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Sample records for complement factors induced

  1. Small Molecule-Induced Complement Factor D (Adipsin) Promotes Lipid Accumulation and Adipocyte Differentiation.

    PubMed

    Song, No-Joon; Kim, Suji; Jang, Byung-Hyun; Chang, Seo-Hyuk; Yun, Ui Jeong; Park, Ki-Moon; Waki, Hironori; Li, Dean Y; Tontonoz, Peter; Park, Kye Won

    2016-01-01

    Adipocytes are differentiated by various transcriptional cascades integrated on the master regulator, Pparγ. To discover new genes involved in adipocyte differentiation, preadipocytes were treated with three newly identified pro-adipogenic small molecules and GW7845 (a Pparγ agonist) for 24 hours and transcriptional profiling was analyzed. Four genes, Peroxisome proliferator-activated receptor γ (Pparγ), human complement factor D homolog (Cfd), Chemokine (C-C motif) ligand 9 (Ccl9), and GIPC PDZ Domain Containing Family Member 2 (Gipc2) were induced by at least two different small molecules but not by GW7845. Cfd and Ccl9 expressions were specific to adipocytes and they were altered in obese mice. Small hairpin RNA (shRNA) mediated knockdown of Cfd in preadipocytes inhibited lipid accumulation and expression of adipocyte markers during adipocyte differentiation. Overexpression of Cfd promoted adipocyte differentiation, increased C3a production, and led to induction of C3a receptor (C3aR) target gene expression. Similarly, treatments with C3a or C3aR agonist (C4494) also promoted adipogenesis. C3aR knockdown suppressed adipogenesis and impaired the pro-adipogenic effects of Cfd, further suggesting the necessity for C3aR signaling in Cfd-mediated pro-adipogenic axis. Together, these data show the action of Cfd in adipogenesis and underscore the application of small molecules to identify genes in adipocytes. PMID:27611793

  2. Small Molecule-Induced Complement Factor D (Adipsin) Promotes Lipid Accumulation and Adipocyte Differentiation

    PubMed Central

    Jang, Byung-Hyun; Chang, Seo-Hyuk; Yun, Ui Jeong; Park, Ki-Moon; Waki, Hironori; Li, Dean Y.; Tontonoz, Peter; Park, Kye Won

    2016-01-01

    Adipocytes are differentiated by various transcriptional cascades integrated on the master regulator, Pparγ. To discover new genes involved in adipocyte differentiation, preadipocytes were treated with three newly identified pro-adipogenic small molecules and GW7845 (a Pparγ agonist) for 24 hours and transcriptional profiling was analyzed. Four genes, Peroxisome proliferator-activated receptor γ (Pparγ), human complement factor D homolog (Cfd), Chemokine (C-C motif) ligand 9 (Ccl9), and GIPC PDZ Domain Containing Family Member 2 (Gipc2) were induced by at least two different small molecules but not by GW7845. Cfd and Ccl9 expressions were specific to adipocytes and they were altered in obese mice. Small hairpin RNA (shRNA) mediated knockdown of Cfd in preadipocytes inhibited lipid accumulation and expression of adipocyte markers during adipocyte differentiation. Overexpression of Cfd promoted adipocyte differentiation, increased C3a production, and led to induction of C3a receptor (C3aR) target gene expression. Similarly, treatments with C3a or C3aR agonist (C4494) also promoted adipogenesis. C3aR knockdown suppressed adipogenesis and impaired the pro-adipogenic effects of Cfd, further suggesting the necessity for C3aR signaling in Cfd-mediated pro-adipogenic axis. Together, these data show the action of Cfd in adipogenesis and underscore the application of small molecules to identify genes in adipocytes. PMID:27611793

  3. Complement fixation by rheumatoid factor.

    PubMed Central

    Tanimoto, K; Cooper, N R; Johnson, J S; Vaughan, J H

    1975-01-01

    The capacity for fixation and activation of hemolytic complement by polyclonal IgM rheumatoid factors (RF) isolated from sera of patients with rheumatoid arthritis and monoclonal IgM-RF isolated from the cryoprecipitates of patients with IgM-IgG mixed cryoglobulinemia was examined. RF mixed with aggregated, reduced, and alkylated human IgG (Agg-R/A-IgG) in the fluid phase failed to significantly reduce the level of total hemolytic complement, CH50, or of individual complement components, C1, C2, C3, and C5. However, sheep erythrocytes (SRC) coated with Agg-R/A-IgG or with reduced and alkylated rabbit IgG anti-SRC antibody were hemolyzed by complement in the presence of polyclonal IgM-RF. Human and guinea pig complement worked equally well. The degree of hemolysis was in direct proportion to the hemagglutination titer of the RF against the same coated cells. Monoclonal IgM-RF, normal human IgM, and purified Waldenström macroglobulins without antiglobulin activity were all inert. Hemolysis of coated SRC by RF and complement was inhibited by prior treatment of the complement source with chelating agents, hydrazine, cobra venom factor, specific antisera to C1q, CR, C5, C6, or C8, or by heating at 56 degrees C for 30 min. Purified radiolabeled C4, C3, and C8 included in the complement source were bound to hemolysed SRC in direct proportion to the degree of hemolysis. These data indicate that polyclonal IgM-RF fix and activate complement via the classic pathway. The system described for assessing complement fixation by isolated RF is readily adaptable to use with whole human serum. PMID:1078825

  4. Heat differentiated complement factor profiling.

    PubMed

    Hamsten, Carl; Skattum, Lillemor; Truedsson, Lennart; von Döbeln, Ulrika; Uhlén, Mathias; Schwenk, Jochen M; Hammarström, Lennart; Nilsson, Peter; Neiman, Maja

    2015-08-01

    Complement components and their cascade of reactions are important defense mechanisms within both innate and adaptive immunity. Many complement deficient patients still remain undiagnosed because of a lack of high throughput screening tools. Aiming towards neonatal proteome screening for immunodeficiencies, we used a multiplex profiling approach with antibody bead arrays to measure 9 complement proteins in serum and dried blood spots. Several complement components have been described as heat sensitive, thus their heat-dependent detectability was investigated. Using sera from 16 patients with complement deficiencies and 23 controls, we confirmed that the proteins C1q, C2, C3, C6, C9 and factor H were positively affected by heating, thus the identification of deficient patients was improved when preheating samples. Measurements of C7, C8 and factor I were negatively affected by heating and non-heated samples should be used in analysis of these components. In addition, a proof of concept study demonstrated the feasibility of labeling eluates from dried blood spots to perform a subsequent correct classification of C2-deficiencies. Our study demonstrates the potential of using multiplexed single binder assays for screening of complement components that open possibilities to expand such analysis to other forms of deficiencies.

