Triple-fusion protein (TriFu): A potent, targeted, enzyme-like inhibitor of all three complement activation pathways.

The introduction of a therapeutic anti-C5 antibody into clinical practice in 2007 inspired a surge into the development of complement-targeted therapies. This has led to the recent approval of a C3 inhibitory peptide, an antibody directed against C1s and a full pipeline of several complement inhibitors in preclinical and clinical development. However, no inhibitor is available that efficiently inhibits all three complement initiation pathways and targets host cell surface markers as well as complement opsonins. To overcome this, we engineered a novel fusion protein combining selected domains of the three natural complement regulatory proteins decay accelerating factor, factor H and complement receptor 1. Such a triple fusion complement inhibitor (TriFu) was recombinantly expressed and purified alongside multiple variants and its building blocks. We analyzed these proteins for ligand binding affinity and decay acceleration activity by surface plasmon resonance. Additionally, we tested complement inhibition in several in vitro/ex vivo assays using standard classical and alternative pathway restricted hemolysis assays next to hemolysis assays with paroxysmal nocturnal hemoglobinuria erythrocytes. A novel in vitro model of the alternative pathway disease C3 glomerulopathy was established to evaluate the potential of the inhibitors to stop C3 deposition on endothelial cells. Next to the novel engineered triple fusion variants which inactivate complement convertases in an enzyme-like fashion, stoichiometric complement inhibitors targeting C3, C5, factor B, and factor D were tested as comparators. The triple fusion approach yielded a potent complement inhibitor that efficiently inhibits all three complement initiation pathways while targeting to surface markers.

Complement-membrane regulatory proteins are absent from the nodes of Ranvier in the peripheral nervous system.

BACKGROUND: Homozygous CD59-deficient patients manifest with recurrent peripheral neuropathy resembling Guillain-Barré syndrome (GBS), hemolytic anemia and recurrent strokes. Variable mutations in CD59 leading to loss of function have been described and, overall, 17/18 of patients with any mutation presented with recurrent GBS. Here we determine the localization and possible role of membrane-bound complement regulators, including CD59, in the peripheral nervous systems (PNS) of mice and humans. METHODS: We examined the localization of membrane-bound complement regulators in the peripheral nerves of healthy humans and a CD59-deficient patient, as well as in wild-type (WT) and CD59a-deficient mice. Cross sections of teased sciatic nerves and myelinating dorsal root ganglia (DRG) neuron/Schwann cell cultures were examined by confocal and electron microscopy. RESULTS: We demonstrate that CD59a-deficient mice display normal peripheral nerve morphology but develop myelin abnormalities in older age. They normally express myelin protein zero (P0), ankyrin G (AnkG), Caspr, dystroglycan, and neurofascin. Immunolabeling of WT nerves using antibodies to CD59 and myelin basic protein (MBP), P0, and AnkG revealed that CD59 was localized along the internode but was absent from the nodes of Ranvier. CD59 was also detected in blood vessels within the nerve. Finally, we show that the nodes of Ranvier lack other complement-membrane regulatory proteins, including CD46, CD55, CD35, and CR1-related gene-y (Crry), rendering this area highly exposed to complement attack. CONCLUSION: The Nodes of Ranvier lack CD59 and are hence not protected from complement terminal attack. The myelin unit in human PNS is protected by CD59 and CD55, but not by CD46 or CD35. This renders the nodes and myelin in the PNS vulnerable to complement attack and demyelination in autoinflammatory Guillain-Barré syndrome, as seen in CD59 deficiency.

Human papillomavirus-mediated expression of complement regulatory proteins in human cervical cancer cells.

OBJECTIVES: This study aimed to evaluate the expression pattern of complement regulatory proteins (CRPs) CD46, CD59, and CD55 in HPV-positive (HPV+) & negative (HPV-) cervical cancer cell lines in search of a reliable differential biomarker. STUDY DESIGN: We analysed the expression of CRPs in HPV 16-positive SiHa cell line, HPV 18-positive HeLa cell line, and HPV-negative cell line C33a using RT-qPCR, Western blotting, flow cytometry, and confocal microscopy. RESULTS: We observed a differential expression profile of CRPs in HPV+ and HPV- cervical cancer cell lines. The mRNA level of CD59 & CD55 showed a higher expression pattern in HPV+ cells when compared to HPV- cancer cells. However, flow cytometry-based experiments revealed that CD46 was preferentially expressed more in HPV 16-positive SiHa cells followed by HPV 18-positive HeLa cells when compared to HPV- C33a cells. Interestingly, confocal microscopy revealed a high level of CD59 expression in Hela cells and SiHa cells but low expression in HPV- C33a cells. In addition, HPV 18-positive HeLa cells expressed more CD55, which was lower in SiHa cells and very weak in C33a cells. CONCLUSION: The study demonstrates the differential expression of CRPs in both HPV+ and HPV- cervical cancer cells for the first time, and their potential to serve as an early diagnostic marker for cervical carcinogenesis.

