Increased plasma level of terminal complement complex in AMD patients: potential functional consequences for RPE cells.

Purpose: Polymorphisms in complement genes are risk-associated for age-related macular degeneration (AMD). Functional analysis revealed a common deficiency to control the alternative complement pathway by risk-associated gene polymorphisms. Thus, we investigated the levels of terminal complement complex (TCC) in the plasma of wet AMD patients with defined genotypes and the impact of the complement activation of their plasma on second-messenger signaling, gene expression, and cytokine/chemokine secretion in retinal pigment epithelium (RPE) cells. Design: Collection of plasma from patients with wet AMD (n = 87: 62% female and 38% male; median age 77 years) and controls (n = 86: 39% female and 61% male; median age 58 years), grouped for risk factor smoking and genetic risk alleles CFH 402HH and ARMS2 rs3750846, determination of TCC levels in the plasma, in vitro analysis on RPE function during exposure to patients' or control plasma as a complement source. Methods: Genotyping, measurement of TCC concentrations, ARPE-19 cell culture, Ca2+ imaging, gene expression by qPCR, secretion by multiplex bead analysis of cell culture supernatants. Main outcome measures: TCC concentration in plasma, intracellular free Ca2+, relative mRNA levels, cytokine secretion. Results: TCC levels in the plasma of AMD patients were five times higher than in non-AMD controls but did not differ in plasma from carriers of the two risk alleles. Complement-evoked Ca2+ elevations in RPE cells differed between patients and controls with a significant correlation between TCC levels and peak amplitudes. Comparing the Ca2+ signals, only between the plasma of smokers and non-smokers, as well as heterozygous (CFH 402YH) and CFH 402HH patients, revealed differences in the late phase. Pre-stimulation with complement patients' plasma led to sensitization for complement reactions by RPE cells. Gene expression for surface molecules protective against TCC and pro-inflammatory cytokines increased after exposure to patients' plasma. Patients' plasma stimulated the secretion of pro-inflammatory cytokines in the RPE. Conclusion: TCC levels were higher in AMD patients but did not depend on genetic risk factors. The Ca2+ responses to patients' plasma as second-messenger represent a shift of RPE cells to a pro-inflammatory phenotype and protection against TCC. We conclude a substantial role of high TCC plasma levels in AMD pathology.

An ex vivo test to investigate genetic factors conferring susceptibility to atypical haemolytic uremic syndrome.

Introduction: Comprehensive genetic analysis is essential to clinical care of patients with atypical haemolytic uremic syndrome (aHUS) to reinforce diagnosis, and to guide treatment. However, the characterization of complement gene variants remains challenging owing to the complexity of functional studies with mutant proteins. This study was designed: 1) To identify a tool for rapid functional determination of complement gene variants; 2) To uncover inherited complement dysregulation in aHUS patients who do not carry identified gene variants. Methods: To address the above goals, we employed an ex-vivo assay of serum-induced C5b-9 formation on ADP-activated endothelial cells in 223 subjects from 60 aHUS pedigrees (66 patients and 157 unaffected relatives). Results: Sera taken from all aHUS patients in remission induced more C5b-9 deposition than control sera, independently from the presence of complement gene abnormalities. To avoid the possible confounding effects of chronic complement dysregulation related to aHUS status, and considering the incomplete penetrance for all aHUS-associated genes, we used serum from unaffected relatives. In control studies, 92.7% of unaffected relatives with known pathogenic variants exhibited positive serum-induced C5b-9 formation test, documenting a high sensitivity of the assay to identify functional variants. The test was also specific, indeed it was negative in all non-carrier relatives and in relatives with variants non-segregating with aHUS. All but one variants in aHUS-associated genes predicted in-silico as likely pathogenic or of uncertain significance (VUS) or likely benign resulted as pathogenic in the C5b-9 assay. At variance, variants in putative candidate genes did not exhibit a functional effect, with the exception of a CFHR5 variant. The C5b-9 assay in relatives was helpful in defining the relative functional effect of rare variants in 6 pedigrees in which the proband carried more than one genetic abnormality. Finally, for 12 patients without identified rare variants, the C5b-9 test in parents unmasked a genetic liability inherited from an unaffected parent. Discussion: In conclusion, the serum-induced C5b-9 formation test in unaffected relatives of aHUS patients may be a tool for rapid functional evaluation of rare complement gene variants. When combined with exome sequencing the assay might be of help in variant selection, to identify new aHUS-associated genetic factors.