  5. Flicker-induced retinal vasodilatation is not dependent on complement factor H polymorphism in healthy young subjects

    PubMed Central

    Told, Reinhard; Palkovits, Stefan; Boltz, Agnes; Schmidl, Doreen; Napora, Katarzyna J; Werkmeister, René M; Haslacher, Helmuth; Frantal, Sophie; Popa-Cherecheanu, Alina; Schmetterer, Leopold; Garhöfer, Gerhard

    2014-01-01

    Purpose The complement factor H (CFH) tyrosine 402 histidine (Y402H, rs1061170) variant is known to be significantly associated with age-related macular degeneration (AMD). Whether this genetic variant may impact retinal blood flow regulation is largely unknown. This study investigated whether flicker-induced vasodilation, an indicator for the coupling between neural activity and blood flow, is altered in subjects carrying the rs1061170 risk allele. Methods One hundred healthy subjects (aged between 18 and 45 years) were included in this study. Retinal blood flow regulation was tested by assessing retinal vessel calibres in response to stimulation with diffuse flicker light. Retinal vascular flicker responses were determined with a Dynamic Vessel Analyzer (DVA). In addition, genotyping for rs1061170 was performed. Results Eighteen subjects were homozygous for the risk allele C, 50 were homozygous for the ancestral allele T, and 31 subjects were heterozygous (CT). One subject had to be excluded from data evaluation, as no genetic analysis could be performed due to technical difficulties. Baseline diameters of retinal arteries (p = 0.39) and veins (p = 0.64) were comparable between the three groups. Flicker-induced vasodilation in both retinal arteries (p = 0.38) and retinal veins (p = 0.62) was also comparable between the three studied groups. Conclusions Our data indicate that homozygous healthy young carriers of the C risk allele at rs1061170 do not show abnormal flicker-induced vasodilation in the retina. This suggests that the high-risk genetic variant of CFH polymorphism does not impact neuro-vascular coupling in healthy subjects. PMID:24863099

  6. Association Between Microglia, Inflammatory Factors, and Complement with Loss of Hippocampal Mossy Fiber Synapses Induced by Trimethyltin.

    PubMed

    Kraft, Andrew D; McPherson, Christopher A; Harry, G Jean

    2016-07-01

    Complement-associated factors are implicated in pathogen presentation, neurodegeneration, and microglia resolution of tissue injury. To characterize complement activation with microglial clearance of degenerating mossy fiber boutons, hippocampal dentate granule neurons were ablated in CD-1 mice with trimethyltin (TMT; 2.2 mg/kg, i.p.). Neuronal apoptosis was accompanied by amoeboid microglia and elevations in tumor necrosis factor [Tnfa], interleukin 1β [Il1b], and Il6 mRNA and C1q protein. Inos mRNA levels were unaltered. Silver degeneration and synaptophysin staining indicated loss of synaptic innervation to CA3 pyramidal neurons. Reactive microglia with thickened bushy morphology showed co-localization of synaptophysin+ fragments. The initial response at 2 days post-TMT included transient elevations in Tnfa, Il1b, Il6, and Inos mRNA levels. A concurrent increase at 2 days was observed in arginase-1 [Arg1], Il10, transforming growth factor β1 [Tgfb1], and chitinase 3 like-3 [Ym1] mRNA levels. At 2 days, C1q protein was evident in the CA3 with elevated C1qa, C1qb, C3, Cr3a, and Cr3b mRNA levels. mRNA levels remained elevated at 5 days, returning to control by 14 days, corresponding to silver degeneration. mRNA levels for pentraxin3 (Ptx3) were elevated on day 2 and Ptx1 was not altered. Our data suggest an association between microglia reactivity, the induction of anti-inflammatory genes concurrent with pro-inflammatory genes and the expression of complement-associated factors with the degeneration of synapses following apoptotic neuronal loss.

  7. The alternative complement component factor B regulates UV-induced oedema, systemic suppression of contact and delayed hypersensitivity, and mast cell infiltration into the skin.

    PubMed

    Byrne, Scott N; Hammond, Kirsten J L; Chan, Carling Y-Y; Rogers, Linda J; Beaugie, Clare; Rana, Sabita; Marsh-Wakefield, Felix; Thurman, Joshua M; Halliday, Gary M

    2015-04-01

    Ultraviolet (UV) wavelengths in sunlight are the prime cause of skin cancer in humans with both the UVA and UVB wavebands making a contribution to photocarcinogenesis. UV has many different biological effects on the skin that contribute to carcinogenesis, including suppression of adaptive immunity, sunburn and altering the migration of mast cells into and away from irradiated skin. Many molecular mechanisms have been identified as contributing to skin responses to UV. Recently, using gene set enrichment analysis of microarray data, we identified the alternative complement pathway with a central role for factor B (fB) in UVA-induced immunosuppression. In the current study we used mice genetically deficient in fB (fB-/- mice) to study the functional role of the alternative complement pathway in skin responses to UV. We found that fB is required for not only UVA but also UVB-induced immunosuppression and solar-simulated UV induction of the oedemal component of sunburn. Factor B-/- mice had a larger number of resident skin mast cells than control mice, but unlike the controls did not respond to UV by increasing mast cell infiltration into the skin. This study provides evidence for a function role for fB in skin responses to UV radiation. Factor B regulates UVA and UVB induced immunosuppression, UV induced oedema and mast cell infiltration into the skin. The alternative complement pathway is therefore an important regulator of skin responses to UV.