Knockdown of membrane-bound complement regulatory proteins suppresses colon cancer growth in mice through inducing tumor cell apoptosis.

CD46, CD55 and CD59 are membrane-bound complement regulatory proteins (mCRPs) and highly expressed in many tumor tissues. Our analysis by RNA sequencing and qRT-PCR revealed that the expression of mCRPs was significantly elevated in cancer tissues of 15 patients with colon cancer. To further investigate the role of mCRPs in the development of colon cancer, we suppressed the expression of mCRPs by CD46-shRNA, CD55-shRNA and CD59-shRNA in colon cancer cell lines, SW620 and HT-29 cells. The results indicated that CD46-shRNA, CD55-shRNA and CD59-shRNA effectively reduced the expression of mCRPs, accompanied with the increased LDH release and the percentage of Annexin V + 7-AAD- early phase of apoptotic cells. The similar cytotoxic effects were also observed in the cells treated with CD46 neutralizing antibody (aCD46), associated with the increased C5b-9 deposition, cleaved caspase-3 and Bax expression in the treated cells. The cytotoxic effects by mCRPs knock-down were potentiated in the cells co-treated with doxorubicin (Dox). In addition, STAT3, STAT6, and p38 MAPK inhibitors, including C188-9, AS1517499 and SB203580 effectively reduced the expression of CD46 in the treated colon cells, associated with increased cell apoptosis and LDH release. Further study with mouse model revealed that mCRPs knockdown by mCRPs-shRNA significantly reduced colon cancer growth, associated with increased expression of Bax, cleaved caspase-3 and C5b-9 deposition, but reduced expression of Bcl-2, IL-6 and IL-1beta in tumor tissues of nude mice transplanted with SW620 cells. Thereby, mCRPs expression in human colon cancer cells were upregulated by STAT3/STAT6/p38 MAPK signaling and mCRPs knockdown reduced colon cancer growth in mice through inducing tumor cell apoptosis.

miR-132-3p regulates antibody-mediated complement-dependent cytotoxicity in colon cancer cells by directly targeting CD55.

The overexpression of membrane-bound complement regulatory proteins (mCRPs) on tumour cells helps them survive complement attacks by suppressing antibody-mediated complement-dependent cytotoxicity (CDC). Consequently, mCRP overexpression limits monoclonal antibody drug immune efficacy. CD55, an mCRP, plays an important role in inhibiting antibody-mediated CDC. However, the mechanisms regulating CD55 expression in tumour cells remain unclear. Here, the aim was to explore CD55-targeting miRNAs. We previously constructed an in vitro model comprising cancer cell lines expressing α-gal and serum containing natural antibodies against α-gal and complement. This was used to simulate antibody-mediated CDC in colon cancer cells. We screened microRNAs that directly target CD55 using LoVo and Ls-174T colon cell lines, which express CD55 at low and high levels, respectively. miR-132-3p expression was dramatically lower in Ls-174T cells than in LoVo cells. miR-132-3p overexpression or inhibition transcriptionally regulated CD55 expression by specifically targeting its mRNA 3'-untranslated regions. Further, miR-132-3p modulation regulated colon cancer cell sensitivity to antibody-mediated CDC through C5a release and C5b-9 deposition. Moreover, miR-132-3p expression was significantly reduced, whereas CD55 expression was increased, in colon cancer tissues compared to levels in adjacent normal tissues. CD55 protein levels were negatively correlated with miR-132-3p expression in colon cancer tissues. Our results indicate that miR-132-3p regulates colon cancer cell sensitivity to antibody-mediated CDC by directly targeting CD55. In addition, incubating the LoVo human tumour cell line, stably transfected with the xenoantigen α-gal, with human serum containing natural antibodies comprises a stable and cheap in vitro model to explore the mechanisms underlying antibody-mediated CDC.

