT-Fatigue scores, while maintaining a favourable safety profile. Improvements in anemia, inhibition of hemolysis and favourable effects on QoL were rapidly achieved and sustained to a similar extent in those patients administered SUT throughout the study and those who switched to SUT treatment in Part B. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Complement C1s as a diagnostic marker and therapeutic target: Progress and propective.

The molecules of the complement system connect the effectors of innate and adaptive immunity and play critical roles in maintaining homeostasis. Among them, the C1 complex, composed of C1q, C1r, and C1s (C1qr2s2), is the initiator of the classical complement activation pathway. While deficiency of C1s is associated with early-onset systemic lupus erythematosus and increased susceptibility to bacteria infections, the gain-of- function variants of C1r and C1s may lead to periodontal Ehlers Danlos syndrome. As C1s is activated under various pathological conditions and associated with inflammation, autoimmunity, and cancer development, it is becoming an informative biomarker for the diagnosis and treatment of a variety of diseases. Thus, more sensitive and convenient methods for assessing the level as well as activity of C1s in clinic samples are highly desirable. Meanwhile, a number of small molecules, peptides, and monoclonal antibodies targeting C1s have been developed. Some of them are being evaluated in clinical trials and one of the antibodies has been approved by US FDA for the treatment of cold agglutinin disease, an autoimmune hemolytic anemia. In this review, we will summarize the biological properties of C1s, its association with development and diagnosis of diseases, and recent progress in developing drugs targeting C1s. These progress illustrate that the C1s molecule is an effective biomarker and promising drug target.

Autoantibodies against Complement Classical Pathway Components C1q, C1r, C1s and C1-Inh in Patients with Lupus Nephritis.

Autoantibodies against the complement component C1q (anti-C1q) are among the main biomarkers in lupus nephritis (LN) known to contribute to renal injury. C1q, the recognition subcomponent of the complement classical pathway, forms a heterotetrameric complex with C1r and C1s, and can also associate a central complement regulator and C1 Inhibitor (C1-Inh). However, the frequency and the pathogenic relevance of anti-C1r, anti-C1s and anti-C1-Inh autoantibodies remain poorly studied in LN. In this paper, we screened for anti-C1q, anti-C1r, anti-C1s and anti-C1-Inh autoantibodies and evaluated their association with disease activity and severity in 74 LN patients followed up for 5 years with a total of 266 plasma samples collected. The presence of anti-C1q, anti-C1r, anti-C1s and anti-C1-Inh was assessed by ELISA. IgG was purified by Protein G from antigen-positive plasma and their binding to purified C1q, C1r and C1s was examined by surface plasmon resonance (SPR). The abilities of anti-C1q, anti-C1r and anti-C1s binding IgG on C1 complex formation were analyzed by ELISA. The screening of LN patients’ plasma revealed 14.9% anti-C1q positivity; only 4.2%, 6.9% and 0% were found to be positive for anti-C1r, anti-C1s and anti-C1-Inh, respectively. Significant correlations were found between anti-C1q and anti-dsDNA, and anti-nuclear antibodies, C3 and C4, respectively. High levels of anti-C1q antibodies were significantly associated with renal histologic lesions and correlated with histological activity index. Patients with the most severe disease (A class according to BILAG Renal score) had higher levels of anti-C1q antibodies. Anti-C1r and anti-C1s antibodies did not correlate with the clinical characteristics of the LN patients, did not interfere with the C1 complex formation, and were not measurable via SPR. In conclusion, the presence of anti-C1q, but not anti-C1s or anti-C1r, autoantibodies contribute to the autoimmune pathology and the severity of LN.

Transcriptome Analysis of Monocytes and Fibroblasts Provides Insights Into the Molecular Features of Periodontal Ehlers-Danlos Syndrome.