  8. Iron-induced Local Complement Component 3 (C3) Up-regulation via Non-canonical Transforming Growth Factor (TGF)-β Signaling in the Retinal Pigment Epithelium*

    PubMed Central

    Li, Yafeng; Song, Delu; Song, Ying; Zhao, Liangliang; Wolkow, Natalie; Tobias, John W.; Song, Wenchao; Dunaief, Joshua L.

    2015-01-01

    Dysregulation of iron homeostasis may be a pathogenic factor in age-related macular degeneration (AMD). Meanwhile, the formation of complement-containing deposits under the retinal pigment epithelial (RPE) cell layer is a pathognomonic feature of AMD. In this study, we investigated the molecular mechanisms by which complement component 3 (C3), a central protein in the complement cascade, is up-regulated by iron in RPE cells. Modulation of TGF-β signaling, involving ERK1/2, SMAD3, and CCAAT/enhancer-binding protein-δ, is responsible for iron-induced C3 expression. The differential effects of spatially distinct SMAD3 phosphorylation sites at the linker region and at the C terminus determined the up-regulation of C3. Pharmacologic inhibition of either ERK1/2 or SMAD3 phosphorylation decreased iron-induced C3 expression levels. Knockdown of SMAD3 blocked the iron-induced up-regulation and nuclear accumulation of CCAAT/enhancer-binding protein-δ, a transcription factor that has been shown previously to bind the basic leucine zipper 1 domain in the C3 promoter. We show herein that mutation of this domain reduced iron-induced C3 promoter activity. In vivo studies support our in vitro finding of iron-induced C3 up-regulation. Mice with a mosaic pattern of RPE-specific iron overload demonstrated co-localization of iron-induced ferritin and C3d deposits. Humans with aceruloplasminemia causing RPE iron overload had increased RPE C3d deposition. The molecular events in the iron-C3 pathway represent therapeutic targets for AMD or other diseases exacerbated by iron-induced local complement dysregulation. PMID:25802332

  9. SIGN-R1 and complement factors are involved in the systemic clearance of radiation-induced apoptotic cells in whole-body irradiated mice

    SciTech Connect

    Park, Jin-Yeon; Loh, SoHee; Cho, Eun-hee; Choi, Hyeong-Jwa; Na, Tae-Young; Nemeno, Judee Grace E.; Lee, Jeong Ik; Yoon, Taek Joon; Choi, In-Soo; Lee, Minyoung; Lee, Jae-Seon; Kang, Young-Sun

    2015-08-07

    Although SIGN-R1-mediated complement activation pathway has been shown to enhance the systemic clearance of apoptotic cells, the role of SIGN-R1 in the clearance of radiation-induced apoptotic cells has not been characterized and was investigated in this study. Our data indicated that whole-body γ-irradiation of mice increased caspase-3{sup +} apoptotic lymphocyte numbers in secondary lymphoid organs. Following γ-irradiation, SIGN-R1 and complements (C4 and C3) were simultaneously increased only in the mice spleen tissue among the assessed tissues. In particular, C3 was exclusively activated in the spleen. The delayed clearance of apoptotic cells was markedly prevalent in the spleen and liver of SIGN-R1 KO mice, followed by a significant increase of CD11b{sup +} cells. These results indicate that SIGN-R1 and complement factors play an important role in the systemic clearance of radiation-induced apoptotic innate immune cells to maintain tissue homeostasis after γ-irradiation. - Highlights: • Splenic SIGN-R1{sup +} macrophages are activated after γ-irradiation. • C3 and C4 levels increased and C3 was activated in the spleen after γ-irradiation. • SIGN-R1 mediated the systemic clearance of radiation-induced apoptotic cells in spleen and liver.

  10. Dimerization of complement factor H-related proteins modulates complement activation in vivo.

    PubMed

    Goicoechea de Jorge, Elena; Caesar, Joseph J E; Malik, Talat H; Patel, Mitali; Colledge, Matthew; Johnson, Steven; Hakobyan, Svetlana; Morgan, B Paul; Harris, Claire L; Pickering, Matthew C; Lea, Susan M

    2013-03-19

    The complement system is a key component regulation influences susceptibility to age-related macular degeneration, meningitis, and kidney disease. Variation includes genomic rearrangements within the complement factor H-related (CFHR) locus. Elucidating the mechanism underlying these associations has been hindered by the lack of understanding of the biological role of CFHR proteins. Here we present unique structural data demonstrating that three of the CFHR proteins contain a shared dimerization motif and that this hitherto unrecognized structural property enables formation of both homodimers and heterodimers. Dimerization confers avidity for tissue-bound complement fragments and enables these proteins to efficiently compete with the physiological complement inhibitor, complement factor H (CFH), for ligand binding. Our data demonstrate that these CFHR proteins function as competitive antagonists of CFH to modulate complement activation in vivo and explain why variation in the CFHRs predisposes to disease.

  11. Factor H-related proteins determine complement-activating surfaces.

    PubMed

    Józsi, Mihály; Tortajada, Agustin; Uzonyi, Barbara; Goicoechea de Jorge, Elena; Rodríguez de Córdoba, Santiago

    2015-06-01

    Complement factor H-related proteins (FHRs) are strongly associated with different diseases involving complement dysregulation, which suggests a major role for these proteins regulating complement activation. Because FHRs are evolutionarily and structurally related to complement inhibitor factor H (FH), the initial assumption was that the FHRs are also negative complement regulators. Whereas weak complement inhibiting activities were originally reported for these molecules, recent developments indicate that FHRs may enhance complement activation, with important implications for the role of these proteins in health and disease. We review these findings here, and propose that FHRs represent a complex set of surface recognition molecules that, by competing with FH, provide improved discrimination of self and non-self surfaces and play a central role in determining appropriate activation of the complement pathway.