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.

Consumption of complement in a 26-year-old woman with severe thrombotic thrombocytopenia after ChAdOx1 nCov-19 vaccination.

Extremely rare reactions characterized by thrombosis and thrombocytopenia have been described in subjects that received ChAdOx1 nCoV-19 vaccination 5-16 days earlier. Although patients with vaccine-induced thrombotic thrombocytopenia (VITT) have high levels of antibodies to platelet factor 4 (PF4)-polyanion complexes, the exact mechanism of the development of thrombosis is still unknown. Here we reported serum studies as well as proteomics and genomics analyses demonstrating a massive complement activation potentially linked to the presence of anti-PF4 antibodies in a patient with severe VITT. At admission, complement activity of the classical and lectin pathways were absent (0% for both) with normal levels of the alternative pathway (73%) in association with elevated levels of the complement activation marker sC5b-9 (630 ng/mL [n.v. 139-462 ng/mL]) and anti-PF4 IgG (1.918 OD [n.v. 0.136-0.300 OD]). The immunoblotting analysis of C2 showed the complete disappearance of its normal band at 110 kDa. Intravenous immunoglobulin treatment allowed to recover complement activity of the classical pathway (91%) and lectin pathway (115%), to reduce levels of sC5b-9 (135 ng/mL) and anti-PF4 IgG (0.681 OD) and to normalize the C2 pattern at immunoblotting. Proteomics and genomics analyses in addition to serum studies showed that the absence of complement activity during VITT was not linked to alterations of the C2 gene but rather to a strong complement activation leading to C2 consumption. Our data in a single patient suggest monitoring complement parameters in other VITT patients considering also the possibility to target complement activation with specific drugs.

Sublytic C5b-9 triggers glomerular mesangial cell proliferation via enhancing FGF1 and PDGFα gene transcription mediated by GCN5-dependent SOX9 acetylation in rat Thy-1 nephritis.

Rat Thy-1 nephritis (Thy-1N) is an animal model of human mesangioproliferative glomerulonephritis (MsPGN), accompanied by glomerular mesangial cell (GMC) proliferation and extracellular matrix (ECM) deposition. Although sublytic C5b-9 formed on GMC membrane could induce cell proliferation, the mechanism is still unclear. In this study, we first demonstrated that the level of SRY related HMG-BOX gene 9 (SOX9), general control nonderepressible 5 (GCN5), fibroblast growth factor 1 (FGF1) and platelet-derived growth factor α (PDGFα) was all elevated both in the renal tissues of Thy-1N rats (in vivo) and in the GMCs (in vitro) with sublytic C5b-9 stimulation. Then, we not only discovered that sublytic C5b-9 caused GMC proliferation through increasing SOX9, GCN5, FGF1 and PDGFα expression, but also proved that SOX9 and GCN5 formed a complex and combined with FGF1 and PDGFα promoters, leading to FGF1 and PDGFα gene transcription. More importantly, GCN5 could mediate SOX9 acetylation at lysine 62 (K62) to enhance SOX9 binding to FGF1 or PDGFα promoter and promote FGF1 or PDGFα synthesis and GMC proliferation. Besides, the experiments in vivo also showed that FGF1 and PDGFα expression, GMC proliferation and urinary protein secretion in Thy-1N rats were greatly reduced by silencing renal SOX9, GCN5, FGF1 or PDGFα gene. Furthermore, the renal tissues of MsPGN patients also exhibited positive expression of these genes mentioned above. Collectively, our findings indicate that GCN5, SOX9 and FGF1/PDGFα can form an axis and play an essential role in sublytic C5b-9-triggered GMC proliferation, which might provide a novel insight into the pathogenesis of Thy-1N and MsPGN.

Bioinformatic investigation for candidate genes and molecular mechanism in the pathogenesis of membranous nephropathy.

