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Complement is increasingly being recognized as an important driver of human disease, including many hemolytic anemias. Paroxysmal nocturnal hemoglobinuria (PNH) cells are susceptible to hemolysis because of a loss of the complement regulatory proteins CD59 and CD55. Patients with atypical hemolytic uremic syndrome (aHUS) develop a thrombotic microangiopathy (TMA) that in most cases is attributable to mutations that lead to activation of the alternative pathway of complement. For optimal therapy, it is critical, but often difficult, to distinguish aHUS from other TMAs, such as thrombotic thrombocytopenic purpura; however, novel bioassays are being developed. In cold agglutinin disease (CAD), immunoglobulin M autoantibodies fix complement on the surface of red cells, resulting in extravascular hemolysis by the reticuloendothelial system. Drugs that inhibit complement activation are increasingly being used to treat these diseases. This article discusses the pathophysiology, diagnosis, and therapy for PNH, aHUS, and CAD., (© 2015 by The American Society of Hematology. All rights reserved.)

Recent thymic emigrants (RTEs) must undergo phenotypic and functional maturation to become long-lived mature naive T cells. In CD4-cre NKAP conditional knockout mice, NKAP-deficient RTEs fail to complete T cell maturation. In this study, we demonstrate that NKAP-deficient immature RTEs do not undergo apoptosis, but are eliminated by complement. C3, C4, and C1q are bound to NKAP-deficient peripheral T cells, demonstrating activation of the classical arm of the complement pathway. As thymocytes mature and exit to the periphery, they increase sialic acid incorporation into cell surface glycans. This is essential to peripheral lymphocyte survival, as stripping sialic acid with neuraminidase leads to the binding of natural IgM and complement fixation. NKAP-deficient T cells have a defect in sialylation on cell surface glycans, leading to IgM recruitment. We demonstrate that the defect in sialylation is due to aberrant α2,8-linked sialylation, and the expression of three genes (ST8sia1, ST8sia4, and ST8sia6) that mediate α2,8 sialylation are downregulated in NKAP-defcient RTEs. The maturation of peripheral NKAP-deficient T cells is partially rescued in a C3-deficient environment. Thus, sialylation during T cell maturation is critical to protect immature RTEs from complement in the periphery., (Copyright © 2014 by The American Association of Immunologists, Inc.)

Objective: To understand the expression of complement regulatory proteins CD55 and CD59, which secreted by epithelial cells of normal and chronic sinusitis patients., Methods: Cell culture and flow cytometry were used to detect the expression of complement regulatory proteins CD55 and CD59.SPSS 18.0 software was used to analyze the data., Results: CD55 was expressed both in normal nasal mucosa and mucosa of chronic sinusitis. The expression in normal group was 0.001 ± 0.001, significantly lower than that in CRS group which was 0.067 ± 0.028 (t = -10.535, P < 0.01). CD59 was also expressed in the two groups . In normal group, the expression of CD59 was 0.879 ± 0.005, significantly higher than that in CRS group which was 0.238 ± 0.034 (t = 83.416, P < 0.01)., Conclusions: The nasal mucosa in CRS patients showed low expression of CD55 and high expression of CD59. This mechanism may be involved in the occurrence of CRS.

Given their localization and important role in regulating complement, complement regulatory proteins may act as target antigens and their antibodies as biomarkers in demyelinating neuropathies. We investigated the binding of autoantibodies to complement regulatory proteins (CD46, 55 and 59) in demyelinating diseases. In 42 acute inflammatory demyelinating polyneuropathy, 23 chronic inflammatory demyelinating polyneuropathy, 13 acute motor axonal neuropathy, 71 multiple sclerosis, and 19 neuromyelitis optica patients as well as 55 healthy controls, we were unable to detect significant titers of antibodies to CD46, CD55 and CD59. These autoantibodies are unlikely to be biomarkers in acute and chronic inflammatory demyelinating polyneuropathies., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The complement system is an ancient and critical effector mechanism of the innate immune system as it senses, kills, and clears infectious and/or dangerous particles and alerts the immune system to the presence of the infection and/or danger. Interestingly, an increasing number of reports have demonstrated a clear role for complement in the adaptive immune system as well. Of note, a number of recent studies have identified previously unknown roles for complement proteins, receptors, and regulators in T cell function. Here, we will review recent data demonstrating the influence of complement proteins C1q, C3b/iC3b, C3a (and C3aR), and C5a (and C5aR) and complement regulators DAF (CD55) and CD46 (MCP) on T cell function during homeostasis and disease. Although new concepts are beginning to emerge in the field of complement regulation of T cell function, future experiments should focus on whether complement is interacting directly with the T cell or is having an indirect effect on T cell function via APCs, the cytokine milieu, or downstream complement activation products. Importantly, the identification of the pivotal molecular pathways in the human systems will be beneficial in the translation of concepts derived from model systems to therapeutic targeting for treatment of human disorders., (© 2014 Society for Leukocyte Biology.)

The epithelial complement inhibitory proteins (CIPs) cluster of differentiation 46 and 55 (CD46 and CD55) regulate circulating immune complex-mediated complement activation in idiopathic pulmonary fibrosis (IPF). Our previous studies demonstrated that IL-17A mediates epithelial injury via transforming growth factor β1 (TGF-β1) and down-regulates CIPs. In the current study, we examined the mechanistic role of TGF-β1 in complement activation-mediated airway epithelial injury in IPF pathogenesis. We observed lower epithelial CIP expression in IPF lungs compared to normal lungs, associated with elevated levels of complement component 3a and 5a (C3a and C5a), locally and systemically. In normal primary human small airway epithelial cells (SAECs) treated with TGF-β1 (10 ng/ml), C3a, or C5a (100 nM), we observed loss of CIPs and increased poly(ADP-ribose) polymerase (PARP) activation [also observed with RNA interference (RNAi) of CD46/CD55]. TGF-β1-mediated loss of CIPs and Snail induction [SNAI1; a transcriptional repressor of E-cadherin (E-CAD)] was blocked by inhibiting mitogen-activated protein kinase (p38MAPK; SB203580) and RNAi silencing of SNAI1. C3a- and C5a-mediated loss of CIPs was also blocked by p38MAPK inhibition. While C3a upregulated TGFb transcripts, both C3a and C5a down-regulated SMAD7 (negative regulator of TGF-β), and whereas TGF-β1 induced C3a/C5a receptor (C3aR/C5aR) expression, pharmacologic C3aR/C5aR inhibition protected against C3a-/C5a-mediated loss of CIPs. Taken together, our results suggest that epithelial injury in IPF can be collectively amplified as a result of TGF-β1-induced loss of CIPs leading to complement activation that down-regulates CIPs and induces TGF-β1 expression, (© FASEB.)

Spontaneous abortion in early pregnancy due to unknown reasons is a common problem. The excess complement activation and consequent placental inflammation and anti-angiogenic milieu is emerging as an important associated factor in many pregnancy-related complications. In the present study we sought to examine the expression of complement inhibitory proteins at the feto-maternal interface and levels of complement split products in the circulation to understand their role in spontaneous abortion. Consenting pregnant women who either underwent elective abortion due to non-clinical reasons (n = 13) or suffered miscarriage (n = 14) were recruited for the study. Systemic levels of complement factors C3a and C5a were measured by enzyme-linked immunosorbent assay (ELISA). Plasma C5 and C3 protein levels were examined by Western blot. Expressions of complement regulatory proteins such as CD46 and CD55 in the decidua were investigated by quantitative polymerase chain reaction (PCR) and Western blot. The median of plasma C3a level was 82·83 ng/ml and 66·17 ng/ml in elective and spontaneous abortion patients, respectively. Medians of plasma C5a levels in elective and spontaneous abortion patients were 0·96 ng/ml and 1·14 ng/ml, respectively. Only plasma C5a levels but not C3a levels showed significant elevation in spontaneous abortion patients compared to elective abortion patients. Further, there was a threefold decrease in the mRNA expressions of complement inhibitory proteins CD46 and CD55 in the decidua obtained from spontaneous abortion patients compared to that of elective abortion patients. These data suggested that dysregulated complement cascade may be associated with spontaneous abortion., (© 2014 British Society for Immunology.)