  12. Identification and characterization of complement factor H in Branchiostoma belcheri.

    PubMed

    Cai, Lu; Zhu, Jiu; Yin, Denghua; Chen, Liming; Jin, Ping; Ma, Fei

    2014-12-10

    Complement factor H (CFH) is an essential regulator of the complement system and plays very important roles in animal innate immunity. Although the complement system of amphioxus has been extensively studied, the expression in amphioxus and evolution of CFH gene remain unknown. In this study, we identified and characterized an amphioxus (Branchiostoma belcheri) CFH gene (designated as AmphiCFH). Our results showed that the full-length cDNA of AmphiCFH gene consists of 1295 bp nucleotides containing an 855 bp open reading frame (ORF) that was predicted to encode a 284 amino acid protein. The putative AmphiCFH protein possessed the characteristic of the CFH protein family, including typical CCP (complement control protein) domain. Real-time PCR analysis showed that the AmphiCFH was ubiquitously and differentially expressed in five investigated tissues (intestine, gills, notochord, muscles, and hepatic cecum). The expression level of the AmphiCFH gene was induced upon lipopolysaccharide stimulation, indicating that the AmphiCFH gene might be involved in innate immunity. In addition, phylogenetic analysis showed that the AmphiCFH gene was located between that of invertebrates and vertebrates, suggesting that the AmphiCFH gene is a member of the CFH gene family. In conclusion, our findings provided an insight into animal innate immunity and evolution of the CFH gene family.

  13. Identification and characterization of complement factor H in Branchiostoma belcheri.

    PubMed

    Cai, Lu; Zhu, Jiu; Yin, Denghua; Chen, Liming; Jin, Ping; Ma, Fei

    2014-12-10

    Complement factor H (CFH) is an essential regulator of the complement system and plays very important roles in animal innate immunity. Although the complement system of amphioxus has been extensively studied, the expression in amphioxus and evolution of CFH gene remain unknown. In this study, we identified and characterized an amphioxus (Branchiostoma belcheri) CFH gene (designated as AmphiCFH). Our results showed that the full-length cDNA of AmphiCFH gene consists of 1295 bp nucleotides containing an 855 bp open reading frame (ORF) that was predicted to encode a 284 amino acid protein. The putative AmphiCFH protein possessed the characteristic of the CFH protein family, including typical CCP (complement control protein) domain. Real-time PCR analysis showed that the AmphiCFH was ubiquitously and differentially expressed in five investigated tissues (intestine, gills, notochord, muscles, and hepatic cecum). The expression level of the AmphiCFH gene was induced upon lipopolysaccharide stimulation, indicating that the AmphiCFH gene might be involved in innate immunity. In addition, phylogenetic analysis showed that the AmphiCFH gene was located between that of invertebrates and vertebrates, suggesting that the AmphiCFH gene is a member of the CFH gene family. In conclusion, our findings provided an insight into animal innate immunity and evolution of the CFH gene family. PMID:25281822

  14. Complement

    MedlinePlus

    ... in: Cancer Certain infections Ulcerative colitis Decreased complement activity may be seen in: Cirrhosis Glomerulonephritis Hereditary angioedema Hepatitis Kidney transplant rejection Lupus nephritis Malnutrition Systemic lupus erythematosis

  15. Shiga toxin activates complement and binds factor H: evidence for an active role of complement in hemolytic uremic syndrome.

    PubMed

    Orth, Dorothea; Khan, Abdul Basit; Naim, Asma; Grif, Katharina; Brockmeyer, Jens; Karch, Helge; Joannidis, Michael; Clark, Simon J; Day, Anthony J; Fidanzi, Sonja; Stoiber, Heribert; Dierich, Manfred P; Zimmerhackl, Lothar B; Würzner, Reinhard

    2009-05-15

    Infections with enterohemorrhagic Escherichia coli (EHEC) are a major cause of hemolytic uremic syndrome (HUS). Shiga toxins (Stxs), especially Stx2, are believed to represent major virulence factors of EHEC, contributing to HUS pathogenesis. Beside EHEC-associated HUS, there are hereditary atypical forms of HUS, which are mostly caused by mutations of complement regulators. The aim of the present study was to investigate whether or not complement is also involved in the pathogenesis of EHEC-induced typical HUS, by being activated either directly or indirectly by involvement of its inhibitors. Purified Stx2 markedly activated complement via the alternative pathway and was found to bind to factor H (FH), however, only when it was active. No apparent cleavage or destruction of FH was visible, and cofactor activity in fluid phase was unaffected, but clearly delayed for surface-attached FH, where it is essential for host cell protection. Binding studies using FH constructs revealed that Stx2 binds to short consensus repeats (SCRs) 6-8 and SCRs18-20, but not to SCRs16-17, i.e., to regions involved in the surface recognition function of FH. In conclusion, complement, and in particular FH, not only plays an important role in atypical HUS, but most probably also in EHEC-induced HUS.