AIM: We aimed to explore the detailed molecular mechanism of immune-associated genes in membranous nephropathy (MN). METHODS: A microarray data set (GSE133288) was retrieved from the Gene Expression Omnibus database. Differentially expressed mRNAs (DEMs) in MN vs control groups were identified, and MN-related DEMs (MN-DEMs) were further verified and screened using the comparative toxicogenomics database (CTD) database. The publicly available database, InnateDB was used to investigate immune genes, and the overlapped genes between MN-DEMs and the immune genes were considered as MN-related immune genes (iDEMs). A protein-protein interaction network (PPI) was constructed based on these iDEMs, followed by function and pathway enrichment analysis. Finally, microRNAs (miRNAs) and long noncoding RNAs (lncRNAs) associated with iDEMs were predicted, followed by a lncRNA-miRNA-mRNA (competing endogenous RNAs, ceRNA) network construction. RESULTS: A total of 327 DEMs and 48 iDEMs were revealed; a PPI network was constructed with 100 PPI pairs and 37 iDEMs. iDEMs including JUN and FOS were mainly enriched in pathways such as osteoclast differentiation and function including response to immobilization stress, respectively. Based on mRNA-associated miRNA and lncRNA prediction, 30 ceRNA interactions including KCNQ1OT1-miR-204-5p-SRY-Box Transcription Factor 4 (SOX4) were explored. CONCLUSION: mRNAs including FOS and JUN might participate in MN development via response to immobilization stress function and the osteoclast differentiation pathway. The mRNA SOX4 might contribute to MN progression via sponging KCNQ1OT1-miR-204-5p interaction.

Complement C6 deficiency exacerbates pathophysiology after spinal cord injury.

Historically, the membrane attack complex, composed of complement components C5b-9, has been connected to lytic cell death and implicated in secondary injury after a CNS insult. However, studies to date have utilized either non-littermate control rat models, or mouse models that lack significant C5b-9 activity. To investigate what role C5b-9 plays in spinal cord injury and recovery, we generated littermate PVG C6 wildtype and deficient rats and tested functional and histological recovery after moderate contusion injury using the Infinite Horizon Impactor. We compare the effect of C6 deficiency on recovery of locomotor function and histological injury parameters in PVG rats under two conditions: (1) animals maintained as separate C6 WT and C6-D homozygous colonies; and (2) establishment of a heterozygous colony to generate C6 WT and C6-D littermate controls. The results suggest that maintenance of separate homozygous colonies is inadequate for testing the effect of C6 deficiency on locomotor and histological recovery after SCI, and highlight the importance of using littermate controls in studies involving genetic manipulation of the complement cascade.

An Ancient Molecular Arms Race: Chlamydia vs. Membrane Attack Complex/Perforin (MACPF) Domain Proteins.

Dynamic interactions that govern the balance between host and pathogen determine the outcome of infection and are shaped by evolutionary pressures. Eukaryotic hosts have evolved elaborate and formidable defense mechanisms that provide the basis for innate and adaptive immunity. Proteins containing a membrane attack complex/Perforin (MACPF) domain represent an important class of immune effectors. These pore-forming proteins induce cell killing by targeting microbial or host membranes. Intracellular bacteria can be shielded from MACPF-mediated killing, and Chlamydia spp. represent a successful paradigm of obligate intracellular parasitism. Ancestors of present-day Chlamydia likely originated at evolutionary times that correlated with or preceded many host defense pathways. We discuss the current knowledge regarding how chlamydiae interact with the MACPF proteins Complement C9, Perforin-1, and Perforin-2. Current evidence indicates a degree of resistance by Chlamydia to MACPF effector mechanisms. In fact, chlamydiae have acquired and adapted their own MACPF-domain protein to facilitate infection.

Evolution and Expression of the Membrane Attack Complex and Perforin Gene Family in the Poaceae.

