Complement C7 and clusterin form a complex in circulation.

Introduction: The complement system is part of innate immunity and is comprised of an intricate network of proteins that are vital for host defense and host homeostasis. A distinct mechanism by which complement defends against invading pathogens is through the membrane attack complex (MAC), a lytic structure that forms on target surfaces. The MAC is made up of several complement components, and one indispensable component of the MAC is C7. The role of C7 in MAC assembly is well documented, however, inherent characteristics of C7 are yet to be investigated. Methods: To shed light on the molecular characteristics of C7, we examined the properties of serum-purified C7 acquired using polyclonal and novel monoclonal antibodies. The properties of serum­purified C7 were investigated through a series of proteolytic analyses, encompassing Western blot and mass spectrometry. The nature of C7 protein-protein interactions were further examined by a novel enzyme-linked immunosorbent assay (ELISA), as well as size­exclusion chromatography. Results: Protein analyses showcased an association between C7 and clusterin, an inhibitory complement regulator. The distinct association between C7 and clusterin was also demonstrated in serum-purified clusterin. Further assessment revealed that a complex between C7 and clusterin (C7-CLU) was detected. The C7-CLU complex was also identified in healthy serum and plasma donors, highlighting the presence of the complex in circulation. Discussion: Clusterin is known to dissociate the MAC structure by binding to polymerized C9, nevertheless, here we show clusterin binding to the native form of a terminal complement protein in vivo. The presented data reveal that C7 exhibits characteristics beyond that of MAC assembly, instigating further investigation of the effector role that the C7-CLU complex plays in the complement cascade.

The first case report of complement component 7 deficiency in Qatar and a 10-year follow-up.

Introduction: Neisseria meningitidis is a significant cause of bacterial meningitis and septicemia worldwide. Recurrent Neisseria meningitidis is frequently associated with terminal complement protein deficiency, including Complement component 7. This report discusses the first case of C7 deficiency in Qatar. Case report: A 30-year-old Qatari man presented with a meningococcal infection, which was verified by a blood culture. He experienced two episodes of meningitis caused by an undetermined organism. His blood tests revealed low levels of CH50 and C7. His C7 gene testing revealed a homozygous mutation in exon 10 (c.1135G>C p.Gly379Arg), a mutation that has not been previously documented in Qatar. However, it has been observed in 1% of Moroccan-origin Israeli Jews who also exhibit C7 deficiency. Regular prophylactic quadrivalent vaccinations against types A, C, Y, and W-135 with azithromycin tabs were administered. Over the last 10 years of follow-up, he remained in good health, with no further meningitis episodes. Conclusion: To our knowledge, this is the first confirmed case of C7 deficiency reported in the Arabian Gulf countries. Such rare diseases should be a public health priority. Awareness among medical practitioners and the community should help with early detection of C7 deficiency and the prevention of its consequences.

Androgen-regulated stromal complement component 7 (C7) suppresses prostate cancer growth.

The complement system is a major component of the innate immune system that works through the cytolytic effect of the membrane attack complex (MAC). Complement component 7 (C7) is essential for MAC assembly and its precisely regulated expression level is crucial for the cytolytic activity of MAC. We show that C7 is specifically expressed by the stromal cells in both mouse and human prostates. The expression level of C7 inversely correlates with clinical outcomes in prostate cancer. C7 is positively regulated by androgen signaling in the mouse prostate stromal cells. The androgen receptor directly transcriptionally regulates the mouse and human C7. Increasing C7 expression in the C57Bl/6 syngeneic RM-1 and Pten-Kras allografts suppresses tumor growth in vivo. Conversely, C7 haploinsufficiency promotes tumor growth in the transgenic adenocarcinoma of the mouse prostate (TRAMP) model. Interestingly, replenishing C7 in androgen-sensitive Pten-Kras tumors during androgen depletion only slightly enhances cellular apoptosis, highlighting the diverse mechanisms employed by tumors to counteract complement activity. Collectively, our research indicates that augmenting complement activity could be a promising therapeutic approach to impede the development of castration resistance in prostate cancer.

Effects of salinity stress on methylation of the liver genome and complement gene in large yellow croaker (Larimichthys crocea).