  16. Bullous pemphigoid autoantibodies directly induce blister formation without complement activation.

    PubMed

    Ujiie, Hideyuki; Sasaoka, Tetsumasa; Izumi, Kentaro; Nishie, Wataru; Shinkuma, Satoru; Natsuga, Ken; Nakamura, Hideki; Shibaki, Akihiko; Shimizu, Hiroshi

    2014-11-01

    Complement activation and subsequent recruitment of inflammatory cells at the dermal/epidermal junction are thought to be essential for blister formation in bullous pemphigoid (BP), an autoimmune blistering disease induced by autoantibodies against type XVII collagen (COL17); however, this theory does not fully explain the pathological features of BP. Recently, the involvement of complement-independent pathways has been proposed. To directly address the question of the necessity of the complement activation in blister formation, we generated C3-deficient COL17-humanized mice. First, we show that passive transfer of autoantibodies from BP patients induced blister formation in neonatal C3-deficient COL17-humanized mice without complement activation. By using newly generated human and murine mAbs against the pathogenic noncollagenous 16A domain of COL17 with high (human IgG1, murine IgG2), low (murine IgG1), or no (human IgG4) complement activation abilities, we demonstrate that the deposition of Abs, and not complements, is relevant to the induction of blister formation in neonatal and adult mice. Notably, passive transfer of BP autoantibodies reduced the amount of COL17 in lesional mice skin, as observed in cultured normal human keratinocytes treated with the same Abs. Moreover, the COL17 depletion was associated with a ubiquitin/proteasome pathway. In conclusion, the COL17 depletion induced by BP autoantibodies, and not complement activation, is essential for the blister formation under our experimental system.

  17. Complement factor H–related hybrid protein deregulates complement in dense deposit disease

    PubMed Central

    Chen, Qian; Wiesener, Michael; Eberhardt, Hannes U.; Hartmann, Andrea; Uzonyi, Barbara; Kirschfink, Michael; Amann, Kerstin; Buettner, Maike; Goodship, Tim; Hugo, Christian; Skerka, Christine; Zipfel, Peter F.

    2013-01-01

    The renal disorder C3 glomerulopathy with dense deposit disease (C3G-DDD) pattern results from complement dysfunction and primarily affects children and young adults. There is no effective treatment, and patients often progress to end-stage renal failure. A small fraction of C3G-DDD cases linked to factor H or C3 gene mutations as well as autoantibodies have been reported. Here, we examined an index family with 2 patients with C3G-DDD and identified a chromosomal deletion in the complement factor H–related (CFHR) gene cluster. This deletion resulted in expression of a hybrid CFHR2-CFHR5 plasma protein. The recombinant hybrid protein stabilized the C3 convertase and reduced factor H–mediated convertase decay. One patient was refractory to plasma replacement and exchange therapy, as evidenced by the hybrid protein quickly returning to pretreatment plasma levels. Subsequently, complement inhibitors were tested on serum from the patient for their ability to block activity of CFHR2-CFHR5. Soluble CR1 restored defective C3 convertase regulation; however, neither eculizumab nor tagged compstatin had any effect. Our findings provide insight into the importance of CFHR proteins for C3 convertase regulation and identify a genetic variation in the CFHR gene cluster that promotes C3G-DDD. Monitoring copy number and sequence variations in the CFHR gene cluster in C3G-DDD and kidney patients with C3G-DDD variations will help guide treatment strategies. PMID:24334459

  18. Von Willebrand factor regulates complement on endothelial cells.

    PubMed

    Noone, Damien G; Riedl, Magdalena; Pluthero, Fred G; Bowman, Mackenzie L; Liszewski, M Kathryn; Lu, Lily; Quan, Yi; Balgobin, Steve; Schneppenheim, Reinhard; Schneppenheim, Sonja; Budde, Ulrich; James, Paula; Atkinson, John P; Palaniyar, Nades; Kahr, Walter H A; Licht, Christoph

    2016-07-01

    Atypical hemolytic uremic syndrome and thrombotic thrombocytopenic purpura have traditionally been considered separate entities. Defects in the regulation of the complement alternative pathway occur in atypical hemolytic uremic syndrome, and defects in the cleavage of von Willebrand factor (VWF)-multimers arise in thrombotic thrombocytopenic purpura. However, recent studies suggest that both entities are related as defects in the disease-causing pathways overlap or show functional interactions. Here we investigate the possible functional link of VWF-multimers and the complement system on endothelial cells. Blood outgrowth endothelial cells (BOECs) were obtained from 3 healthy individuals and 2 patients with Type 3 von Willebrand disease lacking VWF. Cells were exposed to a standardized complement challenge via the combination of classical and alternative pathway activation and 50% normal human serum resulting in complement fixation to the endothelial surface. Under these conditions we found the expected release of VWF-multimers causing platelet adhesion onto BOECs from healthy individuals. Importantly, in BOECs derived from patients with von Willebrand disease complement C3c deposition and cytotoxicity were more pronounced than on BOECs derived from normal individuals. This is of particular importance as primary glomerular endothelial cells display a heterogeneous expression pattern of VWF with overall reduced VWF abundance. Thus, our results support a mechanistic link between VWF-multimers and the complement system. However, our findings also identify VWF as a new complement regulator on vascular endothelial cells and suggest that VWF has a protective effect on endothelial cells and complement-mediated injury. PMID:27236750

  19. LPS induces pulp progenitor cell recruitment via complement activation.

    PubMed

    Chmilewsky, F; Jeanneau, C; Laurent, P; About, I

    2015-01-01

    Complement system, a major component of the natural immunity, has been recently identified as an important mediator of the dentin-pulp regeneration process through STRO-1 pulp cell recruitment by the C5a active fragment. Moreover, it has been shown recently that under stimulation with lipoteichoic acid, a complex component of the Gram-positive bacteria cell wall, human pulp fibroblasts are able to synthesize all proteins required for complement activation. However, Gram-negative bacteria, which are also involved in tooth decay, are known as powerful activators of complement system and inflammation. Here, we investigated the role of Gram-negative bacteria-induced complement activation on the pulp progenitor cell recruitment using lipopolysaccharide (LPS), a major component of all Gram-negative bacteria. Our results show that incubating pulp fibroblasts with LPS induced membrane attack complex formation and C5a release in serum-free fibroblast cultures. The produced C5a binds to the pulp progenitor cells' membrane and induces their migration toward the LPS stimulation chamber, as revealed by the dynamic transwell migration assays. The inhibition of this migration by the C5aR-specific antagonist W54011 indicates that the pulp progenitor migration is mediated by the interaction between C5a and C5aR. Our findings demonstrate, for the first time, a direct interaction between the recruitment of progenitor pulp cells and the activation of complement system generated by pulp fibroblast stimulation with LPS.