Membrane Attack Complex and Perforin (MACPF) proteins play crucial roles in plant development and plant responses to environmental stresses. To date, only four MACPF genes have been identified in Arabidopsis thaliana, and the functions of the MACPF gene family members in other plants, especially in important crop plants, such as the Poaceae family, remain largely unknown. In this study, we identified and analyzed 42 MACPF genes from six completely sequenced and well annotated species representing the major Poaceae clades. A phylogenetic analysis of MACPF genes resolved four groups, characterized by shared motif organizations and gene structures within each group. MACPF genes were unevenly distributed along the Poaceae chromosomes. Moreover, segmental duplications and dispersed duplication events may have played significant roles during MACPF gene family expansion and functional diversification in the Poaceae. In addition, phylogenomic synteny analysis revealed a high degree of conservation among the Poaceae MACPF genes. In particular, Group I, II, and III MACPF genes were exposed to strong purifying selection with different evolutionary rates. Temporal and spatial expression analyses suggested that Group III MACPF genes were highly expressed relative to the other groups. In addition, most MACPF genes were highly expressed in vegetative tissues and up-regulated by several biotic and abiotic stresses. Taken together, these findings provide valuable information for further functional characterization and phenotypic validation of the Poaceae MACPF gene family.

Genomic Profiling Reveals Distinct Routes To Complement Resistance in Klebsiella pneumoniae.

The serum complement system is a first line of defense against bacterial invaders. Resistance to killing by serum enhances the capacity of Klebsiella pneumoniae to cause infection, but it is an incompletely understood virulence trait. Identifying and characterizing the factors responsible for preventing activation of, and killing by, serum complement could inform new approaches to treatment of K. pneumoniae infections. Here, we used functional genomic profiling to define the genetic basis of complement resistance in four diverse serum-resistant K. pneumoniae strains (NTUH-K2044, B5055, ATCC 43816, and RH201207), and explored their recognition by key complement components. More than 90 genes contributed to resistance in one or more strains, but only three, rfaH, lpp, and arnD, were common to all four strains. Deletion of the antiterminator rfaH, which controls the expression of capsule and O side chains, resulted in dramatic complement resistance reductions in all strains. The murein lipoprotein gene lpp promoted capsule retention through a mechanism dependent on its C-terminal lysine residue; its deletion led to modest reductions in complement resistance. Binding experiments with the complement components C3b and C5b-9 showed that the underlying mechanism of evasion varied in the four strains: B5055 and NTUH-K2044 appeared to bypass recognition by complement entirely, while ATCC 43816 and RH201207 were able to resist killing despite being associated with substantial levels of C5b-9. All rfaH and lpp mutants bound C3b and C5b-9 in large quantities. Our findings show that, even among this small selection of isolates, K. pneumoniae adopts differing mechanisms and utilizes distinct gene sets to avoid complement attack.

Crucial role of the terminal complement complex in chondrocyte death and hypertrophy after cartilage trauma.

OBJECTIVE: Innate immune response and particularly terminal complement complex (TCC) deposition are thought to be involved in the pathogenesis of posttraumatic osteoarthritis. However, the possible role of TCC in regulated cell death as well as chondrocyte hypertrophy and senescence has not been unraveled so far and was first addressed using an ex vivo human cartilage trauma-model. DESIGN: Cartilage explants were subjected to blunt impact (0.59 J) and exposed to human serum (HS) and cartilage homogenate (HG) with or without different potential therapeutics: RIPK1-inhibitor Necrostatin-1 (Nec), caspase-inhibitor zVAD, antioxidant N-acetyl cysteine (NAC) and TCC-inhibitors aurintricarboxylic acid (ATA) and clusterin (CLU). Cell death and hypertrophy/senescence-associated markers were evaluated on mRNA and protein level. RESULTS: Addition of HS resulted in significantly enhanced TCC deposition on chondrocytes and decrease of cell viability after trauma. This effect was potentiated by HG and was associated with expression of RIPK3, MLKL and CASP8. Cytotoxicity of HS could be prevented by heat-inactivation or specific inhibitors, whereby combination of Nec and zVAD as well as ATA exhibited highest cell protection. Moreover, HS+HG exposition enhanced the gene expression of CXCL1, IL-8, RUNX2 and VEGFA as well as secretion of IL-6 after cartilage trauma. CONCLUSIONS: Our findings imply crucial involvement of the complement system and primarily TCC in regulated cell death and phenotypic changes of chondrocytes after cartilage trauma. Inhibition of TCC formation or downstream signaling largely modified serum-induced pathophysiologic effects and might therefore represent a therapeutic target to maintain the survival and chondrogenic character of cartilage cells.