Leptospirosis is a global zoonosis caused by pathogenic Leptospira, which can colonize the proximal renal tubules and persist for long periods in the kidneys of infected hosts. Here, we characterized the infection of C57BL/6J wild-type and Daf1-/- mice, which have an enhanced host response, with a virulent Leptospira interrogans strain at 14 days post-infection, its persistence in the kidney, and its link to kidney fibrosis at 90 days post-infection. We found that Leptospira interrogans can induce acute moderate nephritis in wild-type mice and is able to persist in some animals, inducing fibrosis in the absence of mortality. In contrast, Daf1-/- mice showed acute mortality, with a higher bacterial burden. At the chronic stage, Daf1-/- mice showed greater inflammation and fibrosis than at 14 days post-infection and higher levels at all times than the wild-type counterpart. Compared with uninfected mice, infected wild-type mice showed higher levels of IL-4, IL-10 and IL-13, with similar levels of α-smooth muscle actin, galectin-3, TGF-β1, IL-17, IFN-γ, and lower IL-12 levels at 90 days post-infection. In contrast, fibrosis in Daf1-/- mice was accompanied by high expression of α-smooth muscle actin, galectin-3, IL-10, IL-13, and IFN-γ, similar levels of TGF-β1, IL-12, and IL-17 and lower IL-4 levels. This study demonstrates the link between Leptospira-induced murine chronic nephritis with renal fibrosis and shows a protective role of Daf1.

A defining feature in proliferative retinopathies is the formation of pathological neovessels. In these diseases, the balance between neovessel formation and regression determines blindness, making the modulation of neovessel growth highly desirable. The role of the immune system in these retinopathies is of increasing interest, but it is not completely understood. We investigated the role of the alternative complement pathway during the formation and resolution of aberrant neovascularization. We used alternative complement pathway-deficient (Fb(-/-)) mice and age- and strain-matched control mice to assess neovessel development and regression in an oxygen-induced retinopathy (OIR) mouse model. In the control mice, we found increased transcription of Fb after OIR treatment. In the Fb(-/-) mice, we prepared retinal flatmounts and identified an increased number of neovessels, peaking at postnatal day 17 (P17; P=0.001). Subjecting human umbilical vein endothelial cells (HUVECs) to low oxygen, mimicking a characteristic of neovessels, decreased the expression of the complement inhibitor Cd55. Finally, using laser capture microdissection (LCM) to isolate the neovessels after OIR, we found decreased expression of Cd55 (P=0.005). Together, our data implicate the alternative complement pathway in facilitating neovessel clearance by down-regulating the complement inhibitor Cd55 specifically on neovessels, allowing for their targeted removal while leaving the established vasculature intact.-Sweigard, J. H., Yanai, R., Gaissert, P., Saint-Geniez, M., Kataoka, K., Thanos, A., Stahl, G. L., Lambris, J. D., Connor, K. M. The alternative complement pathway regulates pathological angiogenesis in the retina., (© FASEB.)

Context: The expression of membrane-bound complement regulatory proteins (mCRPs) that inhibit the complement system in normal tissues is essential for self-protection against an autologous immune reaction. However, the expression patterns of mCRPs, including CD46, CD55, and CD59, are inconsistent in different types of cancer cells., Objectives: To determine whether CD46, CD55, and CD59 are differentially expressed in neoplastic and adjacent normal colon tissues and to assess their clinical significance., Design: Immunohistochemistry was performed on tissue microarrays of cancerous and adjacent normal colon tissues., Results: The expression levels of CD46, CD55, and CD59 were significantly higher in colon cancer tissues compared with the normal adjacent colon tissues. We found that the expression levels of CD55 and CD59 correlated with the grade of differentiation in colon cancers. In addition, the expression of CD55 and CD59 was greater in stage III and stage IV colon cancers than in stage I and stage II cancers according to staging by the TNM classification., Conclusions: CD46, CD55, and CD59 are up-regulated in colon cancer. Specifically, CD55 and CD59 are of clinical relevance to differentiation and TNM staging of colon cancer. These data suggest that CD46, CD55, and CD59 have the potential to be used for molecular staging diagnoses and for colon cancer therapies.

Engineering cancer cells to express heterologous antigen α-gal and induce the destruction of tumor cells depending on the complement cascade may be a promising strategy of tumor therapy. However, the feasibility and effect of using α-gal to induce colorectal adenocarcinoma cell line cytolysis is not yet known. In this study, we evaluated α-gal expression's ability to sensitize human colorectal adenocarcinoma cell lines to complement attack in cell lines LoVo, SW620, and Ls-174T. Nearly all α-gal-expressing LoVo and SW620 cells were killed by normal human serum (NHS), but α-gal-expressing Ls-174T cells showed no significant lysis. We analyzed the expression levels of membrane-bound complement regulatory proteins (mCRPs) on the three cell lines, and their protective role in α-gal-mediated activation of the complement. LoVo showed no expression of any of the three proteins. CD59 was strongly expressed by SW620 and Ls-174T. CD46 and CD55 varied between the two cell lines. CD46 on SW620 was only half the intensity of CD46 on Ls-174T. Ls-174T showed a notable expression of CD55, while expression of CD55 on SW620 was not detected. The sensitivity of Ls-174T expressing α-gal to NHS greatly increased following the downregulation of CD46 and CD55 with short hairpin RNA (shRNA). However, there is no increase in cell killing when CD59 expression was diminished. Our findings suggest that the use of α-gal as antigen to induce tumor cell killing may be a potential therapeutic strategy in colon cancer and that CD55 plays a primary role in conferring resistance to lysis., (Copyright © 2014 the American Physiological Society.)

Decay accelerating factor (DAF) expression at the muscle endplate is an important defence against complement-mediated damage in myasthenia gravis. Previously we implicated the c.-198C>G DAF polymorphism with the development of treatment-resistant myasthenia-associated ophthalmoplegia by showing that the C>G DAF polymorphism prevented lipopolysaccharide-induced upregulation of lymphoblast DAF. We postulated that drugs used in myasthenia gravis may increase the susceptibility of extraocular muscles to complement-mediated damage and studied their effects on endogenous DAF using patient-derived lymphoblasts as well as mouse myotubes. We show that prednisone repressed C>G DAF expression in lymphoblasts and increased their susceptibility to cytotoxicity. Methotrexate, but not azathioprine or cyclosporine, increased DAF in C>G lymphoblasts. In mouse myotubes expressing wild-type Daf, prednisone also repressed Daf expression. Although cyclosporine, azathioprine, and methotrexate increased muscle Daf levels when used alone, upon co-treatment with prednisone only azathioprine maintained myotube Daf levels close to basal. Therefore, prednisone negatively influences DAF expression in C>G lymphoblasts and in myotubes expressing wild-type Daf. We speculate that myasthenic individuals at risk of developing the ophthalmoplegic complication, such as those with C>G DAF, may have inadequate endogenous levels of complement regulatory protein protection in their extraocular muscle in response to prednisone, increasing their susceptibility to complement-mediated damage., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Preterm birth (PTB) is a major public health problem, with a global prevalence of 9.6% and over a million annual neonatal deaths. In a mouse model of preterm labor (PTL) induced by intravaginal administration of a subclinical dose of lipopolysaccharide (LPS), we previously demonstrated that LPS ascends to the cervix, inducing complement activation, cervical remodeling and PTL. Here we show that complement activation also plays a role in myometrial contractions during PTL in this model. Increased levels of C5a were detected in the myometrium of LPS-treated mice but not in age-matched control or term myometrium. Human and mouse myometrium incubated with C5a showed increased frequency of contractions and expression of connexin 43, suggesting that C5a is an uterotonic molecule. Statins, which showed beneficial effects in preventing complement-mediated pregnancy complications, prevented cervical remodeling, myometrial contractions and PTL in the LPS model. The protective effects of statins in PTL were associated with increased synthesis, expression and activity of heme oxygenase (HO-1) in myometrium and cervix. Coadministration of HO-1 inhibitor tin-protoporphyrin-IX with pravastatin abrogated the protective effects of pravastatin on cervical remodeling and myometrial contractions leading to PTB. In addition, pravastatin inhibited complement activation in the cervix by increasing the synthesis and expression of complement inhibitor decay-accelerating factor. This study in mice suggests that statins might be useful to prevent PTL in humans. Clinical trials in humans are needed and if these results are confirmed, they may form the basis for a new clinical approach to prevent PTB., (© The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background: Decay Accelerating Factor (DAF) and Coxsackievirus-Adenovirus Receptor (CAR) have been identified as cellular receptors for Coxsackie B viruses (CV-B). The aim of this study is to elucidate the different binding properties of CV-B serotypes and to find out if there are any amino acid changes that could be associated to the different phenotypes.Twenty clinical CV-B isolates were tested on CaCo-2 cell line using anti-DAF (BRIC216) and anti-CAR (RmcB) antibodies. CV-B3 Nancy prototype strain and a recombinant strain (Rec, CV-B3/B4) were tested in parallel. The P1 genomic region of 12 CV-B isolates from different serotypes was sequenced and the Trans-Epithelial Electrical Resistance (TEER) along with the virus growth cycle was measured., Results: Infectivity assays revealed clear differences between CV-B isolates with regard to their interactions with DAF and CAR. All tested CV-B isolates showed an absolute requirement for CAR but varied in their binding to DAF. We also reported that for some isolates of CV-B, DAF attachment was not adapted. Genetic analysis of the P1 region detected multiple differences in the deduced amino acid sequences., Conclusion: Within a given serotype, variations exist in the capacity of virus isolates to bind to specific receptors, and variants with different additional ligands may arise during infection in humans as well as in tissue culture.