Periodontal Ehlers–Danlos syndrome (pEDS) is a rare hereditary disorder characterized by severe early-onset periodontitis with premature tooth loss, pretibial hyperpigmentation, and skin fragility. It is caused by mutant variants in the C1R and C1S genes that result in C4 cleavage and local complement cascade activation, as well as other possible consequences. However, the exact functional consequences of this activation remain unclear. To shed light on molecular mechanisms underlying pEDS and to identify novel molecular targets that may expand treatment strategies, we performed transcriptome profiling by RNA sequencing of monocytes and gingival fibroblasts from two patients with pEDS. Compared to normal controls, differential expression of genes was found only in monocytes but not gingival fibroblasts. Most of the significant genes were enriched in biological processes such as neutrophil-mediated immunity, response to bacterium, TNF-α and IL-17 pathway which are related to inflammation response and immune response. In disease ontology enrichment analysis, genes related to periodontal host defense, inflammatory response, skin disease, and vascular development, including MMP9, VEGFA, IL10, IL1A, IL1B, IL2RA, and IL6, were significantly enriched and also validated by qPCR and ELISA. Overall, the present study provides the transcriptomic data of pEDS for the first time and the distinct molecular features in monocytes of pEDS might serve as a tool to better understand the disease.

Complement C1s inhibition with sutimlimab results in durable response in cold agglutinin disease: CARDINAL study 1-year interim follow-up results.

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P053 The intestinal complement system and its role in dysregulation of the intestinal B-cell compartment in Crohn’s Disease patients

Abstract Background For several decades, there is growing evidence that a hyperactive B-cell compartment may be involved in the disturbed mucosal immune response in Crohn’s disease (CD) patients. This is reflected by approximately one third of all CD patients displaying an excessive production of autoantibodies against the intestinal M-cell expressed FimH receptor glycoprotein 2 (GP2) which inhibits induction of adaptive immunity against adherent-invasive flagellated bacteria. However, underlying mechanisms of an imbalanced B-cell compartment in CD patients still remain elusive. Given that the complement system plays a critical role in the detection and clearance of bacteria and promotes B-cell immunity, we asked if the complement system contributes to a dysregulated B-cell response in CD. Methods Systematical expression analyses of the intestinal complement components were performed in IBD patients, including CD and ulcerative colitis (UC) patients, versus non-IBD controls. Additionally, activity of the three activation pathways of complement system was studied in serum samples of CD patients and controls. Further, immunoglobulin levels in intestinal biopsies, sera and fecal samples were analyzed. Results CD patients, but no UC patients, showed increased expression of the early classical complement pathway components C1QA, C1QB, C1S, C2, C3, C4A, CR1 and CR2 in sigmoidal biopsy samples that does not differ significantly between remission and active disease state. This could be verified by immunohistochemistry, displaying an over-representation of mucosal C1q, C3 and B-cell expressed CR2 (also CD21) in colon biopsies of CD patients in remission. In line with this, CD patients in remission present with enhanced activity of the classical as well as alternative pathway of complement activation in serum compared to non-IBD controls. Accordingly, high protein levels of C3 cleavage fragments were detected in fecal samples from CD patients in contrast to non-IBD controls. While there were no differences in systemic immunoglobulin levels in serum, intestinal IgM was increased in CD patients in remission. Notably, fecal IgA levels were strongly diminished in these patients, while expression of the polymeric immunoglobulin receptor was not affected. Conclusion In summary, our findings demonstrate an augmented activation of the intestinal complement system that may potentially be involved in the etiology of CD. Expansion of mucosal IgM+/CR2+ B cells together with a reduced fecal IgA level, points to a locally disturbed induction of intestinal IgA secreting plasma cells in CD patients.

Complement regulation in ovine lymph nodes during early pregnancy.

A fetus changes immune responses in the uterus and the maternal immune system, and lymph nodes are associated with regulating maternal adaptive immunity. Complement activation is associated with abnormal pregnancy in mice and humans. The aim of the present study was to explore the expression levels of complement components in maternal lymph nodes during early pregnancy in sheep. Maternal inguinal lymph nodes were sampled on day 16 of the estrous cycle, and days 13, 16 and 25 of gestation in ewes. Reverse transcription-quantitative PCR, western blotting and immunohistochemical analyses were used to detect the expression levels of complement components C1q, C1r, C1s, C2, C3, C4a, C5b and C9 in the lymph nodes. The results revealed that the protein and mRNA levels of C1q, C1s and C5b were enhanced during early pregnancy, and that C1r and C4a were upregulated at day 25 of pregnancy. The mRNA and protein levels of C2 and C9 peaked at day 16 of pregnancy, but C3 was decreased at day 25 of pregnancy. C3 protein was located in the subcapsular sinuses and lymph sinuses of the maternal lymph node. In summary, the present study detected changes in the expression levels of complement components in maternal lymph nodes, which may be associated with maternal immune regulation during early pregnancy in sheep.