Plasma levels of soluble membrane attack complex are elevated despite viral suppression in HIV patients with poor immune reconstitution.

The complement system is now a therapeutic target for the management of serious and life-threatening conditions such as paroxysmal nocturnal hemoglobinuria, atypical hemolytic uremic syndrome, glomerulonephritis and other diseases caused by complement deficiencies or genetic variants. As complement therapeutics expand into more clinical conditions, monitoring complement activation is increasingly important, as is the baseline levels of complement activation fragments in blood or other body fluid levels. Although baseline complement levels have been reported in the literature, the majority of these data were generated using non-standard assays and with variable sample handling, potentially skewing results. In this study, we examined the plasma and serum levels of the soluble membrane attack complex of complement (sMAC). sMAC is formed in the fluid phase when complement is activated through the terminal pathway. It binds the regulatory proteins vitronectin and/or clusterin and cannot insert into cell membranes, and can serve as a soluble diagnostic marker in infectious disease settings, as previously shown for intraventricular shunt infections. Here we show that in healthy adults, serum sMAC levels were significantly higher than those in plasma, that plasma sMAC levels were similar between in African Americans and Caucasians and that plasma sMAC levels increase with age. Plasma sMAC levels were significantly higher in virally suppressed people living with HIV (PLWH) compared to non-HIV infected healthy donors. More specifically, PLWH with CD4+ T cell counts below 200 had even greater sMAC levels, suggesting diagnostic value in monitoring sMAC levels in this group.

Functional studies of aegerolysin and MACPF-like proteins in Aspergillus niger.

Proteins of the aegerolysin family have a high abundance in Fungi. Due to their specific binding to membrane lipids, and their membrane-permeabilization potential in concert with protein partner(s) belonging to a membrane-attack-complex/perforin (MACPF) superfamily, they were proposed as useful tools in different biotechnological and biomedical applications. In this work, we performed functional studies on expression of the genes encoding aegerolysin and MACPF-like proteins in Aspergillus niger. Our results suggest the sporulation process being crucial for strong induction of the expression of all these genes. However, deletion of either of the aegerolysin genes did not influence the growth, development, sporulation efficiency and phenotype of the mutants, indicating that aegerolysins are not key factors in the sporulation process. In all our expression studies we noticed a strong correlation in the expression of one aegerolysin and MACPF-like gene. Aegerolysins were confirmed to be secreted from the fungus. We also showed the specific interaction of a recombinant A. niger aegerolysin with an invertebrate-specific membrane sphingolipid. Moreover, using this protein labelled with mCherry we successfully stained insect cells membranes containing this particular sphingolipid. Our combined results suggest, that aegerolysins in this species, and probably also in other aspergilli, could be involved in defence against predators.

Glycine, serine and threonine metabolism confounds efficacy of complement-mediated killing.

Serum resistance is a poorly understood but common trait of some difficult-to-treat pathogenic strains of bacteria. Here, we report that glycine, serine and threonine catabolic pathway is down-regulated in serum-resistant Escherichia coli, whereas exogenous glycine reverts the serum resistance and effectively potentiates serum to eliminate clinically-relevant bacterial pathogens in vitro and in vivo. We find that exogenous glycine increases the formation of membrane attack complex on bacterial membrane through two previously unrecognized regulations: 1) glycine negatively and positively regulates metabolic flux to purine biosynthesis and Krebs cycle, respectively. 2) α-Ketoglutarate inhibits adenosine triphosphate synthase, which in together promote the formation of cAMP/CRP regulon to increase the expression of complement-binding proteins HtrE, NfrA, and YhcD. The results could lead to effective strategies for managing the infection with serum-resistant bacteria, an especially valuable approach for treating individuals with weak acquired immunity but a normal complement system.

Complement activation assessed by the plasma terminal complement complex and future risk of venous thromboembolism.