CD55-deficiency in Jews of Bukharan descent is caused by the Cromer blood type Dr(a-) variant.

The complement system regulator CD55 was initially found to carry the Cromer blood group system antigens, and its complete loss of function was subsequently revealed to cause a severe monogenic gastrointestinal syndrome characterized by protein-losing enteropathy and susceptibility to venous thrombosis. Here we present homozygosity to the CD55 c.596C>T; p.Ser199Leu variant, which was previously described as the Cromer Dr(a-) genotype, in two Bukharan Jewish CD55-deficiency patients with variable disease severity. We confirm that this missense variant causes aberrant splicing and deletion of 44 bp in exon 5, leading to premature termination and low expression of the CD55 protein. Furthermore, Patient 1 exhibited a mildly abnormal B cell immunophenotyping profile. By population screening we established that this variant is highly prevalent in the Bukharan Jewish population, with a carrier frequency of 1:17, suggesting that many similar patients are un- or mis-diagnosed. The phenotypic variability, ranging from abdominal pain when eating a high-fat diet to the full CD55-deficiency phenotype, is likely related to modifiers affecting the proportion of the variant that is able to escape aberrant splicing. Establishing the diagnosis of CD55-deficiency in a timely manner, even in patients with milder symptoms, may have a critical effect on their management and quality-of-life since treatment with the complement inhibitor eculizumab is highly effective in ameliorating disease manifestations. Awareness of founder mutations within certain populations can further guide genetic testing and prevent a diagnostic odyssey, by placing this CD55 variant high on the differential diagnosis.

Silencing EGFR-upregulated expression of CD55 and CD59 activates the complement system and sensitizes lung cancer to checkpoint blockade.

The complement system is a critical immune component, yet its role in tumor immune evasion and CD8+ T cell activation is not clearly defined. Here, we demonstrate that epidermal growth factor receptor (EGFR)/Wnt signaling induces ß-catenin-mediated long noncoding RNA (lncRNA) LINC00973 expression to sponge CD55-targeting miR-216b and CD59-targeting miR-150. The consequently upregulated CD55/CD59 expression suppresses the complement system and cytokine secretion required for CD8+ T cell activation. CD55/CD59-neutralizing antibody treatment or mutation of the LINC00973 promoter activates the complement and CD8+ T cells, inhibiting tumor growth. Importantly, combined anti-CD55/CD59 and anti-programmed death 1 (anti-PD-1) antibody treatments elicit a synergistic tumor-inhibiting effect. In addition, CD55/CD59 levels are inversely correlated with infiltration of M1 macrophages and CD8+ T cells in human lung cancer specimens and predict patient outcome. These findings underscore the critical role of EGFR/Wnt/ß-catenin-upregulated CD55/CD59 expression in inhibiting the complement and CD8+ T cell activation for tumor immune evasion and immune checkpoint blockade resistance and identify a potential combination therapy to overcome these effects.

Overexpression of Decay Accelerating Factor Mitigates Fibrotic Responses to Lung Injury.

CD55 or decay accelerating factor (DAF), a ubiquitously expressed glycosylphosphatidylinositol (GPI)-anchored protein, confers a protective threshold against complement dysregulation which is linked to the pathogenesis of idiopathic pulmonary fibrosis (IPF). Since lung fibrosis is associated with downregulation of DAF, we hypothesize that overexpression of DAF in fibrosed lungs will limit fibrotic injury by restraining complement dysregulation. Normal primary human alveolar type II epithelial cells (AECs) exposed to exogenous complement 3a or 5a, and primary AECs purified from IPF lungs demonstrated decreased membrane-bound DAF expression with concurrent increase in the endoplasmic reticulum (ER) stress protein, ATF6. Increased loss of extracellular cleaved DAF fragments was detected in normal human AECs exposed to complement 3a or 5a, and in lungs of IPF patients. C3a-induced ATF6 expression and DAF loss was inhibited using pertussis toxin (an enzymatic inactivator of G-protein coupled receptors), in murine AECs. Treatment with soluble DAF abrogated tunicamycin-induced C3a secretion and ER stress (ATF6 and BiP expression) and restored epithelial cadherin. Bleomycin-injured fibrotic mice subjected to lentiviral overexpression of DAF demonstrated diminished levels of local collagen deposition and complement activation. Further analyses showed diminished release of DAF fragments, as well as reduction in apoptosis (TUNEL and caspase 3/7 activity), and ER stress-related transcripts. Loss-of-function studies using Daf1 siRNA demonstrated worsened lung fibrosis detected by higher mRNA levels of Col1a1 and epithelial injury-related Muc1 and Snai1, with exacerbated local deposition of C5b-9. Our studies provide a rationale for rescuing fibrotic lungs via DAF induction that will restrain complement dysregulation and lung injury.