Inhibition of Complement C1s By Sutimlimab in Patients with Cold Agglutinin Disease (CAD): Efficacy and Safety Results from the Randomized, Placebo-Controlled Phase 3 CADENZA Study

Inhibition of Complement C1s By Sutimlimab in Patients with Cold Agglutinin Disease (CAD): Efficacy and Safety Results from the Randomized, Placebo-Controlled Phase 3 CADENZA Study

Effects of early pregnancy on the complement system in the ovine thymus.

The complement system is crucial for the innate immune system, and complement activation is related to abnormal pregnancy in mice and humans. It is hypothesized that the complement system participates in maternal thymic immune regulation during early pregnancy in sheep. In this study, maternal thymuses were sampled on day 16 of the estrous cycle, and days 13, 16 and 25 of gestation in sheep. Quantitative real-time PCR, Western blot and immunohistochemistry analyses were used to analyze the expression of the complement components C1q, C1r, C1s, C2, C3, C4a, C5b and C9 in the maternal thymus. The results revealed that the mRNA and protein expression of C1r, C1s, C2, C3 and C4a was inhibited by early pregnancy, and the pregnancy recognition signal induced upregulation of C1q, C5b and C9 expression at day 16 of gestation. Furthermore, C3 protein was mostly located in epithelial reticular cells and thymic corpuscles, which may be involved in immune regulation. In summary, early pregnancy inhibits the complement system in the maternal thymus, which may be essential for the maternal immune regulation and successful pregnancy in sheep.

Transcriptome Analysis of Post-Mortem Brain Tissue Reveals Up-Regulation of the Complement Cascade in a Subgroup of Schizophrenia Patients.

Schizophrenia is a genetically complex neuropsychiatric disorder with largely unresolved mechanisms of pathology. Identification of genes and pathways associated with schizophrenia is important for understanding the development, progression and treatment of schizophrenia. In this study, pathways associated with schizophrenia were explored at the level of gene expression. The study included post-mortem brain tissue samples from 68 schizophrenia patients and 44 age and sex-matched control subjects. Whole transcriptome poly-A selected paired-end RNA sequencing was performed on tissue from the prefrontal cortex and orbitofrontal cortex. RNA expression differences were detected between case and control individuals, focusing both on single genes and pathways. The results were validated with RT-qPCR. Significant differential expression between patient and controls groups was found for 71 genes. Gene ontology analysis of differentially expressed genes revealed an up-regulation of multiple genes in immune response among the patients (corrected p-value = 0.004). Several genes in the category belong to the complement system, including C1R, C1S, C7, FCN3, SERPING1, C4A and CFI. The increased complement expression is primarily driven by a subgroup of patients with increased expression of immune/inflammatory response genes, pointing to important differences in disease etiology within the patient group. Weighted gene co-expression network analysis highlighted networks associated with both synaptic transmission and activation of the immune response. Our results demonstrate the importance of immune-related pathways in schizophrenia and provide evidence for elevated expression of the complement cascade as an important pathway in schizophrenia pathology.

Blood Plasma Biomarkers for Woody Breast Disease in Commercial Broilers.

Woody breast (WB) myopathy results in poor muscle quality. The increasing incidence of WB over the last several years indicates a need for improved prediction or early diagnosis. We hypothesized that the use of body fluids, including blood, may be more suitable than breast muscle tissue in developing a minimally invasive diagnostic tool for WB detection. To identify potential early-age-biomarkers that may represent the potential onset of WB, blood samples were collected from 100, 4 wks old commercial male broilers. At 8 wks of age, WB conditions were scored by manual palpation. A total of 32 blood plasma samples (eight for each group of WB and non-WB control birds at two time points, 4 wks and 8 wks) were subjected to shotgun proteomics and untargeted metabolomics to identify differentially abundant plasma proteins and metabolites in WB broilers compared to non-WB control (Con) broilers. From the proteomics assay, 25 and 16 plasma proteins were differentially abundant (p < 0.05) in the 4 and 8 wks old samples, respectively, in WB compared with Con broilers. Of those, FRA10A associated CGG repeat 1 (FRAG10AC1) showed >2-fold higher abundance in WB compared with controls. In the 8 wks old broilers, 4 and 12 plasma proteins displayed higher and lower abundances, respectively, in WB compared with controls. Myosin heavy chain 9 (MYH9) and lipopolysaccharide binding protein (LBP) showed more than 2-fold higher abundances in WB compared with controls, while transferrin (TF) and complement C1s (C1S) showed more than 2-fold lower abundances compared with controls. From the untargeted metabolomics assay, 33 and 19 plasma metabolites were differentially abundant in birds at 4 and 8 wks of age, respectively, in WB compared with controls. In 4 wks old broilers, plasma 3-hydroxybutyric acid (3-HB) and raffinose concentrations showed the highest and lowest fold changes, respectively, in WB compared with controls. The blood plasma 3-HB and raffinose concentrations were confirmed with targeted biochemical assays. Blood biomarkers, such as 3-HB and raffinose, may be suitable candidate targets in the prediction of WB onset at early ages.