  20. Role of complement in porphyrin-induced photosensitivity

    SciTech Connect

    Lim, H.W.; Gigli, I.

    1981-01-01

    Addition of porphyrins to sera of guinea pigs in vitro, followed by irradiation with 405 nm light, resulted in dose-dependent inhibitions of hemolytic activity of complement. With guinea pig as an animal model, we also found that systemically administered porphyrins, followed by irradiation with 405 nm light, resulted in dose-dependent inhibition of CH50 in vivo. The erythrocytes from porphyrin-treated guinea pigs showed an increased susceptibility to hemolysis induced by 405 nm irradiation in vitro. Clinical changes in these animals were limited to light-exposed areas and consisted of erythema, crusting, and delayed growth of hair. Histologically, dermal edema, dilation of blood vessels, and infiltration of mononuclear and polymorphonuclear cells were observed. Guinea pigs irradiated with ultraviolet-B developed erythema, but had no alteration of their complement profiles. It is suggested that complement products may play a specific role in the pathogenesis of the cutaneous lesions of some porphyrias.

  1. Complement factor H polymorphism and age-related macular degeneration.

    PubMed

    Edwards, Albert O; Ritter, Robert; Abel, Kenneth J; Manning, Alisa; Panhuysen, Carolien; Farrer, Lindsay A

    2005-04-15

    Age-related macular degeneration (AMD) is a common, late-onset, and complex trait with multiple risk factors. Concentrating on a region harboring a locus for AMD on 1q25-31, the ARMD1 locus, we tested single-nucleotide polymorphisms for association with AMD in two independent case-control populations. Significant association (P = 4.95 x 10(-10)) was identified within the regulation of complement activation locus and was centered over a tyrosine-402 --> histidine-402 protein polymorphism in the gene encoding complement factor H. Possession of at least one histidine at amino acid position 402 increased the risk of AMD 2.7-fold and may account for 50% of the attributable risk of AMD.

  2. Complement-related proteins control the flavivirus infection of Aedes aegypti by inducing antimicrobial peptides.

    PubMed

    Xiao, Xiaoping; Liu, Yang; Zhang, Xiaoyan; Wang, Jing; Li, Zuofeng; Pang, Xiaojing; Wang, Penghua; Cheng, Gong

    2014-04-01

    The complement system functions during the early phase of infection and directly mediates pathogen elimination. The recent identification of complement-like factors in arthropods indicates that this system shares common ancestry in vertebrates and invertebrates as an immune defense mechanism. Thioester (TE)-containing proteins (TEPs), which show high similarity to mammalian complement C3, are thought to play a key role in innate immunity in arthropods. Herein, we report that a viral recognition cascade composed of two complement-related proteins limits the flaviviral infection of Aedes aegypti. An A. aegypti macroglobulin complement-related factor (AaMCR), belonging to the insect TEP family, is a crucial effector in opposing the flaviviral infection of A. aegypti. However, AaMCR does not directly interact with DENV, and its antiviral effect requires an A. aegypti homologue of scavenger receptor-C (AaSR-C), which interacts with DENV and AaMCR simultaneously in vitro and in vivo. Furthermore, recognition of DENV by the AaSR-C/AaMCR axis regulates the expression of antimicrobial peptides (AMPs), which exerts potent anti-DENV activity. Our results both demonstrate the existence of a viral recognition pathway that controls the flaviviral infection by inducing AMPs and offer insights into a previously unappreciated antiviral function of the complement-like system in arthropods.

  3. Inhibition of the alternative complement pathway by antisense oligonucleotides targeting complement factor B improves lupus nephritis in mice.

    PubMed

    Grossman, Tamar R; Hettrick, Lisa A; Johnson, Robert B; Hung, Gene; Peralta, Raechel; Watt, Andrew; Henry, Scott P; Adamson, Peter; Monia, Brett P; McCaleb, Michael L

    2016-06-01

    Systemic lupus erythematosus is an autoimmune disease that manifests in widespread complement activation and deposition of complement fragments in the kidney. The complement pathway is believed to play a significant role in the pathogenesis and in the development of lupus nephritis. Complement factor B is an important activator of the alternative complement pathway and increasing evidence supports reducing factor B as a potential novel therapy to lupus nephritis. Here we investigated whether pharmacological reduction of factor B expression using antisense oligonucleotides could be an effective approach for the treatment of lupus nephritis. We identified potent and well tolerated factor B antisense oligonucleotides that resulted in significant reductions in hepatic and plasma factor B levels when administered to normal mice. To test the effects of factor B antisense oligonucleotides on lupus nephritis, we used two different mouse models, NZB/W F1 and MRL/lpr mice, that exhibit lupus nephritis like renal pathology. Antisense oligonucleotides mediated reductions in circulating factor B levels were associated with significant improvements in renal pathology, reduced glomerular C3 deposition and proteinuria, and improved survival. These data support the strategy of using factor B antisense oligonucleotides for treatment of lupus nephritis in humans.