The active form of Helicobacter pylori vacuolating cytotoxin induces decay-accelerating factor CD55 in association with intestinal metaplasia in the human gastric mucosa.

High-level expression of decay-accelerating factor, CD55, has previously been found in human gastric cancer (GC) and intestinal metaplasia (IM) tissues. Therapeutic effects of CD55 inhibition in cancer have been reported. However, the role of Helicobacter pylori infection and virulence factors in the induction of CD55 and its association with histological changes of the human gastric mucosa remain incompletely understood. We hypothesised that CD55 would be increased during infection with more virulent strains of H. pylori, and with more marked gastric mucosal pathology. RT-qPCR and immunohistochemical analyses of gastric biopsy samples from 42 H. pylori-infected and 42 uninfected patients revealed that CD55 mRNA and protein were significantly higher in the gastric antrum of H. pylori-infected patients, and this was associated with the presence of IM, but not atrophy, or inflammation. Increased gastric CD55 and IM were both linked with colonisation by vacA i1-type strains independently of cagA status, and in vitro studies using isogenic mutants of vacA confirmed the ability of VacA to induce CD55 and sCD55 in gastric epithelial cell lines. siRNA experiments to investigate the function of H. pylori-induced CD55 showed that CD55 knockdown in gastric epithelial cells partially reduced IL-8 secretion in response to H. pylori, but this was not due to modulation of bacterial adhesion or cytotoxicity. Finally, plasma samples taken from the same patients were analysed for the soluble form of CD55 (sCD55) by ELISA. sCD55 levels were not influenced by IM and did not correlate with gastric CD55 mRNA levels. These results suggest a new link between active vacA i1-type H. pylori, IM, and CD55, and identify CD55 as a molecule of potential interest in the management of IM as well as GC treatment. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

3D analysis of CROMER (DAF) and a new antigen CRAG.

BACKGROUND: Cromer antigens are carried on decay accelerating factor (DAF, CD55), for which the crystal structure is available. We investigated two samples with an unidentified antibody to a high prevalence antigen and evaluate the location and characteristics of amino acids associated with antigens on the CD55 by 3D modelling. MATERIALS AND METHODS: Antigen typing and antibody identification were by standard methods. CD55 was sequenced, and Cromer variants were generated using the protein's crystal structure (1OK3, chain A). Antigen-associated residues and intraprotein interactions were investigated in 3D (Naccess, Protein Interactions Calculator). RESULTS: The antibody in the sample from a woman of Kashmiri descent was identified as anti-IFC (anti-CROM7). Her RBCs were negative for high-prevalence Cromer antigens including IFC. CD55 sequencing revealed a silent c.147G>A (p.Leu49=) and c.148G>T (p.Glu50Ter) changes, designated CROM*01N.05. The antibody in the sample from a woman of Greek ancestry was only compatible with IFC- RBCs but her RBCs were positive for known high-prevalence Cromer antigens. CD55 sequencing found she was homozygous for c.173A>G (p.Asp58Gly). The high prevalence antigen was named CRAG (ISBT CROM18 or 021018) and the allele designated CROM*01.-18. By 3D analysis, all known antigen-associated residues, including the new CRAG antigen, were exposed at the protein surface. Interactions between antigen-associated residues within the same CD55 domains were identified. DISCUSSION: Identification of antibodies to high prevalence Cromer antigens can be challenging. The surface exposure of antigen-associated residues likely accounts for their immunogenicity. 3D analysis of CD55 provides insight into previous serologic observations regarding the influence of some Cromer antigens on the expression of others.

Upregulation of Checkpoint Ligand Programmed Death-Ligand 1 in Patients with Paroxysmal Nocturnal Hemoglobinuria Explained by Proximal Complement Activation.