Purpose: Endothelial keratoplasty (EKP) has become increasingly popular in the treatment of corneal disease. However, the global shortage of human donor corneas limits clinical corneal transplantation. Genetically engineered (GE) pigs may provide an alternative source of corneas for EKP. The aim of this study was to evaluate corneal endothelial cells (CECs) from wild-type (WT) and GE pigs., Methods: Density, size of CECs, and the percentage of hexagonal cells (as a measure of heterogeneity) were measured by ex vivo confocal microscopy in corneas from WT and GE pigs of different ages - neonatal (4-5 days), young (5-15 weeks), adult (5-15 months), and old (20-42 months). α1,3-galactosyltransferase gene-knockout (GTKO) pigs transgenic for the human complement-regulatory protein(s), CD46 (GTKO/CD46) +/- CD55 (GTKO/CD46/CD55) were used as sources of GE corneas., Results: Mean CEC densities (cells/mm²) were neonatal (5968), young (3789), adult (2589), and old (2070). As with human corneas, there was an age-dependent decrease in pig CEC density and increase in pig CEC size. However, unlike human corneas, there was no correlation between the percentage of hexagonal cells (approximately 50% in all pig corneas) and age, suggesting that heterogeneity is intrinsic for pig corneas. Genetic modification did not affect CEC density, size, or morphology compared to WT pigs., Conclusion: Because of the availability of young pigs and their greater CEC density (and the protection afforded against the human immune response), GE pigs could provide an unlimited source of corneas for clinical EKP.

Endothelial injury and dysfunction precede accelerated arterial disease in allograft vasculopathy and systemic autoimmune diseases and involve pathogenic Abs and complement. Recent reports suggest that switching to rapamycin from calcineurin antagonists reduces posttransplant vasculopathy and prolongs survival following cardiac transplantion. The majority of these patients also receive statin therapy. We examined potential mechanisms underlying this protective response in human endothelial cells and identified synergy between rapamycin and atorvastatin. Mechanistically, atorvastatin and rapamycin activated a protein kinase Cα, AMP-activated kinase, and CREB-dependent vasculoprotective pathway, which induced decay-accelerating factor (DAF) promoter activity via binding to the cAMP response element, mutation of which attenuated promoter activity. This response significantly increased endothelial cell surface DAF and enhanced protection against complement-mediated injury. Synergy with rapamycin was reproduced by simvastatin, whereas combining atorvastatin with cyclosporine or mycophenolate in place of rapamycin was ineffective. Importantly, synergy was reproduced in vivo, in which only atorvastatin and rapamycin therapy in combination was sufficient to induce DAF on murine aortic endothelium. We believe this pathway represents an important therapeutically inducible vasculoprotective mechanism for diseases mediated by pathogenic Abs and complement, including posttransplant vasculopathy and systemic lupus erythematosus. Although our study focuses on the vascular endothelium, the findings are likely to be broadly applicable, given the diverse cellular expression of DAF.

Background: Promising developments in porcine islet xenotransplantation could resolve the donor pancreas shortage for patients with type 1 diabetes. Using α1,3-galactosyltransferase gene knockout (GTKO) donor pigs with multiple transgenes should extend xenoislet survival via reducing complement activation, thrombus formation, and the requirement for exogenous immune suppression. Studying the xenoantibody response to GTKO/hCD55/hCD59/hHT islets in the pig-to-baboon model, and comparing it with previously analyzed responses, would allow the development of inhibitory reagents capable of targeting conserved idiotypic regions., Methods: We generated IgM heavy and light chain gene libraries from 10 untreated baboons and three baboons at 28 days following transplantation of GTKO/hCD55/hCD59/hHT pig neonatal islet cell clusters with immunosuppression. Flow cytometry was used to confirm the induction of a xenoantibody response. IgM germline gene usage was compared pre- and post-transplant. Homology modeling was used to compare the structure of xenoantibodies elicited after transplantation of GTKO/hCD55/hCD59/hHT pig islets with those induced by GTKO and wild-type pig endothelial cells without further genetic modification., Results: IgM xenoantibodies that bind to GTKO pig cells and wild-type pig cells were induced after transplantation. These anti-non-Gal antibodies were encoded by the IGHV3-66*02 (Δ28%) and IGKV1-12*02 (Δ25%) alleles, for the immunoglobulin heavy and light chains, respectively. IGHV3-66 is 86.7% similar to IGHV3-21 which was elicited by rhesus monkeys in response to GTKO endothelial cells. Heavy chain genes most similar to IGHV3-66 were found to utilize the IGHJ4 gene in 85% of V-D regions analyzed. However, unlike the wild-type response, a consensus complementary determining region 3 was not identified., Conclusions: Additional genetic modifications in transgenic GTKO pigs do not substantially modify the structure of the restricted group of anti-non-Gal xenoantibodies that mediate induced xenoantibody responses with or without immunosuppression. The use of this information to develop new therapeutic agents to target this restricted response will likely be beneficial for long-term islet cell survival and for developing targeted immunosuppressive regimens with less toxicity., (© 2014 John Wiley & Sons A/S.)

Background:  Escherichia coli-bearing Dr-adhesins (Dr+ E. coli) cause chronic pyelonephritis in pregnant women and animal models. This chronic renal infection correlates with the capacity of bacteria to invade epithelial cells expressing CD55. The mechanism of infection remains unknown., Methods:  CD55 amino acids in the vicinity of binding pocket-Ser155 for Dr-adhesin were mutated to alanine and subjected to temporal gentamicin-invasion/gentamicin-survival assay in Chinese hamster ovary cells. CD55/microtubule (MT) responses were studied using confocal/electron microscopy, and 3-dimensional structure analysis., Results:  Mutant analysis revealed that complement-protective CD55-Ser165 and CD55-Phe154 epitopes control E. coli invasion by coregulating CD55-MT complex expression. Single-point CD55 mutations changed E. coli to either a minimally invasive (Ser165Ala) or a hypervirulent pathogen (Phe154Ala). Thus, single amino acid modifications with no impact on CD55 structure and bacterial attachment can have a profound impact on E. coli virulence. While CD55-Ser165Ala decreased E. coli invasion and led to dormant intracellular persistence, intracellular E. coli in CD55-Phe154Ala developed elongated forms (multiplying within vacuoles), upregulated CD55-MT complexes, acquired CD55 coat, and escaped phagolysosomal fusion., Conclusions:  E. coli target complement-protective CD55 epitopes for invasion and exploit CD55-MT complexes to escape phagolysosomal fusion, leading to a nondestructive parasitism that allows bacteria to persist intracellularly.