  4. Factor C acts as a lipopolysaccharide-responsive C3 convertase in horseshoe crab complement activation.

    PubMed

    Ariki, Shigeru; Takahara, Shusaku; Shibata, Toshio; Fukuoka, Takaaki; Ozaki, Aya; Endo, Yuichi; Fujita, Teizo; Koshiba, Takumi; Kawabata, Shun-ichiro

    2008-12-01

    The complement system in vertebrates plays an important role in host defense against and clearance of invading microbes, in which complement component C3 plays an essential role in the opsonization of pathogens, whereas the molecular mechanism underlying C3 activation in invertebrates remains unknown. In an effort to understand the molecular activation mechanism of invertebrate C3, we isolated and characterized an ortholog of C3 (designated TtC3) from the horseshoe crab Tachypleus tridentatus. Flow cytometric analysis using an Ab against TtC3 revealed that the horseshoe crab complement system opsonizes both Gram-negative and Gram-positive bacteria. Evaluation of the ability of various pathogen-associated molecular patterns to promote the proteolytic conversion of TtC3 to TtC3b in hemocyanin-depleted plasma indicated that LPS, but not zymosan, peptidoglycan, or laminarin, strongly induces this conversion, highlighting the selective response of the complement system to LPS stimulation. Although originally characterized as an LPS-sensitive initiator of hemolymph coagulation stored within hemocytes, we identified factor C in hemolymph plasma. An anti-factor C Ab inhibited various LPS-induced phenomena, including plasma amidase activity, the proteolytic activation of TtC3, and the deposition of TtC3b on the surface of Gram-negative bacteria. Moreover, activated factor C present on the surface of Gram-negative bacteria directly catalyzed the proteolytic conversion of the purified TtC3, thereby promoting TtC3b deposition. We conclude that factor C acts as an LPS-responsive C3 convertase on the surface of invading Gram-negative bacteria in the initial phase of horseshoe crab complement activation.

  5. The quantitative role of alternative pathway amplification in classical pathway induced terminal complement activation.

    PubMed

    Harboe, M; Ulvund, G; Vien, L; Fung, M; Mollnes, T E

    2004-12-01

    Complement activation with formation of biologically potent mediators like C5a and the terminal C5b-9 complex (TCC) contributes essentially to development of inflammation and tissue damage in a number of autoimmune and inflammatory conditions. A particular role for complement in the ischaemia/reperfusion injury of the heart, skeletal muscle, central nervous system, intestine and kidney has been suggested from animal studies. Previous experiments in C3 and C4 knockout mice suggested an important role of the classical or lectin pathway in initiation of complement activation during intestinal ischaemia/reperfusion injury while later use of factor D knockout mice showed the alternative pathway to be critically involved. We hypothesized that alternative pathway amplification might play a more critical role in classical pathway-induced C5 activation than previously recognized and used pathway-selective inhibitory mAbs to further elucidate the role of the alternative pathway. Here we demonstrate that selective blockade of the alternative pathway by neutralizing factor D in human serum diluted 1 : 2 with mAb 166-32 inhibited more than 80% of C5a and TCC formation induced by solid phase IgM and solid- and fluid-phase human aggregated IgG via the classical pathway. The findings emphasize the influence of alternative pathway amplification on the effect of initial classical pathway activation and the therapeutic potential of inhibiting the alternative pathway in clinical conditions with excessive and uncontrolled complement activation. PMID:15544620

  6. Complement factor B activation in patients with preeclampsia.

    PubMed

    Velickovic, Ivan; Dalloul, Mudar; Wong, Karen A; Bakare, Olufunke; Schweis, Franz; Garala, Maya; Alam, Amit; Medranda, Giorgio; Lekovic, Jovana; Shuaib, Waqas; Tedjasukmana, Andreas; Little, Perry; Hanono, Daniel; Wijetilaka, Ruvini; Weedon, Jeremy; Lin, Jun; Toledano, Roulhac d'Arby; Zhang, Ming

    2015-06-01

    Preeclampsia is a leading cause of maternal and fetal morbidity and mortality. Bb, the active fragment of complement factor B (fB), has been reported to be a predictor of preeclampsia. However, conflicting results have been found by some investigators. We hypothesized that the disagreement in findings may be due to the racial/ethnic differences among various study groups, and that fB activation is significant in women of an ethnic minority with preeclampsia. We investigated the maternal and fetal levels of Bb (the activated fB fragment) in pregnant women of an ethnic minority with or without preeclampsia. We enrolled 291 pregnant women (96% of an ethnic minority, including 78% African-American). Thirteen percent of these were diagnosed with preeclampsia. Maternal venous blood was collected from all participants together with fetal umbilical cord blood samples from 154 deliveries in the 291 women. The results were analyzed using the Mann-Whitney U test and multivariate analyses. Maternal Bb levels were significantly higher in the preeclamptic group than in the nonpreeclamptic group. Levels of Bb in fetal cord blood were similar in both groups. Subgroup analyses of African-American patients' results confirmed the study hypothesis that there would be a significant increase in Bb in the maternal blood of the preeclamptic group and no increase in Bb in the fetal cord blood of this group. These results suggest that a maternal immune response through complement fB might play a role in the development of preeclampsia, particularly in African-American patients.

  7. Shiga toxin-induced complement-mediated hemolysis and release of complement-coated red blood cell-derived microvesicles in hemolytic uremic syndrome.

    PubMed

    Arvidsson, Ida; Ståhl, Anne-Lie; Hedström, Minola Manea; Kristoffersson, Ann-Charlotte; Rylander, Christian; Westman, Julia S; Storry, Jill R; Olsson, Martin L; Karpman, Diana

    2015-03-01

    Shiga toxin (Stx)-producing Escherichia coli (STEC) cause hemolytic uremic syndrome (HUS). This study investigated whether Stx2 induces hemolysis and whether complement is involved in the hemolytic process. RBCs and/or RBC-derived microvesicles from patients with STEC-HUS (n = 25) were investigated for the presence of C3 and C9 by flow cytometry. Patients exhibited increased C3 deposition on RBCs compared with controls (p < 0.001), as well as high levels of C3- and C9-bearing RBC-derived microvesicles during the acute phase, which decreased after recovery. Stx2 bound to P1 (k) and P2 (k) phenotype RBCs, expressing high levels of the P(k) Ag (globotriaosylceramide), the known Stx receptor. Stx2 induced the release of hemoglobin and lactate dehydrogenase in whole blood, indicating hemolysis. Stx2-induced hemolysis was not demonstrated in the absence of plasma and was inhibited by heat inactivation, as well as by the terminal complement pathway Ab eculizumab, the purinergic P2 receptor antagonist suramin, and EDTA. In the presence of whole blood or plasma/serum, Stx2 induced the release of RBC-derived microvesicles coated with C5b-9, a process that was inhibited by EDTA, in the absence of factor B, and by purinergic P2 receptor antagonists. Thus, complement-coated RBC-derived microvesicles are elevated in HUS patients and induced in vitro by incubation of RBCs with Stx2, which also induced hemolysis. The role of complement in Stx2-mediated hemolysis was demonstrated by its occurrence only in the presence of plasma and its abrogation by heat inactivation, EDTA, and eculizumab. Complement activation on RBCs could play a role in the hemolytic process occurring during STEC-HUS.