Salinity is an important environmental factor that affects the yield and quality of large yellow croaker (Larimichthys crocea) during aquaculture. Here, whole-genome bisulfite sequencing (WGBS), RNA-seq, bisulfite sequencing PCR (BSP), quantitative real-time PCR (qPCR), and dual luciferase reporter gene detection technologies were used to analyze the DNA methylation characteristics and patterns of the liver genome, the expression and methylation levels of important immune genes in large yellow croaker in response to salinity stress. The results of WGBS showed that the cytosine methylation of CG type was dominant, CpGIsland and repeat regions were important regions where DNA methylation occurred, and the DNA methylation in upstream 2k (2000bp upstream of the promoter) and repeat regions had different changes in the liver tissue of large yellow croaker in the response to the 12‰, 24‰, 36‰ salinity stress of 4 w (weeks). In the combined analysis of WGBS and transcriptome, the complement and coagulation cascade pathways were significantly enriched, in which the complement-related genes C7, C3, C5, C4, C1R, MASP1, and CD59 were mainly changed in response to salinity stress. In the studied area of MASP1 gene promoter, the methylation levels of many CpG sites as well as total cytosine were strongly negatively correlated with mRNA expression level. Methylation function analysis of MASP1 promoter further proved that DNA methylation could inhibit the activity of MASP1 promoter, indicating that salinity may affect the expressions of complement-related genes by DNA methylation of gene promoter region.

Comprehensive Update and Revision of Nomenclature on Complement C6 and C7 Variants.

Complement genes encompass a wide array of variants, giving rise to numerous protein isoforms that have often been shown to exhibit clinical significance. Given that these variants have been discovered over a span of 50 y, one challenging consequence is the inconsistency in the terminology used to classify them. This issue is prominently evident in the nomenclature used for complement C6 and C7 variants, for which we observed a great discrepancy between previously published works and variants described in current genome browsers. This report discusses the causes for the discrepancies in C6 and C7 nomenclature and seeks to establish a classification system that would unify existing and future variants. The inconsistency in the methods used to annotate amino acids and the modifications pinpointed in the C6 and C7 primers are some of the factors that contribute greatly to the discrepancy in the nomenclature. Several variants that were classified incorrectly are highlighted in this report, and we showcase first-hand how a unified classification system is important to match previous with current genetic information. Ultimately, we hope that the proposed classification system of nomenclature becomes an incentive for studies on complement variants and their physiological and/or pathological effects.

Birth and death in terminal complement pathway.

The cytolytic activity of the membrane attack complex (MAC) is pivotal in the complement-mediated elimination of pathogens. Terminal complement pathway (TCP) genes encode the proteins that form the MAC. Although the TCP genes are well conserved within most vertebrate species, the early evolution of the TCP genes is poorly understood. Based on the comparative genomic analysis of the early evolutionary history of the TCP homologs, we evaluated four possible scenarios that could have given rise to the vertebrate TCP. Currently available genomic data support a scheme of complex sequential protein domain gains that may be responsible for the birth of the vertebrate C6 gene. The subsequent duplication and divergence of this vertebrate C6 gene formed the C7, C8α, C8ß, and C9 genes. Compared to the widespread conservation of TCP components within vertebrates, we discovered that C9 has disintegrated in the genomes of galliform birds. Publicly available genome and transcriptome sequencing datasets of chicken from Illumina short read, PacBio long read, and Optical mapping technologies support the validity of the genome assembly at the C9 locus. In this study, we have generated a > 120X coverage whole-genome Chromium 10x linked-read sequencing dataset for the chicken and used it to verify the loss of the C9 gene in the chicken. We find multiple CR1 (chicken repeat 1) element insertions within and near the remnant exons of C9 in several galliform bird genomes. The reconstructed chronology of events shows that the CR1 insertions occurred after C9 gene loss in an early galliform ancestor. Loss of C9 in galliform birds, in contrast to conservation in other vertebrates, may have implications for host-pathogen interactions. Our study of C6 gene birth in an early vertebrate ancestor and C9 gene death in galliform birds provides insights into the evolution of the TCP.

Functional genetic variants in complement component 7 confer susceptibility to gastric cancer.