Lipid rafts that are enriched in glycosylphosphatidylinositol (GPI)-anchored proteins serve as a platform for important biological events. To elucidate the molecular mechanisms of these events, identification of co-clustering molecules in individual raft domains is required. Here we describe an approach to this issue using the recently developed method termed enzyme-mediated activation of radical source (EMARS), by which molecules in the vicinity within 300 nm from horseradish peroxidase (HRP) set on the probed molecule are labeled. GPI-anchored HRP fusion proteins (HRP-GPIs), in which the GPI attachment signals derived from human decay accelerating factor and Thy-1 were separately connected to the C-terminus of HRP, were expressed in HeLa S3 cells, and the EMARS reaction was catalyzed by these expressed HRP-GPIs under a living condition. As a result, these different HRP-GPIs had differences in glycosylation and localization and formed distinct clusters. This novel approach distinguished molecular clusters associated with individual GPI-anchored proteins, suggesting that it can identify co-clustering molecules in individual raft domains.

Decay accelerating factor (DAF/CD55) is targeted by many pathogens for cell entry. It has been implicated as a co-receptor for hantaviruses. To examine the binding of hantaviruses to DAF, we describe the use of Protein G beads for binding human IgG Fc domain-functionalized DAF ((DAF)₂-Fc). When mixed with Protein G beads the resulting DAF beads can be used as a generalizable platform for measuring kinetic and equilibrium binding constants of DAF binding targets. The hantavirus interaction has high affinity (24-30 nM; k(on) ~ 10⁵ M⁻¹ s⁻¹, k(off) ~ 0.0045 s⁻¹). The bivalent (DAF)₂-Fc/SNV data agree with hantavirus binding to DAF expressed on Tanoue B cells (K(d) = 14.0 nM). Monovalent affinity interaction between SNV and recombinant DAF of 58.0 nM is determined from competition binding. This study serves a dual purpose of presenting a convenient and quantitative approach of measuring binding affinities between DAF and the many cognate viral and bacterial ligands and providing new data on the binding constant of DAF and Sin Nombre hantavirus. Knowledge of the equilibrium binding constant allows for the determination of the relative fractions of bound and free virus particles in cell entry assays. This is important for drug discovery assays for cell entry inhibitors.

CD226, an activating receptor that interacts with the ligands CD155 and CD112, activates natural killer (NK) cells via its immunoreceptor tyrosine-based activatory motif (ITAM). There are two extracellular domains of CD226; however, the comparative functional relevance of these domains remains unknown. In this study, two different deletion mutants, rCD226-ECD1 (the first extracellular domain) and rCD226-ECD (full extracellular domains), were recombinantly expressed. We observed that rCD226-ECD1, similar to rCD226-ECD, specifically bound to ligand-positive cell lines and that this interaction could be competitively blocked by an anti-CD226 mAb. In addition, rCD226-ECD1 was able to block the binding of CD112 mAb to tumor cells in a competitive binding assay. Importantly, based on surface plasmon resonance (SPR), we determined that rCD226-ECD1, similar to rCD226-ECD, directly bound to its ligand CD155 on a protein chip. Functionally, NK cell cytotoxicity against K562 or HeLa cells was blocked by rCD226-ECD1 by reducing the expression of CD69 and granzyme B, indicating the critical role of ECD1 in NK cell activation. We also examined the role of rCD226-ECD1 in effector/target interactions by using rCD226-ECD to block these interactions. Using flow cytometry, we found that the number of conjugates between IL-2-dependent NKL cells and HeLa cells was reduced and observed that the formation of immune synapses was also decreased under confocal microscopy. In addition, we prepared two anti-rCD226-ECD1 agonistic antibodies, 2E6 and 3B9. Both 2E6 and 3B9 antibodies could induce the phosphorylation of ERK in NK-92 cells. Taken together, our results show that CD226 functions via its first extracellular domain.

The fully human monoclonal antibody PAT-SC1 is specific for an isoform of CD55 (decay-accelerating factor) designated CD55PAT-SC1. This antigen is expressed in the majority (80%) of gastric cancers (GCs), and the antibody induces tumour cell-specific apoptosis in vitro as well as in vivo. PAT-SC1, therefore, has been deemed promising as a therapeutic agent. Here, we describe the results of an academic clinical study performed in a neoadjuvant setting with resectable GC patients. Patients undergoing treatment for GC between 1997 and 2001 were tested for CD55PAT-SC1 expression. Fifty-one resectable patients that tested positively received a single administration of 20 mg PAT-SC1 48 h prior to surgery. They underwent standard surgery with either subtotal or total gastrectomy with bursectomy, omentectomy and a modified D2-lymphadenectomy, aimed at R0 resection. Primary endpoints of the present study were to evaluate side-effects of the PAT-SC1 antibody treatment and to evaluate histopathological effects such as tumour regression and induction of apoptosis. Long-term survival was a secondary endpoint. Administration of PAT-SC1 appeared safe with only reversible side-effects according to WHO grade I and II. Despite the low‑dose of the antibody, 81.6% of the patients showed signs of increased apoptosis within the primary tumour and 60% showed signs of tumour cell regression. Comparison of the 10-year survival rates of the R0-resected CD55PAT-SC1-positive patients treated with the PAT-SC1 antibody with a historical collective of R0-resected CD55PAT-SC1-positive patients not treated with PAT-SC1 indicated a survival benefit in the treated patients. Furthermore, comparison of the patient survival of CD55PAT‑SC1-positive vs. CD55PAT-SC1-negative groups suggested that CD55PAT-SC1 antigen expression is an independent predictor of poor survival in a Cox regression analysis. Antibody PAT-SC1 may be a useful additive therapeutic agent in the treatment of patients with CD55PAT-SC1-expressing GCs. In combination with radical standard surgery, PAT-SC1 given as an adjuvant or neoadjuvant immunotherapeutic agent induces apoptosis in tumour cells which may improve survival of these patients. Because of the human origin and its specific binding to the CD55PAT-SC1 antigen, PAT-SC1 was well tolerated in this trial.

Background: Complement has the potential to provoke severe impairment to host tissues, as shown in autoimmune diseases where complement activation has been associated with diminished CD55 and/or CD59 expression on peripheral blood cell membranes. The aim of this study was to evaluate the presence of CD55- and/or CD59-deficient erythrocytic populations in patients with different rheumatic diseases and to investigate possible correlations with clinical or laboratory parameters., Material and Methods: CD55 and CD59 expression was evaluated in erythrocytes of 113 patients with rheumatic diseases, 121 normal individuals, and 10 patients with paroxysmal nocturnal hemoglobinuria (PNH) using the Sephacryl gel microtyping system. Ham and sucrose tests were also performed., Results: Interestingly, the majority of patients (104/113, 92%) demonstrated CD55- and/or CD59-deficient erythrocytes: 47 (41.6%) with concomitant deficiency of CD55 and CD59, 50 (44.2%) with isolated deficiency of CD55, and 6 (6.2%) with isolated deficiency of CD59. In normal individuals, only 2 (1%) had concomitant CD55/CD59 negativity and 3 (2%) had isolated CD55 or CD59 deficiency. All PNH patients exhibited simultaneous CD55/CD59 deficiency. Positive Ham and sucrose tests were found only in PNH patients. There was no association between the CD55- and/or CD59-deficient erythrocytes and hemocytopenias or undergoing treatment. However, CD55 expression significantly influenced hemoglobin values (F=6.092, p=0.015)., Conclusions: This study provides evidence supporting the presence of erythrocytes with CD55 and/or CD59 deficiency in patients with rheumatic diseases. Moreover, CD55 deficiency on red cells influences hemoglobin concentration. Further studies using molecular techniques will clarify the exact pathophysiological mechanisms of this deficiency.