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare hemolytic disease driven by impaired complement regulation. Mutations in genes encoding the enzymes that build the GPI anchors are causative, with somatic mutations in the PIG-A gene occurring most frequently. As a result, the important membrane-bound complement regulators CD55 and CD59 are missing on the affected hematopoietic stem cells and their progeny, rendering those cells vulnerable to complement attack. Immune escape mechanisms sparing affected PNH stem cells from removal are suspected in the PNH pathogenesis, but molecular mechanisms have not been elucidated. We hypothesized that exuberant complement activity in PNH results in enhanced immune checkpoint interactions, providing a molecular basis for the potential immune escape in PNH. In a series of PNH patients, we found increased expression levels of the checkpoint ligand programmed death-ligand 1 (PD-L1) on granulocytes and monocytes, as well as in the plasma of PNH patients. Mechanistically, we demonstrate that complement activation leading to the decoration of particles/cells with C3- and/or C4-opsonins increased PD-L1 expression on neutrophils and monocytes as shown for different in vitro models of classical or alternative pathway activation. We further establish in vitro that complement inhibition at the level of C3, but not C5, inhibits the alternative pathway-mediated upregulation of PD-L1 and show by means of soluble PD-L1 that this observation translates into the clinical situation when PNH patients are treated with either C3 or C5 inhibitors. Together, the presented data show that the checkpoint ligand PD-L1 is increased in PNH patients, which correlates with proximal complement activation.

The genetic and epigenetic regulation of CD55 and its pathway analysis in colon cancer.

Background: CD55 plays an important role in the development of colon cancer. This study aims to evaluate the expression of CD55 in colon cancer and discover how it is regulated by transcriptional factors and miRNA. Methods: The expression of CD55 was explored by TIMER2.0, UALCAN, and Human Protein Atlas (HPA) databases. TRANSFAC and Contra v3 were used to predict the potential binding sites of transcription factors in the CD55 promoter. TargetScan and starBase v2.0 were used to predict the potential binding ability of miRNAs to the 3' untranslated region (3'UTR) of CD55. SurvivalMeth was used to explore the differentially methylated sites in the CD55 promoter. Western blotting was used to detect the expression of TFCP2 and CD55. Dual-luciferase reporter assay and chromatin immunoprecipitation (ChIP) assay were performed to determine the targeting relationship of TFCP2, NF-κB, or miR-27a-3p with CD55. CD55-related genes were explored by constructing a protein-protein interaction (PPI) network and performing pathway analysis by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). Results: CD55 was highly expressed in colon cancer tissues. The mRNA and protein expression levels of TFCP2 were reduced by si-TFCP2. NF-κB mRNA was obviously reduced by NF-κB inhibitor and increased by NF-κB activator. CD55 protein was also inhibited by miR-27a-3p. Dual-luciferase reporter assays showed that after knocking down TFCP2 or inhibiting NF-κB, the promoter activity of CD55 was decreased by 21% and 70%, respectively; after activating NF-κB, the promoter activity of CD55 increased by 2.3 times. As TFCP2 or NF-κB binding site was mutated, the transcriptional activity of CD55 was significantly decreased. ChIP assay showed that TFCP2 and NF-κB combined to the promoter of CD55. The luciferase activity of CD55 3'UTR decreased after being co-transfected with miR-27a-3p mimics and increased by miR-27a-3p antagomir. As the miR-27a-3p binding site was mutated, we did not find any significant effect of miR-27a-3p on reporter activity. PPI network assay revealed a set of CD55-related genes, which included CFP, CFB, C4A, and C4B. GO and KEGG analyses revealed that the target genes occur more frequently in immune-related pathways. Conclusion: Our results indicated that CD55 is regulated by TFCP2, NF-κB, miR-27a-3p, and several immune-related genes, which in turn affects colon cancer.

The Cromer blood group system: an update.

This update of the Cromer (CROM) blood group system (Storry JR, Reid ME, Yazer MH. The Cromer blood group system: a review. Immunohematology 2010;26:109-17) includes additional variants to the Cromer system (ISBT021), both new antigens and new molecular bases underlying the null phenotype. The molecule on which the Cromer blood group antigens are carried, CD55 (DAF), is an important receptor for the malaria parasite, Plasmodium falciparum, and the role of CD55 in health and disease continues to expand.This update of the Cromer (CROM) blood group system (Storry JR, Reid ME, Yazer MH. The Cromer blood group system: a review. Immunohematology 2010;26:109­17) includes additional variants to the Cromer system (ISBT021), both new antigens and new molecular bases underlying the null phenotype. The molecule on which the Cromer blood group antigens are carried, CD55 (DAF), is an important receptor for the malaria parasite, Plasmodium falciparum, and the role of CD55 in health and disease continues to expand.