BACKGROUND: Complement system plays an important role in innate immunity which involved in the changes tumor immune microenvironment by mediating the inflammatory response. This study aims to explore the relationship between complement component 7 (C7) polymorphisms and the risk of gastric cancer (GC). MATERIALS AND METHODS: All selected SNPs of C7 were genotyped in 471 patients and 471 controls using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated by unconditional Logistic regression to analyze the relationship between each genotype and the genetic susceptibility to gastric cancer. The level of C7 expression in GC was analyzed by Gene Expression Profiling Interactive Analysis (GEPIA) and detected by Enzyme Linked Immunosorbent Assay. Kaplan-Meier plotter were used to reveal C7 of prognostic value in GC. We examined SNPs associated with the expression of C7 using the GTEx database. The effect of C7 polymorphisms on the regulatory activity of C7 was detected by luciferase reporter assay. RESULTS: Unconditional logistic regression showed that individuals with C7 rs1376178 AA or CA genotype had a higher risk of GC with OR (95% CI) of 2.09 (1.43-3.03) and 1.88 (1.35-2.63), respectively. For C7 rs1061429 C > A polymorphism, AA genotype was associated with the elevated risk for developing gastric cancer (OR = 2.16, 95% CI [1.37-3.38]). In stratified analysis, C7 rs1376178 AA genotype increased the risk of GC among males (OR = 2.88, 95% CI [1.81-4.58]), but not among females (OR = 1.06, 95% CI [0.55-2.06]). Individuals carrying rs1061429 AA significantly increased the risk of gastric cancer among youngers (OR = 2.84, 95% CI [1.39-5.80]) and non-smokers (OR = 2.79, 95% CI [1.63-4.77]). C7 was overexpressed in gastric cancer tissues and serum of cancer patients and was significantly associated with the prognosis. C7 rs1061429 C > A variant contributed to reduced protein level of C7 (P = 0.029), but rs1376178 didn't. Luciferase reporter assay showed that rs1376178C-containing plasmid exhibited 2.86-fold higher luciferase activity than rs1376178 A-containing plasmid (P < 0.001). We also found that rs1061429A allele contributed 1.34-fold increased luciferase activity than rs1061429C allele when co-transfected with miR-591 (P = 0.0012). CONCLUSIONS: These findings highlight the role of C7 in the development of gastric cancer.

Complement component 7 is associated with total- and cardiac death in chest-pain patients with suspected acute coronary syndrome.

BACKGROUND: Complement activation has been associated with atherosclerosis, atherosclerotic plaque destabilization and increased risk of cardiovascular events. Complement component 7 (CC7) binds to the C5bC6 complex which is part of the terminal complement complex (TCC/C5b-9). High-sensitivity C-reactive protein (hsCRP) is a sensitive marker of systemic inflammation and may reflect the increased inflammatory state associated with cardiovascular disease. AIM: To evaluate the associations between CC7 and total- and cardiac mortality in patients hospitalized with chest-pain of suspected coronary origin, and whether combining CC7 with hsCRP adds prognostic information. METHODS: Baseline levels of CC7 were related to 60-months survival in a prospective, observational study of 982 patients hospitalized with a suspected acute coronary syndrome (ACS) at 9 hospitals in Salta, Argentina. A cox regression model, adjusting for conventional cardiovascular risk factors, was fitted with all-cause mortality, cardiac death and sudden cardiac death (SCD) as the dependent variables. A similar Norwegian population of 871 patients was applied to test the reproducibility of results in relation to total death. RESULTS: At follow-up, 173 patients (17.7%) in the Argentinean cohort had died, of these 92 (9.4%) were classified as cardiac death and 59 (6.0%) as SCD. In the Norwegian population, a total of 254 patients (30%) died. In multivariable analysis, CC7 was significantly associated with 60-months all-cause mortality [hazard ratio (HR) 1.26 (95% confidence interval (CI), 1.07-1.47) and cardiac death [HR 1.28 (95% CI 1.02-1.60)], but not with SCD. CC7 was only weakly correlated with hsCRP (r = 0.10, p = 0.002), and there was no statistically significant interaction between the two biomarkers in relation to outcome. The significant association of CC7 with total death was reproduced in the Norwegian population. CONCLUSIONS: CC7 was significantly associated with all-cause mortality and cardiac death at 60-months follow-up in chest-pain patients with suspected ACS. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT01377402, NCT00521976.

Similarities and differences in the structures and proteoform profiles of the complement proteins C6 and C7.