Identification of Diagnostic Biomarkers and Their Correlation with Immune Infiltration in Age-Related Macular Degeneration.

Age-related macular degeneration (AMD) is a progressive neurodegenerative disease of the central retina, with no suitable biomarkers for early diagnosis and treatment. This study aimed to find potential diagnostic biomarker candidates for AMD and investigate their immune-related roles in this pathology. Weight gene correlation analysis was first performed based on data from the Gene Expression Omnibus database and 20 hub genes were identified. The functional enrichment analyses showed that the innate immune response, inflammatory response, and complement activation were key pathways associated with AMD. Complement C1s (C1S), adrenomedullin (ADM), and immediate early response 5 like (IER5L) were identified as the crucial genes with favorable diagnostic values for AMD by using LASSO analysis and multiple logistic regression. Furthermore, a 3-gene model was constructed and proved to be of good diagnostic and predictive performance for AMD (AUC = 0.785, 0.840, and 0.810 in training, test, and validation set, respectively). Finally, CIBERSORT was used to evaluate the infiltration of immune cells in AMD tissues. The results showed that the NK cells, CD4 memory T cell activation, and macrophage polarization may be involved in the AMD process. C1S, ADM, and IER5L were correlated with the infiltration of the above immune cells. In conclusion, our study suggests that C1S, ADM, and IER5L are promising diagnostic biomarker candidates for AMD and may regulate the infiltration of immune cells in the occurrence and progression of AMD.

Complement C1s and C4d as Prognostic Biomarkers in Renal Cancer: Emergence of Noncanonical Functions of C1s.

The complement system plays a complex role in cancer. In clear cell renal cell carcinoma (ccRCC), local production of complement proteins drives tumor progression, but the mechanisms by which they do this are poorly understood. We found that complement activation, as reflected by high plasma C4d or as C4d deposits at the tumor site, was associated with poor prognosis in two cohorts of patients with ccRCC. High expression of the C4-activating enzyme C1s by tumor cells was associated with poor prognosis in three cohorts. Multivariate Cox analysis revealed that the prognostic value of C1s was independent from complement deposits, suggesting the possibility of complement cascade-unrelated, protumoral functions for C1s. Silencing of C1s in cancer cell lines resulted in decreased proliferation and viability of the cells and in increased activation of T cells in in vitro cocultures. Tumors expressing high levels of C1s showed high infiltration of macrophages and T cells. Modification of the tumor cell phenotype and T-cell activation were independent of extracellular C1s levels, suggesting that C1s was acting in an intracellular, noncanonical manner. In conclusion, our data point to C1s playing a dual role in promoting ccRCC progression by triggering complement activation and by modulating the tumor cell phenotype and tumor microenvironment in a complement cascade-independent, noncanonical manner. Overexpression of C1s by tumor cells could be a new escape mechanism to promote tumor progression.See related Spotlight by Magrini and Garlanda, p. 855. See article by Daugan et al., p. 909 (40).