Hemopexin Modulates Expression of Complement Regulatory Proteins in Rat Glomeruli.

In systemic hemolysis and in hematuric forms of kidney injury, the major heme scavenging protein, hemopexin (HPX), becomes depleted, and the glomerular microvasculature (glomeruli) is exposed to high concentrations of unbound heme, which, in addition to causing oxidative injury, can activate complement cascades; thus, compounding extent of injury. It is unknown whether unbound heme can also activate specific complement regulatory proteins that could defend against complement-dependent injury. Isolated rat glomeruli were incubated in media supplemented with HPX-deficient (HPX-) or HPX-containing (HPX+) sera as a means of achieving different degrees of heme partitioning between incubation media and glomerular cells. Expression of heme oxygenase (HO)-1 and of the complement activation inhibitors, decay-accelerating factor (DAF), CD59, and complement receptor-related gene Y (Crry), was assessed by western blot analysis. Expression of HO-1 and of the GPI-anchored DAF and CD59 proteins increased in isolated glomeruli incubated with HPX- sera with no effect on Crry expression. Exogenous heme (hemin) did not further induce DAF but increased Crry expression. HPX modulates heme-mediated induction of complement activation controllers in glomeruli. This effect could be of translational relevance in glomerular injury associated with hematuria.

New Insights into Xenotransplantation for Cartilage Repair: Porcine Multi-Genetically Modified Chondrocytes as a Promising Cell Source.

Transplantation of xenogenic porcine chondrocytes could represent a future strategy for the treatment of human articular cartilage defects. Major obstacles are humoral and cellular rejection processes triggered by xenogenic epitopes like α-1,3-Gal and Neu5Gc. Besides knockout (KO) of genes responsible for the biosynthesis of respective epitopes (GGTA1 and CMAH), transgenic expression of human complement inhibitors and anti-apoptotic as well as anti-inflammatory factors (CD46, CD55, CD59, TNFAIP3 and HMOX1) could synergistically prevent hyperacute xenograft rejection. Therefore, chondrocytes from different strains of single- or multi-genetically modified pigs were characterized concerning their protection from xenogeneic complement activation. Articular chondrocytes were isolated from the knee joints of WT, GalTKO, GalT/CMAH-KO, human CD59/CD55//CD46/TNFAIP3/HMOX1-transgenic (TG), GalTKO/TG and GalT/CMAHKO/TG pigs. The tissue-specific effectiveness of the genetic modifications was tested on gene, protein and epitope expression level or by functional assays. After exposure to 20% and 40% normal human serum (NHS), deposition of C3b/iC3b/C3c and formation of the terminal complement complex (TCC, C5b-9) was quantified by specific cell ELISAs, and generation of the anaphylatoxin C5a by ELISA. Chondrocyte lysis was analyzed by Trypan Blue Exclusion Assay. In all respective KO variants, the absence of α -1,3-Gal and Neu5Gc epitope was verified by FACS analysis. In chondrocytes derived from TG animals, expression of CD55 and CD59 could be confirmed on gene and protein level, TNFAIP3 on gene expression level as well as by functional assays and CD46 only on gene expression level whereas transgenic HMOX1 expression was not evident. Complement activation in the presence of NHS indicated mainly effective although incomplete protection against C3b/iC3b/C3c deposition, C5a-generation and C5b-9 formation being lowest in single GalTKO. Chondrocyte viability under exposure to NHS was significantly improved even by single GalTKO and completely preserved by all other variants including TG chondrocytes without KO of xenoepitopes.

Genomic biomarkers in chronic beryllium disease and sarcoidosis.