The human complement system provides a first line of defence against pathogens. It requires a well-orchestrated sequential assembly of an array of terminal complement components (C5, C6, C7, C8, and C9), ultimately forming the membrane attack complex (MAC). Although much information about MAC assembly is available, the structure of the soluble C7 has remained elusive. The complement proteins C7 and C6 share very high sequence homology and exhibit several conserved domains, disulphide bridges, and C-mannosylation sites. Here, we used an integrative structural MS-based approach combining native MS, glycopeptide-centric MS, in-gel cross-linking MS (IGX-MS) and structural modelling to describe structural features, including glycosylation, of human serum soluble C7. We compare this data with structural and glycosylation data for human serum C6. The new structural model for C7 shows that it adopts a compact conformation in solution. Although C6 and C7 share many similarities, our data reveals distinct O-, and N-linked glycosylation patterns in terms of location and glycan composition. Cumulatively, our data provide valuable new insight into the structure and proteoforms of C7, solving an essential piece of the puzzle in our understanding of MAC assembly.

A Distinct Innate Immune Signature of Early Onset Colorectal Cancer.

Despite a decrease in the prevalence of colorectal cancer (CRC) over the last 40 y, the prevalence of CRC in people under 50 y old is increasing around the globe. Early onset (≤50 y old) and late onset (≥65 y old) CRC appear to have differences in their clinicopathological and genetic features, but it is unclear if there are differences in the tumor microenvironment. We hypothesized that the immune microenvironment of early onset CRC is distinct from late onset CRC and promotes tumor progression. We used NanoString immune profiling to analyze mRNA expression of immune genes in formalin-fixed paraffin-embedded surgical specimens from patients with early (n = 40) and late onset (n = 39) CRC. We found three genes, SAA1, C7, and CFD, have increased expression in early onset CRC and distinct immune signatures based on the tumor location. After adjusting for clinicopathological features, increased expression of CFD and SAA1 were associated with worse progression-free survival, and increased expression of C7 was associated with worse overall survival. We also performed gain-of-function experiments with CFD and SAA1 in s.c. tumor models and found that CFD is associated with higher tumor volumes, impacted several immune genes, and impacted three genes in mice that were also found to be differentially expressed in early onset CRC (EGR1, PSMB9, and CXCL9). Our data demonstrate that the immune microenvironment, characterized by a distinct innate immune response signature in early onset CRC, is unique, location dependent, and might contribute to worse outcomes.

Recipient C7 rs9292795 genotype and the risk of hepatocellular carcinoma recurrence after orthotopic liver transplantation in a Han Chinese population.

BACKGROUND: Complement component(C7) gene has been shown to influence the prognosis in Hepatocellular carcinoma (HCC) patients. The association between C7 and HCC recurrence after orthotopic liver transplantation (OLT), however, is still unknown. The purpose of this study was to evaluate whether the donor and recipient C7 gene polymorphisms are related to HCC recurrence after OLT in the Han Chinese population. METHODS: A total of 73 consecutive patients with HCC who had undergone OLT, both donors and recipients, were involved in this research. A single nucleotide polymorphism of C7, rs9292795, was genotyped using Sequenom MassARRAY in the cohort. The expression of C7 and the association between C7 gene polymorphisms and HCC recurrence following OLT were analyzed by bioinformatics and statistical analysis, respectively. RESULTS: As shown in database, the expression of C7 was higher in HCC tissues than that in normal tissues, and represented a worse prognosis. We also found that recipient C7 rs9292795 polymorphism, rather than the donor, was significantly associated with HCC recurrence after OLT. Multivariate logistic regression analysis confirmed that TNM stage (P = 0.001), Milan criteria (P = 0.000) and recipient rs9292795 genotype (TT vs AA/AT, P = 0.008) were independent risk factors for HCC recurrence. Furthermore, the recipient carrying AA/AT showed higher recurrence-free survival (RFS) and overall survival (OS) than that carrying TT (P < 0.05). In Cox proportional hazards model, TNM stage, recipient rs9292795 genotype, and Milan criteria were identified as independent factors for RFS and OS (P < 0.05) as well as pre-OLT serum alpha fetoprotein (AFP) level was associated with OS (P < 0.05). CONCLUSIONS: Recipient C7 rs9292795 gene polymorphism is related to the recurrence of HCC after OLT, which may be a helpful prognostic marker for HCC patients who receive OLT.

Disseminated gonococcal infection in a Japanese man with complement 7 deficiency with compound heterozygous variants: A case report.