Indoles are composed of a common core structure, the indole ring, and are widely used as pharmaceuticals and their precursors. In this study, a newly composed relatively small indole compound, AWT-489 was examined to find a novel specific antagonist for DP receptors; the cognate receptors for prostaglandin D2 (PGD2), to prevent colon cancer malignancy. Here we showed that AWT-489 antagonized DP receptor-mediated cyclic AMP formation, and expression of CD55, an inhibitor of the complement system that correlates with poor survival in patients with colorectal cancer, in LS174T human colon cancer cells. Interestingly, unlike a popular indole compound, indomethacin, AWT-489 did not act on the cyclooxygenases as a non-steroidal anti-inflammatory drug. Moreover, AWT-489 exhibited a better inhibitory effect than that of the well-used DP receptor antagonist, BWA868C when a dose close to the physiological concentration of PGD2 was used. These results suggest that AWT-489 can act as a novel human DP receptor antagonist to reduce the expression of CD55 in LS174T human colon cancer cells. We believe that AWT-489 has potential as a lead compound for designing a new DP receptor antagonist that may help improve PGD2-related diseases, especially colon cancer in the near future., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The aim of the present study was to characterize potential somatic cell nuclear transfer (SCNT) donor cells by comparing two lines of transfected cells with their non‑modified parental controls in culture. Fetal fibroblasts used in the study originated from crossbred Landrace x Yorkshire x Duroc (LYD) or Yucatan mini‑pigs. The LYD fibroblasts were modified by the transfection of a tetracycline on/off gene, whereas Yucatan fibroblasts were triple transfected with the complement regulatory factors, human decay‑accelerating factor and human CD59, as well as H‑transferase. At the 9th doubling passage, parameters associated with senescence and apoptosis, including morphology, mRNA expression (TP53, Bcl‑2, Bax) and reactive oxygen species (ROS) levels, were evaluated. Population doubling (PD) time was calculated by assessing the time required for cell numbers to double by averaging the three cell passages. Quantitative polymerase chain reaction revealed that when comparing LYD with Yucatan fibroblasts, the latter exhibited a lower relative expression of TP53 and a higher relative expression of antiproliferative Bcl‑2, which correlated with the PD time results (26 and 40 h, respectively). Tetracycline on/off transfected cell lines exhibited a lower relative expression of antiapoptotic Bcl‑2 compared with their originating LYD cells. Similarly, triple transgenic cells exhibited higher TP53 and Bax mRNA expression levels than their non‑transgenic counterparts. For ROS measurement, cells were incubated with 2',7'‑dichlorofluorescin diacetate under the same conditions and were analyzed by flow cytometry. Yucatan fibroblasts exhibited higher ROS content than LYD cells. In addition, the two transgenic cell lines produced higher ROS levels than their corresponding non‑transfected cell lines. In conclusion, these results indicate that characteristics associated with senescence and apoptosis in transfected cells during culture may affect the efficiency of SCNT when used as donor cells for the production of transgenic swine.

CD55 is a complement regulatory protein expressed by cells to protect them from bystander lysis by complement. It prevents the formation of C3/C5 convertase. In β-thalassemia (β-thal), the defective hemoglobin (Hb) production makes red blood cells (RBCs) lyse early and frequently. Loss of CD55 expression in those patients compromises the complement regulatory function, thereby accelerating RBC lysis. In this study, we aimed to evaluate the expression of CD55 on erythrocytes of β-thal patients. Flow cytometry analysis of CD55 was conducted on RBCs of 21 β-thalassemia major (β-TM) patients, 11 β-thalassemia intermedia (β-TI) patients and 10 healthy volunteers. The results showed a significant decrease in CD55 expression in β-TM (57.5 ± 16.7%), while there was a slight decrease in β-TI patients (81.8 ± 3.8%) in comparison with that of the normal controls (88.7 ± 0.8%). The diminished expression of CD55 was not accompanied by decrease in CD59 expression in β-thal patients (97.2 ± 2.3%). This could suggest a mechanism (could be genetic) responsible for low CD55 expression. It may be related to defective Hb genes in thalassemia, but it does not relate to cell membrane changes.

During the course of their lifespan, erythrocytes actively shed phospholipid-bound microvesicles (MVs). In stored blood, the number of these erythrocyte-derived MVs has been observed to increase over time, suggesting their potential value as a quality metric for blood products. The lack of sensitive, standardized MV assays, however, poses a significant barrier to implementing MV analyses into clinical settings. Here, we report on a new nanotechnology platform capable of rapid and sensitive MV detection in packed red blood cell (pRBC) units. A filter-assisted microfluidic device was designed to enrich MVs directly from pRBC units, and label them with target-specific magnetic nanoparticles. Subsequent detection using a miniaturized nuclear magnetic resonance system enabled accurate MV quantification as well as the detection of key molecular markers (CD44, CD47, CD55). When the developed platform was applied, MVs in stored blood units could also be monitored longitudinally. Our results showed that MV counts increase over time and, thus, could serve as an effective metric of blood aging. Furthermore, our studies found that MVs have the capacity to generate oxidative stress and consume nitric oxide. By advancing our understanding of MV biology, we expect that the developed platform will lead to improved blood product quality and transfusion safety.

Enteroviruses can frequently target the human central nervous system to induce a variety of neurological diseases. Although enteroviruses are highly cytolytic, emerging evidence has shown that these viruses can establish persistent infections both in vivo and in vitro. Here, we investigated the susceptibility of three human brain cell lines, CCF-STTG1, T98G, and SK-N-SH, to infection with three enterovirus serotypes: coxsackievirus B3 (CVB3), enterovirus 71, and coxsackievirus A9. Persistent infection was observed in CVB3-infected CCF-STTG1 cells, as evidenced by prolonged detection of infectious virions, viral RNA, and viral antigens. Of note, infected CCF-STTG1 cells expressed the nonfunctional canonical viral receptors coxsackievirus-adenovirus receptor and decay-accelerating factor, while removal of cell surface chondroitin sulfate from CCF-STTG1 cells inhibited the replication of CVB3, suggesting that receptor usage was one of the major limiting factors in CVB3 persistence. In addition, CVB3 curtailed the induction of beta interferon in infected CCF-STTG1 cells, which likely contributed to the initiation of persistence. Furthermore, proinflammatory chemokines and cytokines, such as vascular cell adhesion molecule 1, interleukin-8 (IL-8), and IL-6, were upregulated in CVB3-infected CCF-STTG1 cells and human progenitor-derived astrocytes. Our data together demonstrate the potential of CCF-STTG1 cells to be a novel cell model for studying CVB3-central nervous system interactions, providing the basis toward a better understanding of CVB3-induced chronic neuropathogenesis.

Background: Xenotransplantation could provide a solution to the donor shortage that is currently the major barrier to solid-organ transplantation. The ability to breed pigs with multiple genetic modifications provides a unique opportunity to explore the immunologic challenges of pulmonary xenotransplantation., Methods: Explanted lungs from wild-type and 3 groups of genetically modified pigs were studied: (i) α1,3-galactosyltransferase gene knockout (GTKO); (ii) GTKO pigs expressing the human complementary regulatory proteins CD55 and CD59 (GTKO/CD55-59); and (iii) GTKO pigs expressing both CD55-59 and CD39 (GTKO/CD55-59/CD39). The physiologic, immunologic and histologic properties of porcine lungs were evaluated on an ex vivo rig after perfusion with human blood., Results: Lungs from genetically modified pigs demonstrated stable pulmonary vascular resistance and better oxygenation of the perfusate, and survived longer than wild-type lungs. Physiologic function was inversely correlated with the degree of platelet sequestration into the xenograft. Despite superior physiologic profiles, lungs from genetically modified pigs still showed evidence of intravascular thrombosis and coagulopathy after perfusion with human blood., Conclusions: The ability to breed pigs with multiple genetic modifications, and to evaluate lung physiology and histology in real-time on an ex vivo rig, represent significant advances toward better understanding the challenges inherent to pulmonary xenotransplantation., (© 2013 International Society for Heart and Lung Transplantation. All rights reserved.)