Complement C1s cleaves PfEMP1 at interdomain conserved sites inhibiting Plasmodium falciparum cytoadherence

Cytoadhesion of Plasmodium falciparum-infected erythrocytes (IEs) to the endothelial lining of blood vessels protects parasites from splenic destruction, but also leads to detrimental inflammation and vessel occlusion. Surface display of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) adhesion ligands exposes them to host antibodies and serum proteins. PfEMP1 are important targets of acquired immunity to malaria, and through evolution, the protein family has expanded and diversified to bind a select set of host receptors through antigenically diversified receptor-binding domains. Here, we show that complement component 1s (C1s) in serum cleaves PfEMP1 at semiconserved arginine motifs located at interdomain regions between the receptor-binding domains, rendering the IE incapable of binding the two main PfEMP1 receptors, CD36 and endothelial protein C receptor (EPCR). Bioinformatic analyses of PfEMP1 protein sequences from 15 P. falciparum genomes found the C1s motif was present in most PfEMP1 variants. Prediction of C1s cleavage and loss of binding to endothelial receptors was further corroborated by testing of several different parasite lines. These observations suggest that the parasites have maintained susceptibility for cleavage by the serine protease, C1s, and provides evidence for a complex relationship between the complement system and the P. falciparum cytoadhesion virulence determinant.

Periodontal (formerly type VIII) Ehlers–Danlos syndrome: Description of 13 novel cases and expansion of the clinical phenotype

Periodontal Ehlers-Danlos syndrome (pEDS) is a rare condition caused by pathogenic variants in the C1R and C1S genes, encoding subunits C1r and C1s of the first component of the classical complement pathway. It is characterized by early-onset periodontitis with premature tooth loss, pretibial hyperpigmentation and skin fragility. Rare arterial complications have been reported, but venous insufficiency is rarely described. Here we report 13 novel patients carrying heterozygous pathogenic variants in C1R and C1S including three novel C1S variants (c.962G > C, c.961 T > G and c.961 T > A). In addition to the pEDS phenotype, three patients and one relative displayed widespread venous insufficiency leading to persistent varicose leg ulcers. One patient suffered an intracranial aneurysm with familial vascular complications including thoracic and abdominal aortic aneurysm and dissection and intracranial aneurysm rupture. This work confirms that vascular complications can occur, although they are not frequent, which leads us to propose to carry out a first complete non-invasive vascular evaluation at the time of the diagnosis in pEDS patients. However, larger case series are needed to improve our understanding of the link between complement pathway activation and connective tissue alterations observed in these patients, and to better assess the frequency, type and consequences of the vascular complications.

Role of intracellular complement activation in kidney fibrosis.

Increased expression of complement C1r, C1s and C3 in kidney cells plays an important role in the pathogenesis of kidney fibrosis. Our studies suggest that activation of complement in kidney cells with increased generation of C3 and its fragments occurs by activation of classical and alternative pathways. Single nuclei RNA sequencing studies in kidney tissue from unilateral ureteral obstruction mice show that increased synthesis of complement C3 and C5 occurs primarily in renal tubular epithelial cells (proximal and distal), while increased expression of complement receptors C3ar1 and C5ar1 occurs in interstitial cells including immune cells like monocytes/macrophages suggesting compartmentalization of complement components during kidney injury. Although global deletion of C3 and macrophage ablation prevent inflammation and reduced kidney tissue scarring, the development of mice with cell-specific deletion of complement components and their regulators could bring further insights into the mechanisms by which intracellular complement activation leads to fibrosis and progressive kidney disease. LINKED ARTICLES: This article is part of a themed issue on Canonical and non-canonical functions of the complement system in health and disease. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.14/issuetoc.

Construction of a competitive endogenous RNA network and analysis of potential regulatory axis targets in glioblastoma