RATIONALE: Complement deficiency are known to be predisposed to disseminated gonococcal infection (DGI). We herein present a case of DGI involving a Japanese man who latently had a complement 7 deficiency with compound heterozygous variants. PATIENT CONCERNS: A previously healthy 51-year-old Japanese man complained of sudden-onset high fever. Physical examination revealed various skin lesions including red papules on his trunk and extremities, an impetigo-like pustule on left forearm, and tendinitis of his right forefinger. DIAGNOSIS: Blood culture testing detected gram-negative cocci, which was confirmed to be Neisseria gonorrhoeae based on mass spectrometry and a pathogen-specific PCR test. INTERVENTIONS: Screening tests for underlying immunocompromised factors uncovered that complement activities (CH50) was undetectable. With a suspicion of a congenital complement deficiency, genetic analysis revealed rare single nucleotide variants in complement 7 (C7), including c.281-1G>T and a novel variant c.1454C>T (p.A485V). CH50 was normally recovered by adding purified human C7 to the patient's serum, supporting that the patient has C7 deficiency with compound heterozygous variants. OUTCOMES: Under a diagnosis of DGI, the patient underwent an antibiotic treatment with cefotaxime for a week and was discharged without any sequela. LESSONS: DGI is a rare sexually-transmitted infection that potentially induces systemic complications. Complement immunity usually defeats N. gonorrhoeae and prevents the organism from causing DGI. This case highlighted the importance of suspecting a complement deficiency when a person develops DGI.

Rare Genetic Variants in Immune Genes and Neonatal Herpes Simplex Viral Infections.

Neonatal herpes simplex virus (HSV) infection is a devastating disease with high mortality, particularly when disseminated. Studies in adults and children suggest that susceptibility to herpes simplex encephalitis (HSE) may represent phenotypes for inborn errors in toll-like receptor 3 (TLR3) signaling. However, the genetic basis of susceptibility to neonatal HSV including disseminated disease remains unknown. To test the hypothesis that variants in known HSE-susceptible genes as well as genes mediating HSV immunity will be identified in neonatal HSV, we performed an unbiased exome sequencing study in 10 newborns with disseminated, HSE, and skin, eyes, and mouth disease. Determination of potential impact on function was determined by following American College of Medical Genetics and Genomics guidelines. We identified deleterious and potentially deleterious, rare variants in known HSE-related genes including a stop IRF3 variant (disseminated), nonsynonymous variants in TLR3 and TRAF3 (HSE), STAT1 (skin, eyes, and mouth), and DBR1 (disseminated) in our cohort. Novel and rare variants in other immunodeficiency genes or HSV-related immune genes GRB2, RAG2, PRF1, C6, C7, and MSR1 were found in 4 infants. The variant in GRB2, essential for T-lymphocyte cell responses to HSV, is a novel stop variant not found in public databases. In this pilot study, we identified deleterious or potentially deleterious variants in TLR3 pathway and genes that regulate anti-HSV immunity in neonates with HSV including disseminated disease. Larger, definitive studies incorporating functional analysis of genetic variants are required to validate these data and determine the role of immune genetic variants in neonatal HSV susceptibility.

The complement C7 variant rs3792646 is associated with amyotrophic lateral sclerosis in a Han Chinese population.

The complement system has been shown to have a critical pathogenetic role in amyotrophic lateral sclerosis (ALS). Recently a C7 variant in rs3792646 was linked to neurodegenerative diseases in a Chinese population. We used whole exome sequencing to evaluate the role of C7 (rs3792646) in ALS in a Chinese cohort with 1970 individuals. The minor allele frequency in cases was 0.032 while 0.016 in controls, suggesting this variant was associated with ALS. Further analyses showed the prevalence of the variant was significantly higher in Chinese than Caucasian, suggesting its importance in Han individuals. rs3792646-C was significantly associated with a lower onset age in both genders, and a survival analysis revealed a significant relationship between the variant and decreased survival. There was no significant association between the variant and other common ALS-related variants. Our study further elucidated the relationship between the complement system and ALS from a genetic perspective. In addition, the results suggested C7 (rs3792646) could be a potential predictive factor for poor prognosis in ALS.

Evaluation of Serum and Gene Expression of Galectin-4, Interleukin-27, and Complement-7 in Hepatitis C Virus-Infected Egyptian Patients.