Objective: To investigate the activities of cytokine induced killer cells (CIK cells) and specific cytotoxic T lymphocytes (CTL) in killing CD55(high) subgroup in MCF-7 breast cancer cell line., Methods: The killing effects of CIK cells, and specific CTLs in diminishing CD55(high) subgroup cells were analyzed and compared with those of three chemotherapy drugs. First, the MCF-7 breast cancer cells were treated with different concentrations of three chemotherapeutic drugs, docetaxel, epirubicin and fluorouracil. Then immune cells were isolated from human peripheral blood and were induced to become CIK cells. They were then used to treat CD55(high) and CD55(low) subpopulations in MCF-7 cells. Finally, lymphocytes were isolated from surgical specimens of human axially lymph nodes, and were induced to become tumor-specific CTL in vitro. Their activities in killing CD55(high) and CD55(low) subpopulations in MCF-7 cells were measured as well., Results: The killing rates of docetaxel, epirubicin and fluorouracil on CD55(high) subgroup MCF-7 cells were much lower than on total MCF-7 cells. The killing rate of CIK cells on CD55(high) subgroup cells was(42.72 ± 4.36)%. The killing rates of specific CTLs on CD55(high), CD55(low), and total MCF-7 cells were(52.86 ± 4.45)%, (22.41 ± 2.83)%, and(21.67 ± 4.15)%, respectively., Conclusion: Specific CTL possessed more effective killing effects on CD55(high) cells.

Background: The venom of Loxosceles reclusa (Brown Recluse spider) can cause a severe, life-threatening hemolysis in humans for which no therapy is currently available in the USA beyond supportive measures. Because this hemolysis is uncommon, relatively little is known about its clinical manifestation, diagnosis, or management. Here, we aimed to clarify the clinical details of envenomation, to determine the efficacy of the complement inhibitor eculizumab to prevent the hemolysis in vitro, and to investigate markers of exposure to Brown Recluse venom., Study Design and Methods: We performed a 10-year chart review of cases of Brown Recluse spider bite-mediated hemolysis at our institution. We also designed an in vitro assay to test the efficacy of eculizumab to inhibit hemolysis of venom exposed red blood cells. Finally, we compared levels of CD55, CD59 and glycophorin A on venom exposed versus venom-naïve cells., Results: Most victims of severe Brown Recluse spider mediated hemolysis at our institution are children and follow an unpredictable clinical course. Brown Recluse spider bite mediated hemolysis is reduced by 79.2% (SD=18.8%) by eculizumab in vitro. Erythrocyte glycophorin A, but not CD55 or CD59, is reduced after red blood cells are incubated with venom in vitro., Conclusion: Taken together, our laboratory data and clinical observations indicate that L. reclusa venom exposure results in non-specific antibody and complement fixation on red blood cells, resulting in complement mediated hemolysis that is curtailed by the complement inhibitor eculizumab in vitro. Glycophorin A measurement by flow cytometry may help to identify victims of L. reclusa envenomation.

Although the genes involved in the biosynthesis of glycosylphosphatidylinositol (GPI) are well characterized, the regulation of GPI biosynthesis remains unclear. We isolated and characterized a mutant cell line showing decreased surface expression of CD59 and the accumulation of GPI intermediates. The mutant cell line was partially defective in MPDU1, which encodes a protein required for the utilization of dolichol-phosphate mannose. Overexpression of PIGV, which encodes GPI mannosyltransferase II, restored the surface expression of CD59 and normalized the accumulation of GPI intermediates in the mutant cells. Among all known genes involved in GPI biosynthetic pathway, only PIGV had such suppressive activity. PIGV, however, did not restore the abnormality of N-glycosylation caused by MPDU1 mutation. Our results suggest that GPI mannosyltransferase II is the rate-limiting enzyme in GPI biosynthesis under limited dolichol-phosphate mannose availability.

The control of complement activation in the embryo-maternal environment has been demonstrated to be critical for embryo survival. Complement proteins are expressed in the human endometrium; however, the modulation of this expression by embryo signals has not been explored. To assess the expression of complement proteins in response to human chorionic gonadotropin (hCG), we designed an experimental study using in vivo and in vitro models. Twelve fertile women were treated with hCG or left untreated during the mid-luteal phase, and an endometrial biopsy was performed 24 hours later. The localizations of C3, membrane cofactor protein (MCP; CD46), decay-accelerating factor (DAF; CD55), and protectin (CD59) were assessed by immunohistochemistry, and the messenger RNA (mRNA) levels of these proteins were quantified by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) in cells harvested from endometrial compartments using laser capture microdissection. Endometrial explants were cultured with or without hCG for 24 hours, and the C3 and DAF protein levels were measured by Western blotting. Elevated C3 mRNA levels in stromal cells and elevated DAF levels in epithelial luminal cells were detected after hCG treatment. In the endometrial explant model, the progesterone receptor antagonist RU486 inhibited the increases in the levels of C3 and DAF in response to hCG. The findings of this study indicate that hCG plays a role in embryo-endometrium communication and affects the expression of complement proteins in endometrial compartments during the implantation window.

Maternal-fetal transmission of group B coxsackieviruses (CVB) during pregnancy has been associated with a number of diverse pathological outcomes, including hydrops fetalis, fetal myocarditis, meningoencephalitis, neurodevelopmental delays, congenital skin lesions, miscarriage, and/or stillbirth. Throughout pregnancy, the placenta forms a critical antimicrobial protective barrier at the maternal-fetal interface. Despite the severity of diseases accompanying fetal CVB infections, little is known regarding the strategies used by CVB to gain entry into placental trophoblasts. Here we used both a trophoblast cell line and primary human trophoblasts to demonstrate the mechanism by which CVB gains entry into polarized placental trophoblasts. Our studies revealed that the kinetics of CVB entry into placental trophoblasts are similar to those previously described for polarized intestinal epithelial cells. Likewise, CVB entry into placental trophoblasts requires decay-accelerating factor (DAF) binding and involves relocalization of the virus from the apical surface to intercellular tight junctions. In contrast, we have identified a divergent mechanism for CVB entry into polarized trophoblasts that is clathrin, caveolin-1, and dynamin II independent but requires intact lipid rafts. In addition, we found that members of the Src family of tyrosine kinases were required for CVB entry. Our studies highlight the complexity of viral entry into human placental trophoblasts and may serve as a model for mechanisms used by diverse pathogens to penetrate the placental barrier.

We previously reported that aged mice lacking complement factor H (CFH) exhibit visual defects and structural changes in the retina. However, it is not known whether this phenotype is age-related or is the consequence of disturbed development. To address this question we investigated the effect of Cfh gene deletion on the retinal phenotype of young and mid-age mice. Cfh(-/-) mouse eyes exhibited thickening of the retina and reduced nuclear density, but relatively normal scotopic and photopic electroretinograms. At 12 months there was evidence of subtle astroglial activation in the Cfh(-/-) eyes, and significant elevation of the complement regulator, decay-accelerating factor (DAF) in Müller cells. In the retinal pigment epithelium (RPE) of young control and Cfh(-/-) animals mitochondria and melanosomes were oriented basally and apically respectively, whereas the apical positioning of melanosomes was significantly perturbed in the mid-age Cfh(-/-) RPE. We conclude that deletion of Cfh in the mouse leads to defects in the retina that precede any marked loss of visual function, but which become progressively more marked as the animals age. These observations are consistent with a lifelong role for CFH in retinal homeostasis.

Postnatal enlargement of the mammalian intestine comprises cylindrical and luminal growth, associated with crypt fission and crypt/villus hyperplasia, respectively, which subsequently predominate before and after weaning. The bipartite adhesion G protein-coupled receptor CD97 shows an expression gradient along the crypt-villus axis in the normal human intestine. We here report that transgenic mice overexpressing CD97 in intestinal epithelial cells develop an upper megaintestine. Intestinal enlargement involves an increase in length and diameter but does not affect microscopic morphology, as typical for cylindrical growth. The megaintestine is acquired after birth and before weaning, independent of the genotype of the mother, excluding altered availability of milk constituents as driving factor. CD97 overexpression does not regulate intestinal growth factors, stem cell markers, and Wnt signaling, which contribute to epithelial differentiation and renewal, nor does it affect suckling-to-weaning transition. Consistent with augmented cylindrical growth, suckling but not adult transgenic mice show enlarged crypts and thus more crypt fissions caused by a transient increase of the crypt transit-amplifying zone. Intestinal enlargement by CD97 requires its seven-span transmembrane/cytoplasmic C-terminal fragment but not the N-terminal fragment binding partner CD55. In summary, ectopic expression of CD97 in intestinal epithelial cells provides a unique model for intestinal cylindrical growth occurring in breast-fed infants.