BACKGROUND: It remains uncertain whether activation of the complement system, assessed by the soluble terminal C5b-9 complement complex (plasma TCC), is associated with future risk of incident venous thromboembolism (VTE). OBJECTIVES: To investigate the association between plasma levels of TCC and future risk of incident VTE in a nested case-control study, and to explore genetic variants associated with TCC using protein quantitative trait loci analysis of exome sequencing data. METHODS: We sampled 415 VTE cases and 848 age- and sex-matched controls from a population-based cohort, the Tromsø study. Logistic regression models were used to calculate odds ratios with 95% confidence intervals for VTE across quartiles of plasma levels of TCC. Whole exome sequencing was conducted using the Agilent SureSelect 50 Mb capture kit. RESULTS: The risk of VTE increased across increasing quartiles of plasma TCC, particularly for unprovoked VTE. Participants with TCC in the highest quartile (>1.40 complement arbitrary units/mL) had an odds ratio for unprovoked VTE of 1.74 (95% confidence interval: 1.10-2.78) compared with those with TCC in the lowest quartile (≤0.80 complement arbitrary units/mL) in analyses adjusted for age, sex, and body mass index. A substantially higher risk for VTE was observed in samples taken shortly before VTE event. We found no association between genome-wide or complement-related gene variants and plasma levels of TCC. CONCLUSIONS: We found that high levels of plasma TCC were associated with VTE risk, and unprovoked events in particular. There was no genome-wide association between gene variants and plasma levels of TCC.

Differential contribution of C5aR and C5b-9 pathways to renal thrombic microangiopathy and macrovascular thrombosis in mice carrying an atypical hemolytic syndrome-related factor H mutation.

Atypical hemolytic uremic syndrome (aHUS) is a form of thrombotic microangiopathy (TMA) caused by dysregulated complement activation. Clinically, aHUS is effectively treated by an anti-C5 monoclonal antibody (mAb) but whether the disease is mediated by the C5a receptor (C5aR) or C5b-9 pathway, or both, is unknown. Here we address this in a factor H mutant mouse (FHR/R) which developed complement-mediated TMA as well as macrovascular thrombosis caused by an aHUS-related factor H point mutation (mouse W1206R, corresponding to human W1183R). C5 deficiency and anti-C5 mAb treatment blocked all disease manifestations in FHR/R mice. C5aR1 gene deficiency prevented macrovascular thrombosis in various organs but did not improve survival or reduce renal TMA. Conversely, C6 or C9 deficiency significantly improved survival and markedly diminished renal TMA but did not prevent macrovascular thrombosis. Interestingly, as they aged both FHR/R C6-/- and FHR/R C9-/- mice developed glomerular disease reminiscent of C3 glomerulonephritis. Thus, C5aR and C5b-9 pathways drove different aspects of disease in FHR/R mice with the C5aR pathway being responsible for macrovascular thrombosis and chronic inflammatory injury while the C5b-9 pathway caused renal TMA. Our data provide new understanding of the pathogenesis of complement-mediated TMA and macrovascular thrombosis in FHR/R mice and suggest that C5 blockade is more effective for the treatment of aHUS than selectively targeting the C5aR or C5b-9 pathway alone.

Structure-function characterization of an insecticidal protein GNIP1Aa, a member of an MACPF and ß-tripod families.

The crystal structure of the Gram-negative insecticidal protein, GNIP1Aa, has been solved at 2.5-Å resolution. The protein consists of two structurally distinct domains, a MACPF (membrane attack complex/PerForin) and a previously uncharacterized type of domain. GNIP1Aa is unique in being a prokaryotic MACPF member to have both its structure and function identified. It was isolated from a Chromobacterium piscinae strain and is specifically toxic to Diabrotica virgifera virgifera larvae upon feeding. In members of the MACPF family, the MACPF domain has been shown to be important for protein oligomerization and formation of transmembrane pores, while accompanying domains define the specificity of the target of the toxicity. In GNIP1Aa the accompanying C-terminal domain has a unique fold composed of three pseudosymmetric subdomains with shared sequence similarity, a feature not obvious from the initial sequence examination. Our analysis places this domain into a protein family, named here ß-tripod. Using mutagenesis, we identified functionally important regions in the ß-tripod domain, which may be involved in target recognition.