BACKGROUND: Hepatitis C virus (HCV) is considered a major global public health problem. Recently, there are great advances in HCV therapy, but there are some limitations that are creating an urgent need for assessment of some cytokines that have a potent antiviral effect in the immune system and anti-inflammatory effects to provide a potential novel immunotherapeutic target in HCV infection. OBJECTIVE: This study was directed to assess the serum levels and gene expression levels of Galectin-4 (LEG4), Interleukin-27 (IL-27), and Complement-7 (C-7) and their correlation with the viral load in HCV infection. Subjects and Methods. This work was conducted on 80 subjects, Group 1 (n = 40) early detected HCV patients and Group 2 (n = 40) healthy controls. LEG4, IL-27, and C-7 were assessed at the protein levels by ELISA, and their gene expression was assessed by RT-qPCR. The viral load was measured by PCR. RESULTS: There were significant elevations in the mean levels of gene expression and serum levels of all studied parameters LEG4, IL-27, and C-7 in the HCV group compared to the control group. Significant negative correlations between the viral load and each of the serum proteins and gene expressions of both LEG4 and IL-27 in HCV patients were found. The gene expression levels of LEG4, IL-27, and C-7 were positively correlated with their corresponding serum proteins in HCV patients. CONCLUSION: LEG4 and IL-27 showed significant negative correlations with the viral load, which could be an immune response to the control of the extent of hepatic inflammation, thus creating a potential novel immunotherapeutic approach in HCV infection for further studies or therapeutic clinical trials.

Comparing serum protein levels can aid in differentiating HPV-negative and -positive oropharyngeal squamous cell carcinoma patients.

BACKGROUND: The surrogate immunohistochemical marker, p16INK4a, is used in clinical practice to determine the high-risk human papillomavirus (HPV) status of oropharyngeal squamous cell carcinomas (OPSCC). With a specificity of 83%, this will misclassify some patients compared with direct HPV testing. Patients who are p16INK4a-positive but HPV DNA-negative, or RNA-negative, may be unsuitable for treatment de-escalation aimed at reducing treatment-related side effects. We aimed to identify cost-effective serum markers to improve decision making for patients at risk of misclassification by p16INK4a alone. METHODS: Serum proteins from pre-treatment samples of 36 patients with OPSCC were identified and quantified using label-free mass spectrometry-based proteomics. HPV-status was determined using p16INK4a/HPV DNA and E6/E7 mRNA. Serum protein expressions were compared between groups of patients according to HPV status, using the unpaired t-test with a Benjamini-Hochberg correction. ROC curves (AUC) were calculated with SPSS (v25). RESULTS: Of 174 serum proteins identified, complement component C7 (C7), apolipoprotein F (ApoF) and galectin-3-Binding Protein (LGALS3BP) significantly differed between HPV-positive and -negative tumors (AUC ranging from 0.84-0.87). ApoF levels were more than twice as high in the E6/E7 mRNA HPV-positive group than HPV-negative. CONCLUSIONS: Serum C7, ApoF and LGALS3BP levels discriminate between HPV-positive and HPV-negative OPSCC. Further studies are needed to validate these host immunity-related proteins as markers for HPV-associated OPSCC.

Proteomic screening of plasma identifies potential noninvasive biomarkers associated with significant/advanced fibrosis in patients with nonalcoholic fatty liver disease.

Noninvasive biomarkers are clinically useful for evaluating liver fibrosis stage in patients with nonalcoholic fatty liver disease (NAFLD). The aim of the present study was to compare plasma proteins in patients with early nonalcoholic steatohepatitis (NASH) (F0-F1) versus NASH with significant/advanced fibrosis (F2-F4) to determine whether candidate proteins could be used as potential noninvasive biomarkers. Nineteen biopsy-proven NAFLD patients including ten early NASH patients and nine NASH patients with significant/advanced fibrosis were enrolled in the present study. High-resolution proteomics screening of plasma was performed with the SCIEX TripleTOF 5600 System. Proteins were quantified using two different software platforms, Progenesis Qi and Scaffold Q+, respectively. Progenesis Qi analysis resulted in the discovery of 277 proteins compared with 235 proteins in Scaffold Q+. Five consensus proteins (i.e. Complement component C7; α-2-macroglobulin; Complement component C8 γ chain; Fibulin-1; α-1-antichymotrypsin) were identified. Complement component C7 was three-fold higher in the NASH group with significant/advanced fibrosis (F2-F4) compared with the early NASH (F0-F1) group (q-value = 3.6E-6). Complement component C7 and Fibulin-1 are positively correlated with liver stiffness (P=0.000, P=0.002, respectively); whereas, Complement component C8 γ chain is negatively correlated (P=0.009). High levels of Complement C7 are associated with NASH with significant/advanced fibrosis and Complement C7 is a perfect classifier of patients included in this pilot study. Further studies will be needed in a larger validation cohort to confirm the utility of complement proteins as biomarkers or mechanistic determinants of NASH with significant/advanced fibrosis.