BackgroundGlioblastoma is the most common primary malignant brain tumor. Because of the limited understanding of its pathogenesis, the prognosis of glioblastoma remains poor. This study was conducted to explore potential competing endogenous RNA (ceRNA) network chains and biomarkers in glioblastoma by performing integrated bioinformatics analysis.MethodsTranscriptome expression data from The Cancer Genome Atlas database and Gene Expression Omnibus were analyzed to identify differentially expressed genes between glioblastoma and normal tissues. Biological pathways potentially associated with the differentially expressed genes were explored by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis, and a protein-protein interaction network was established using the STRING database and Cytoscape. Survival analysis using Gene Expression Profiling Interactive Analysis was based on the Kaplan–Meier curve method. A ceRNA network chain was established using the intersection method to align data from four databases (miRTarBase, miRcode, TargetScan, and lncBace2.0), and expression differences and correlations were verified by quantitative reverse-transcription polymerase chain reaction analysis and by determining the Pearson correlation coefficient. Additionally, an MTS assay and the wound-healing and transwell assays were performed to evaluate the effects of complement C1s (C1S) on the viability and migration and invasion abilities of glioblastoma cells, respectively.ResultsWe detected 2842 differentially expressed (DE) mRNAs, 2577 DE long non-coding RNAs (lncRNAs), and 309 DE microRNAs (miRNAs) that were dysregulated in glioblastoma. The final ceRNA network consisted of six specific lncRNAs, four miRNAs, and four mRNAs. Among them, four DE mRNAs and one DE lncRNA were correlated with overall survival (p < 0.05). C1S was significantly correlated with overall survival (p= 0.015). In functional assays, knockdown of C1S inhibited the proliferation and invasion of glioblastoma cell lines.ConclusionsWe established four ceRNA networks that may influence the occurrence and development of glioblastoma. Among them, the MIR155HG/has-miR-129-5p/C1S axis is a potential marker and therapeutic target for glioblastoma. Knockdown of C1S inhibited the proliferation, migration, and invasion of glioblastoma cells. These findings clarify the role of the ceRNA regulatory network in glioblastoma and provide a foundation for further research.

A 44-kb deleted-type copy number variation is associated with decreasing complement component activity and calf mortality in Japanese Black cattle

BackgroundCalf mortality generally occurs in calves prior to weaning, which is a serious problem in cattle breeding. Several causative variants of monogenic Mendelian disorders in calf mortality have been identified, whereas genetic factors affecting the susceptibility of calves to death are not well known. To identify variants associated with calf mortality in Japanese Black cattle, we evaluated calf mortality as a categorical trait with a threshold model and performed a genome-wide copy number variation (CNV) association study on calf mortality.ResultsWe identified a 44-kb deleted-type CNV ranging from 103,317,687 to 103,361,802 bp on chromosome 5, which was associated with the mortality of 1–180-day-old calves. The CNV harbored C1RL, a pseudogene, and an IncRNA localized in the C1R and C1S gene cluster, which is a component of the classical complement activation pathway for immune complexes for infectious pathogens. The average complement activity in CNVR_221 homozygotes at postnatal day 7 was significantly lower than that of wild-type animals and heterozygotes. The frequency of the risk allele in dead calves suffering from diarrhea and pneumonia and in healthy cows was 0.35 and 0.28, respectively (odds ratio = 2.2, P = 0.016), suggesting that CNVR_221 was associated with the mortality of Japanese Black calves suffering from an infectious disease.ConclusionsThis study identified a deleted-type CNV associated with the mortality of 1–180-day-old calves. The complement activity in CNVR_221 homozygotes was significantly lower than that in heterozygotes and wild type animals. The frequency of the risk allele was higher in dead calves suffering from an infectious disease than in healthy cows. These results suggest that the existence of CNVR_221 in calves could be attributed to a reduction in complement activity, which in turn leads to susceptibility to infections. Thus, the risk allele could serve as a useful marker to reduce the mortality of infected Japanese Black calves.

Prospective clinical investigations of children with periodontal Ehlers–Danlos syndrome identify generalized lack of attached gingiva as a pathognomonic feature

PurposeWe report prospective clinical investigations of children affected with periodontal Ehlers–Danlos syndrome (pEDS). The main clinical features of pEDS in adults are early severe periodontitis, generalized lack of attached gingiva, and pretibial hemosiderin plaques due to dominant pathogenic variants in the C1R or C1S genes. MethodsNineteen children with a parent diagnosed with molecularly confirmed pEDS underwent physical examination including oral and radiological investigations followed by genetic testing. ResultsThe only consistent manifestation of pEDS in childhood was a characteristic gingival phenotype: generalized lack of attached gingiva. All children with this gingival phenotype had inherited the familial pathogenic variant (n = 12) whereas the gingival phenotype was absent in children without the familial pathogenic variant (n = 7). Easy bruising was reported in eight affected and zero unaffected children. Other manifestations of pEDS were rarely present in children. Only 2/12 affected children aged 8 and 13 years fulfilled the clinical criteria for pEDS. ConclusionGeneralized lack of attached gingiva is a pathognomonic feature of pEDS and the only clinical finding that is consistently present in affected adults and children. This is important because an early diagnosis may facilitate better dental hygiene in childhood, which may be essential to prevent early dental loss.