CD55 limits excessive complement activation on the host cell surface by accelerating the decay of C3 convertases. In this study, we observed that hepatitis C virus (HCV) infection of hepatocytes or HCV core protein expression in transfected hepatocytes upregulated CD55 expression at the mRNA and protein levels. Further analysis suggested that the HCV core protein or full-length (FL) genome enhanced CD55 promoter activity in a luciferase-based assay, which was further augmented in the presence of interleukin-6. Mutation of the CREB or SP-1 binding site on the CD55 promoter impaired HCV core protein-mediated upregulation of CD55. HCV-infected or core protein-transfected Huh7.5 cells displayed greater viability in the presence of CD81 and CD55 antibodies and complement. Biochemical analysis revealed that CD55 was associated with cell culture-grown HCV after purification by sucrose density gradient ultracentrifugation. Consistent with this, a polyclonal antibody to CD55 captured cell culture-grown HCV. Blocking antibodies against CD55 or virus envelope glycoproteins in the presence of normal human serum as a source of complement inhibited HCV infection. The inhibition was enhanced in the presence of both the antibodies and serum complement. Collectively, these results suggest that HCV induces and associates with a negative regulator of the complement pathway, a likely mechanism for immune evasion.

CD55 (decay-accelerating factor, DAF) is overexpressed in several types of cancer, including colorectal cancer. Because of its inhibitory effect on the complement system, it has been suggested as a possible target for cancer immunotherapy. However, CD55 is also expressed in normal tissues, body fluids and stroma, limiting the use of anti-CD55 therapeutic antibodies. Two novel CD55 splice variants or isoforms have recently been identified. These have been shown to contain part or all of intron 7 (CD55(int7+)), in contrast to the previously identified splice variants (CD55(wt)), which do not contain intron 7. Our aim was to determine the pattern of expression of the CD55(int7+) isoforms in normal and cancer tissues and to compare it to CD55(wt). We found that while CD55's isoforms levels vary directly, CD55(wt) is much more abundant (on average 48 times more) than CD55(int7+). Moreover, colon cancers that express high CD55(wt) mRNA levels tend to upregulate CD55(int7+) to a further extent. Finally, we compared the protein levels of CD55(int7+) to CD55(wt) by immunohistochemistry in various colorectal pathological conditions including neoplasia, and found that the levels of both isoforms were elevated in all types of colonic pathology. These results show that the levels of CD55(int7+) in normal tissue are much lower than CD55(wt), while in tumors it is restricted to the epithelial structures unlike CD55(wt). Thus, CD55(int7+) may be a more suitable target for cancer immunotherapy., (© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Nucleotide-binding oligomerization domain (NOD) 2 is a cytosolic protein that plays a defensive role in bacterial infection by sensing peptidoglycans. C5a, which has harmful effects in sepsis, interacts with innate proteins. However, whether NOD2 regulates C5a generation during sepsis remains to be determined. To address this issue, cecal ligation & puncture (CLP)-induced sepsis was compared in wild type and Nod2-/- mice. Nod2-/- mice showed lower levels of C5a, IL-10, and IL-1β in serum and peritoneum, but higher survival rate during CLP-induced sepsis compared to wild type mice. Injection of recombinant C5a decreased survival rates of Nod2-/- mice rate during sepsis, whereas it did not alter those in wild type mice. These findings suggest a novel provocative role for NOD2 in sepsis, in contrast to its protective role during bacterial infection. Furthermore, we found that NOD2-mediated IL-10 production by neutrophils enhanced C5a generation by suppressing CD55 expression on neutrophils in IL-1β-dependent and/or IL-1β-independent manners, thereby aggravating CLP-induced sepsis. SB203580, a receptor-interacting protein 2 (RIP2) inhibitor downstream of NOD2, reduced C5a generation by enhancing CD55 expression on neutrophils, resulting in attenuation of polymicrobial sepsis. Therefore, we propose a novel NOD2-mediated complement cascade regulatory pathway in sepsis, which may be a useful therapeutic target.

Adhesion G protein-coupled receptors (aGPCRs) are two-subunit molecules, consisting of an adhesive extracellular α subunit that couples noncovalently to a seven-transmembrane β subunit. The cooperation between the two subunits and the effect of endogenous ligands on the functioning of aGPCRs is poorly understood. In this study, we investigated the interaction between the pan-leukocyte aGPCR CD97 and its ligand CD55. We found that leukocytes from CD55-deficient mice express significantly increased levels of cell surface CD97 that normalized after transfer into wild-type mice because of contact with CD55 on both leukocytes and stromal cells. Downregulation of both CD97 subunits occurred within minutes after first contact with CD55 in vivo, which correlated with an increase in plasma levels of soluble CD97. In vitro, downregulation of CD97 on CD55-deficient leukocytes cocultured with wild-type blood cells was strictly dependent on shear stress. In vivo, CD55-mediated downregulation of CD97 required an intact circulation and was not observed on cells that lack contact with the blood stream, such as microglia. Notably, de novo ligation of CD97 did not activate signaling molecules constitutively engaged by CD97 in cancer cells, such as ERK and protein kinase B/Akt. We conclude that CD55 downregulates CD97 surface expression on circulating leukocytes by a process that requires physical forces, but based on current evidence does not induce receptor signaling. This regulation can restrict CD97-CD55-mediated cell adhesion to tissue sites.

We previously reported that nitric oxide (NO) reduces the rate of bacteremia and maternal mortality in pregnant rats with uterine infection by Escherichia coli expressing the Dr Fimbria (Dr(+) ). The epithelial invasion of Dr(+) E. coli is dependent on the expression level of its cellular receptor decay accelerating factor (DAF). NO reduces the rate of bacteremia by downregulating the expression of DAF. In this study, we elucidated the role of transcription factor Sp1 and RNA binding protein HuR in the downregulation of human DAF by NO. We generated a series of deletion mutant constructs of DAF gene 5'-untranslated region and mapped the NO-response region upstream to the core promoter region of the DAF gene. One of the several Sp1 binding sites in the DAF 5'-untranslated region was located within the NO-response region. The binding of Sp1 to this site was inhibited by NO. Furthermore, NO also promoted the degradation of DAF mRNA. The 3'-untranslated region of DAF harbors an AU-rich element and this element destabilized the mRNA transcript. NO promoted the rapid degradation of DAF mRNA by inhibiting the binding of mRNA stabilizing protein HuR to this AU-rich region. The inhibition of binding of HuR to the AU-rich region was due to the S-nitrosylation of one or more cysteine residues by NO. Thus, these data reveal the molecular mediators of transcriptional and post-transcriptional regulation of DAF by NO with implications in pathophysiology related to DAF., (© 2012 The Authors Journal compilation © 2012 FEBS.)

Background and Aim: Routine screening for paroxysmal nocturnal hemoglobinuria (PNH) in patients with Budd-Chiari syndrome (BCS) or portal vein thrombosis (PVT) has been recommended in Western countries. However, little is known about whether the routine screening test should be necessary in Chinese patients with BCS or PVT. We conducted a prospective observational study to examine the prevalence of PNH in these patients., Methods: Patients with primary BCS or non-malignant PVT who were consecutively admitted to our department or regularly followed up between September 2009 and December 2011 were eligible for the study and detected the expression of CD55 and CD59 on erythrocytes and granulocytes. The CD55 or CD59 deficiency was considered as the proportion of erythrocytes or granulocytes with normal expression of CD55 or CD59 was less than 90%. PNH was diagnosed by both CD55 and CD59 deficient clone at flow cytometry of peripheral blood cells., Results: CD55 and/or CD59 deficiencies were found in 1.6% (2/127) of patients with primary BCS, 1.0% (1/100) of non-malignant and non-cirrhotic patients with PVT, and 4.7% (4/85) of cirrhotic patients with PVT. Only one patient had both CD55 and CD59 deficiencies on granulocytes. But he had been diagnosed with PNH before BCS., Conclusions: Paroxysmal nocturnal hemoglobinuria was very rare in Chinese patients with BCS or PVT, suggesting that routine screening for PNH should not be indiscriminately performed in such patients., (© 2012 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd.)