A C5a-Immunoglobulin complex in chronic lymphocytic leukemia patients is associated with decreased complement activity.

Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in the Western world. The therapeutic approach to CLL includes chemotherapeutic regimens and immunotherapy. Complement-mediated cytotoxicity, which is one of the mechanisms activated by the therapeutic monoclonal antibodies, depends on the availability and activity of the complement (C) system. The aim was to study the structure of circulating C components and evaluate the importance of C5 structural integrity for C activity in CLL patients. Blood samples were collected from 40 naïve CLL patients and 15 normal controls (NC). The Western blot analysis showed abnormal C5 pattern in some CLL patients, while patterns of C3 and C4 were similar in all subjects. Levels of the C activation markers sC5b-9 and C5a were quantified before and after activation via the classical (CP) and alternative (AP) pathways. In patients with abnormal C5, basal levels of sC5b-9 and C5a were increased while activities of the CP and of the CP C5-convertase, the immediate C5-upstream complex, were decreased compared to NC and to patients with normal C5. The data indicate a link between CP activation and apparent C5 alterations in CLL. This provides a potential prognostic tool that may personalize therapy by identifying a sub-group of CLL patients who display an abnormal C5 pattern, high basal levels of sC5b-9 and C5a, and impaired CP activity, and are likely to be less responsive to immunotherapy due to compromised CP activity.

Complement regulatory protein CD59a plays a protective role in immune liver injury of trichloroethylene-sensitized BALB/c mice.

Trichloroethylene (TCE) is a major occupational and environmental chemical compound which causes occupational dermatitis medicamentosa-like of TCE with severe liver damage. Our previous studies showed that complement activation was a newly recognized mechanism for TCE-induced liver damage. The objective of this study was to explore the role of the key complement regulatory protein, CD59a, in TCE-induced immune liver injury. We firstly evaluated the changes of CD59a expression in liver tissue and then investigated if the changes were associated with membrane attack complex (MAC) formation, nuclear factor kappa B (NF-κB) activation and liver damage in BALB/c mice model of TCE-induced skin sensitization in the absence or presence of soluble recombinant rat CD59-Cys. The results showed that low expression of CD59a accompanied by MAC deposition in the liver of TCE-sensitized BALB/c mice, which was consistent in time. In addition, activation of NF-κB pathway, upregulation of inflammatory cytokine and liver damage also occured. Additional experiment showed that recombinant rat sCD59-Cys alleviated inflammation and liver damage in TCE-sensitized BALB/c mice. Moreover, recombinant rat sCD59-Cys reduced MAC formation and inhibited NF-κB activation measured by P-IκBα and nuclear NF-κB p65 in the liver of TCE-sensitized BALB/c mice. In conclusion, recombinant rat sCD59-Cys plays a protective role in immune liver injury of TCE-sensitized BALB/c mice.

Reduced Terminal Complement Complex Formation in Mice Manifests in Low Bone Mass and Impaired Fracture Healing.

The terminal complement complex (TCC) is formed on activation of the complement system, a crucial arm of innate immunity. TCC formation on cell membranes results in a transmembrane pore leading to cell lysis. In addition, sublytic TCC concentrations can modulate various cellular functions. TCC-induced effects may play a role in the pathomechanisms of inflammatory disorders of the bone, including rheumatoid arthritis and osteoarthritis. In this study, we investigated the effect of the TCC on bone turnover and repair. Mice deficient for complement component 6 (C6), an essential component for TCC assembly, and mice with a knockout of CD59, which is a negative regulator of TCC formation, were used in this study. The bone phenotype was analyzed in vivo, and bone cell behavior was analyzed ex vivo. In addition, the mice were subjected to a femur osteotomy. Under homeostatic conditions, C6-deficient mice displayed a reduced bone mass, mainly because of increased osteoclast activity. After femur fracture, the inflammatory response was altered and bone formation was disturbed, which negatively affected the healing outcome. By contrast, CD59-knockout mice only displayed minor skeletal alterations and uneventful bone healing, although the early inflammatory reaction to femur fracture was marginally enhanced. These results demonstrate that TCC-mediated effects regulate bone turnover and promote an adequate response to fracture, contributing to an uneventful healing outcome.