Background Previous gene expression studies have identified genes IFNγ, TNFα, RNase 3, CXCL9, and CD55 as potential biomarkers for sarcoidosis and/or chronic beryllium disease (CBD). We hypothesized that differential expression of these genes could function as diagnostic biomarkers for sarcoidosis and CBD, and prognostic biomarkers for sarcoidosis. Study Design/Methods We performed RT-qPCR on whole blood samples from CBD (n = 132), beryllium sensitized (BeS) (n = 109), and sarcoidosis (n = 99) cases and non-diseased controls (n = 97) to determine differential expression of target genes. We then performed logistic regression modeling and generated ROC curves to determine which genes could most accurately differentiate: 1) CBD versus sarcoidosis 2) CBD versus BeS 3) sarcoidosis versus controls 4) non-progressive versus progressive sarcoidosis. Results CD55 and TNFα were significantly upregulated, while CXCL9 was significantly downregulated in CBD compared to sarcoidosis (p < 0.05). The ROC curve from the logistic regression model demonstrated high discriminatory ability of the combination of CD55, TNFα, and CXCL9 to distinguish between CBD and sarcoidosis with an AUC of 0.98. CD55 and TNFα were significantly downregulated in sarcoidosis compared to controls (p < 0.05). The ROC curve from the model showed a reasonable discriminatory ability of CD55 and TNFα to distinguish between sarcoidosis and controls with an AUC of 0.86. There was no combination of genes that could accurately differentiate between CBD and BeS or sarcoidosis phenotypes. Interpretation CD55, TNFα and CXCL9 expression levels can accurately differentiate between CBD and sarcoidosis, while CD55 and TNFα expression levels can accurately differentiate sarcoidosis and controls.

Erythrocyte CD55 mediates the internalization of Plasmodium falciparum parasites.

Invasion of human erythrocytes by the malaria parasite Plasmodium falciparum is a multi-step process. Previously, a forward genetic screen for P. falciparum host factors identified erythrocyte CD55 as essential for invasion, but its specific role and how it interfaces with the other factors that mediate this complex process are unknown. Using CRISPR-Cas9 editing, antibody-based inhibition, and live cell imaging, here we show that CD55 is specifically required for parasite internalization. Pre-invasion kinetics, erythrocyte deformability, and echinocytosis were not influenced by CD55, but entry was inhibited when CD55 was blocked or absent. Visualization of parasites attached to CD55-null erythrocytes points to a role for CD55 in stability and/or progression of the moving junction. Our findings demonstrate that CD55 acts after discharge of the parasite's rhoptry organelles, and plays a unique role relative to all other invasion receptors. As the requirement for CD55 is strain-transcendent, these results suggest that CD55 or its interacting partners may hold potential as therapeutic targets for malaria.

Dynamic regulation of B cell complement signaling is integral to germinal center responses.

Maturation of B cells within germinal centers (GCs) generates diversified B cell pools and high-affinity B cell antigen receptors (BCRs) for pathogen clearance. Increased receptor affinity is achieved by iterative cycles of T cell-dependent, affinity-based B cell positive selection and clonal expansion by mechanisms hitherto incompletely understood. Here we found that, as part of a physiologic program, GC B cells repressed expression of decay-accelerating factor (DAF/CD55) and other complement C3 convertase regulators via BCL6, but increased the expression of C5b-9 inhibitor CD59. These changes permitted C3 cleavage on GC B cell surfaces without the formation of membrane attack complex and activated C3a- and C5a-receptor signals required for positive selection. Genetic disruption of this pathway in antigen-activated B cells by conditional transgenic DAF overexpression or deletion of C3a and C5a receptors limited the activation of mechanistic target of rapamycin (mTOR) in response to BCR-CD40 signaling, causing premature GC collapse and impaired affinity maturation. These results reveal that coordinated shifts in complement regulation within the GC provide crucial signals underlying GC B cell positive selection.

Human SND2 mediates ER targeting of GPI-anchored proteins with low hydrophobic GPI attachment signals.

Over 100 glycosylphosphatidylinositol-anchored proteins (GPI-APs) are encoded in the mammalian genome. It is not well understood how these proteins are targeted and translocated to the endoplasmic reticulum (ER). Here, we reveal that many GPI-APs, such as CD59, CD55, and CD109, utilize human SND2 (hSND2)-dependent ER targeting machinery. We also found that signal recognition particle receptors seem to cooperate with hSND2 to target GPI-APs to the ER. Both the N-terminal signal sequence and C-terminal GPI attachment signal of GPI-APs contribute to ER targeting via the hSND2-dependent pathway. Particularly, the hydrophobicity of the C-terminal GPI attachment signal acts as the determinant of hSND2 dependency. Our results explain the route and mechanism of the ER targeting of GPI-APs in mammalian cells.