CryoEM reveals how the complement membrane attack complex ruptures lipid bilayers.

The membrane attack complex (MAC) is one of the immune system's first responders. Complement proteins assemble on target membranes to form pores that lyse pathogens and impact tissue homeostasis of self-cells. How MAC disrupts the membrane barrier remains unclear. Here we use electron cryo-microscopy and flicker spectroscopy to show that MAC interacts with lipid bilayers in two distinct ways. Whereas C6 and C7 associate with the outer leaflet and reduce the energy for membrane bending, C8 and C9 traverse the bilayer increasing membrane rigidity. CryoEM reconstructions reveal plasticity of the MAC pore and demonstrate how C5b6 acts as a platform, directing assembly of a giant ß-barrel whose structure is supported by a glycan scaffold. Our work provides a structural basis for understanding how ß-pore forming proteins breach the membrane and reveals a mechanism for how MAC kills pathogens and regulates cell functions.

The utility of complement assays in clinical immunology: A comprehensive review.

The multi-tasking organ liver, which is the major synthesis site of most serum proteins, supplies humoral components of the innate, - including proteins of the complement system; and, less intensely, also of the acquired immune system. In addition to hepatocyte origins, C1q, factor D, C3, C7 and other protein components of the complement system are produced at various body locations by monocytes/macrophages, lymphocytes, adipocytes, endometrium, enterocytes, keratinocytes and epithelial cells; but the contribution of these alternate sites to the total serum concentrations is slight. The two major exceptions are factor D, which cleaves factor B of the alternative pathway derived largely from adipocytes, and C7, derived largely from polymorphonuclear leukocytes and monocytes/macrophages. Whereas the functional meaning of the extrahepatic synthesis of factor D remains to be elucidated, the local contribution of C7 may up- or downregulate the complement attack. The liver, however, is not classified as part of the immune system but is rather seen as victim of autoimmune diseases, a point that needs apology. Recent histological and cell marker technologies now turn the hands to also conceive the liver as proactive autoimmune disease catalyst. Hosting non-hepatocytic cells, e.g. NK cells, macrophages, dendritic cells as well as T and B lymphocytes, the liver outreaches multiple sites of the immune system. Immunopharmacological follow up of liver transplant recipients teaches us on liver-based presence of ABH-glycan HLA phenotypes and complement mediated ischemia/regeneration processes. In clinical context, the adverse reactions of the complement system can now be curbed by specific drug therapy. This review extends on the involvement of the complement system in liver autoimmune diseases and should allow to direct therapeutic opportunities.

Complement 7 Is Up-Regulated in Human Early Diabetic Kidney Disease.

There is a temporal window from the time diabetes is diagnosed to the appearance of overt kidney disease during which time the disease progresses quietly without detection. Currently, there is no way to detect early diabetic nephropathy (EDN). Herein, we performed an unbiased assessment of gene-expression analysis of postmortem human kidneys to identify candidate genes that may contribute to EDN. We then studied one of the most promising differentially expressed genes in both kidney tissue and blood samples. Differential transcriptome analysis of EDN kidneys and matched nondiabetic controls showed alterations in five canonical pathways, and among them the complement pathway was the most significantly altered. One specific complement pathway gene, complement 7 (C7), was significantly elevated in EDN kidney. Real-time PCR confirmed more than a twofold increase of C7 expression in EDN kidneys compared with controls. Changes in C7 gene product level were confirmed by immunohistochemistry. C7 protein levels were elevated in proximal tubules of EDN kidneys. Serum C7 protein levels were also measured in EDN and control donors. C7 levels were significantly higher in EDN serum than control serum. This latter finding was independently confirmed in a second set of blood samples from a previously collected data set. Together, our data suggest that C7 is associated with EDN, and can be used as a molecular target for detection and/or treatment of EDN.