A study of the complement system in cardiosurgical patients with moderate (40 patients) and marked (18 patients) hemolysis after coronary artery bypass grafting in conditions of cardiopulmonary bypass was carried out. Before and after operation the content of D35+-, D55+-erythrocytes and reticulocytes in blood, free hemoglobin in blood plasma, indicators of the functional state of classical, lectin and alternative pathways of complement activation as well as concentration of its terminal complex in blood serum were analyzed. It was established that development of marked hemolysis was associated with higher (compared with moderate hemolysis) content of terminal complement complex and reticulocytes in blood before operation as well as deficiency of D55+- erythrocytes and low activity of alternative pathway.

The coxsackievirus-adenovirus receptor (CAR) and decay-accelerating factor (DAF) have been identified as cellular receptors for coxsackievirus B3 (CVB3). The first described DAF-binding isolate was obtained during passage of the prototype strain, Nancy, on rhabdomyosarcoma (RD) cells, which express DAF but very little CAR. Here, the structure of the resulting variant, CVB3-RD, has been solved by X-ray crystallography to 2.74 Å, and a cryo-electron microscopy reconstruction of CVB3-RD complexed with DAF has been refined to 9.0 Å. This new high-resolution structure permits us to correct an error in our previous view of DAF-virus interactions, providing a new footprint of DAF that bridges two adjacent protomers. The contact sites between the virus and DAF clearly encompass CVB3-RD residues recently shown to be required for binding to DAF; these residues interact with DAF short consensus repeat 2 (SCR2), which is known to be essential for virus binding. Based on the new structure, the mode of the DAF interaction with CVB3 differs significantly from the mode reported previously for DAF binding to echoviruses.

Background: Epithelio- and endotheliotropic viruses often exert polarized entry and release that may be responsible for viral spread and dissemination. Hantaviruses, mostly rodent-borne members of the Bunyaviridae family infect epithelial and endothelial cells of different organs leading to organ dysfunction or even failure. Endothelial and renal epithelial cells belong to the target cells of Old World hantavirus. Therefore, we examined the release of hantaviruses in several renal epithelial cell culture models. We used Vero cells that are commonly used in hantavirus studies and primary human renal epithelial cells (HREpC). In addition, we analyzed MDCKII cells, an epithelial cell line of a dog kidney, which represents a widely accepted in vitro model of polarized monolayers for their permissiveness for hantavirus infection., Results: Vero C1008 and primary HREpCs were grown on porous-support filter inserts for polarization. Monolayers were infected with hantavirus Hantaan (HTNV) and Puumala (PUUV) virus. Supernatants from the apical and basolateral chamber of infected cells were analyzed for the presence of infectious particles by re-infection of Vero cells. Viral antigen and infectious particles of HTNV and PUUV were exclusively detected in supernatants collected from the apical chamber of infected Vero C1008 cells and HREpCs. MDCKII cells were permissive for hantavirus infection and polarized MDCKII cells released infectious hantaviral particles from the apical surface corresponding to the results of Vero and primary human epithelial cells., Conclusions: Pathogenic Old World hantaviruses are released from the apical surface of different polarized renal epithelial cells. We characterized MDCKII cells as a suitable polarized cell culture model for hantavirus infection studies.

Aspirin-exacerbated respiratory disease (AERD) is a respiratory disease characterized by acute bronchial responses upon the administration of non-steroidal anti‑inflammatory drugs (NSAIDs) and the immune response by mast cells is regarded as one of the noteworthy causes of AERD pathogenesis. The complement cascade is regarded as a key mechanism for clearing pathogens from the host. CD55 is one of the proteins involved in self-recognition, a central component of the complement system and autoimmunity. To investigate the associations between CD55 single nucleotide polymorphisms (SNPs) and the risk of AERD, we carried out logistic analyses with three genetic models and further regression analysis was performed with the fall rate of forced expiratory volume in 1 sec (FEV1) by aspirin provocation. However, our results demonstrate that no CD55 polymorphisms are associated with the risk of AERD and the fall rate of FEV1 (P>0.05). Therefore, our results suggest that CD55 polymorphisms are not genetic markers of aspirin‑induced bronchospasm, including FEV1, in the population studied. Although the genetic role of CD55 has been found to be integral to human immunity, our results indicate that genetic variations of CD55 do not influence the risk of AERD and the fall rate of FEV1 in the population studied.

C3 nephritic factors are autoantibodies that prolong the half-life or prevent regulation of the alternative pathway C3 convertase, resulting in uncontrolled complement activation. They are strongly associated with renal disease but their role in pathogenesis remains controversial. Here we optimized and compared a panel of assays to identify and interrogate nephritic factor activities. Of 101 patients with histologic or clinically evident disease, 48 were positive in some or all assays. In the presence of properdin, binding of autoantibody was detected in 39 samples and convertase stabilization was detected in 36. Forty-two of 48 nephritic factors tested prevented convertase decay by factor H, and most of these by decay accelerating factor (28) and complement receptor 1 (34). Representative properdin-independent nephritic factors had no effect on C5 cleavage and terminal pathway activity, while properdin-dependent nephritic factors enhanced activity. Biacore analysis of four purified IgG samples confirmed resistance to decay and showed that properdin-independent nephritic factors increased convertase half-life over 50-fold, whereas properdin-dependent nephritic factors increased the half-life 10- to 20-fold and also increased activity of the C3 convertase up to 10-fold. Thus, our study provides a rational approach to detect and characterize nephritic factors in patients.

Factors controlling complement activation appear to exert a protective effect on pregnancy. This is particularly important in women with thrombophilia. The aim of this study was to determine the transcript and protein levels of complement decay-accelerating factor (DAF) and membrane cofactor protein (MCP) in the placentas of women with acquired and inherited thrombophilia. Also, we assessed immunohistochemistry staining of inhibitors of the complement cascade, DAF and MCP proteins, in the placentas of thrombophilic women.Placentas were collected from eight women with inherited thrombophilia and ten with acquired thrombophilia.The levels of DAF and MCP transcripts were evaluated by qPCR, the protein level was evaluated by Western blot. We observed a higher transcript (p < 0.05) and protein (p < 0.001) levels of DAF and MCP in the placentas of thrombophilic women than in the control group. DAF and MCP were localized on villous syncytiotrophoblast membranes, but the assessment of staining in all groups did not differ. The observed higher expression level of proteins that control activation of complement control proteins is only seemingly contradictory to the changes observed for example in the antiphospholipid syndrome. However, given the hitherto known biochemical changes associated with thrombophilia, a mechanism in which increased expression of DAF and MCP in the placentas is an effect of proinflammatory cytokines, which accompanies thrombophilia, is probable.

Introduction: Murine infection with Trypanosoma cruzi (Tc) has been used to study the role of T-cells in the pathogenesis of human inflammatory idiopathic myositis. Absence of decay-accelerating factor 1 (Daf1) has been shown to enhance murine T-cell responses and autoimmunity., Methods: To determine whether Daf1 deficiency can exacerbate Tc-induced myositis, C57BL/6 DAF(+/+) and DAF(-/-) mice were inoculated with 5 × 10(4) trypomastigotes, and their morbidity, parasitemia, parasite burden, histopathology, and T-cell expansion were studied in the acute and chronic stages., Results: DAF(-/-) mice had lower parasitemia and parasite burden but higher morbidity, muscle histopathology, and increased number of CD44(+) (activated/memory phenotype) splenic CD4(+) and CD8(+) T-cells., Conclusions: An enhanced CD8(+) T-cell immune-specific response may explain the lower parasitemia and parasite burden levels and the increase in histopathological lesions. We propose that Tc-inoculated DAF(-/-) mice are a useful model to study T-cell mediated immunity in skeletal muscle tissues., (Copyright © 2012 Wiley Periodicals, Inc.)