  8. Abnormal immune complex processing and spontaneous glomerulonephritis in complement factor H-deficient mice with human complement receptor 1 on erythrocytes.

    PubMed

    Alexander, Jessy J; Hack, Bradley K; Jacob, Alexander; Chang, Anthony; Haas, Mark; Finberg, Robert W; Quigg, Richard J

    2010-09-15

    Complement receptor 1 (CR1) on human erythrocytes (Es) and complement factor H (CFH) on rodent platelets perform immune adherence, which is a function that allows the processing of immune complexes (ICs) bearing C3 by the mononuclear phagocyte system. Similar immune adherence occurs in the glomerular podocyte by CR1 in humans and CFH in rodents. As a model for human IC processing, we studied transgenic mice lacking CFH systemically but with human CR1 on Es. These CR1(hu)Tg/CFH(-/-) mice spontaneously developed proliferative glomerulonephritis, which was accelerated in a chronic serum sickness model by active immunization with heterologous apoferritin. ICs containing Ag, IgG and C3 bound to Es in CR1(hu)Tg/CFH(-/-) mice. In this setting, there was increased IC deposition in glomeruli, attributable to the presence of CR1 on Es, together with the absence of CFH on platelets and podocytes. In the absence of plasma CFH, the accumulated ICs activated complement, which led to spontaneous and chronic serum sickness-induced proliferative glomerulonephritis. These findings illustrate the complexities of complement-dependent IC processing by blood cells and in the glomerulus, and the importance of CFH as a plasma complement regulator.

  9. Complement depletion with humanised cobra venom factor: efficacy in preclinical models of vascular diseases.

    PubMed

    Vogel, Carl-Wilhelm; Fritzinger, David C; Gorsuch, W Brian; Stahl, Gregory L

    2015-03-01

    The complement system is an intrinsic part of the immune system and has important functions in both innate and adaptive immunity. On the other hand, inadvertent or misdirected complement activation is also involved in the pathogenesis of many diseases, contributing solely or significantly to tissue injury and disease development. Multiple approaches to develop pharmacological agents to inhibit complement are currently being pursued. We have developed a conceptually different approach of not inhibiting but depleting complement, based on the complement-depleting activities of cobra venom factor (CVF), a non-toxic cobra venom component with structural and functional homology to complement component C3. We developed a humanised version of CVF by creating human complement component C3 derivatives with complement-depleting activities of CVF (humanised CVF) as a promising therapeutic agent for diseases with complement pathogenesis. Here we review the beneficial therapeutic effect of humanised CVF in several murine models of vascular diseases such as reperfusion injury.

  10. Complement Evasion Mediated by Enhancement of Captured Factor H: Implications for Protection of Self-Surfaces from Complement

    PubMed Central

    Herbert, Andrew P.; Makou, Elisavet; Chen, Zhuo A.; Kerr, Heather; Richards, Anna; Rappsilber, Juri

    2015-01-01

    In an attempt to evade annihilation by the vertebrate complement system, many microbes capture factor H (FH), the key soluble complement-regulating protein in human plasma. However, FH is normally an active complement suppressor exclusively on self-surfaces and this selective action of FH is pivotal to self versus non-self discrimination by the complement system. We investigated whether the bacterially captured FH becomes functionally enhanced and, if so, how this is achieved at a structural level. We found, using site-directed and truncation mutagenesis, surface plasmon resonance, nuclear magnetic resonance spectroscopy, and cross-linking and mass spectrometry, that the N-terminal domain of Streptococcus pneumoniae protein PspC (PspCN) not only binds FH extraordinarily tightly but also holds it in a previously uncharacterized conformation. Functional enhancement arises from exposure of a C-terminal cryptic second binding site in FH for C3b, the activation-specific fragment of the pivotal complement component, C3. This conformational change of FH doubles its affinity for C3b and increases 5-fold its ability to accelerate decay of the binary enzyme (C3bBb) responsible for converting C3 to C3b in an amplification loop. Despite not sharing critical FH-binding residues, PspCNs from D39 and Tigr4 S. pneumoniae exhibit similar FH-anchoring and enhancing properties. We propose that these bacterial proteins mimic molecular markers of self-surfaces, providing a compelling hypothesis for how FH prevents complement-mediated injury to host tissue while lacking efficacy on virtually all other surfaces. In hemolysis assays with 2-aminoethylisothiouronium bromide–treated erythrocytes that recapitulate paroxysmal nocturnal hemoglobinuria, PspCN enhanced protection of cells by FH, suggesting a new paradigm for therapeutic complement suppression. PMID:26459349

  11. Enhancement of complement-mediated lysis by a peptide derived from SCR 13 of complement factor H.

    PubMed

    Stoiber, H; Ammann, C; Spruth, M; Müllauer, B; Eberhart, A; Harris, C L; Huber, C G; Longhi, R; Falkensammer, B; Würzner, R; Dierich, M P

    2001-05-01

    Complement factor H (fH) is an important regulator of complement activation. It contributes to protection of cells against homologous complement attack. In this study we tested the effect of fH-depletion of normal human serum (NHS) on lysis of antibody-coated sheep and human erythrocytes (EshA and EhuA). In the absence of fH, lysis of sensitised Esh and Ehu was clearly increased. Addition of fH to fH-depleted serum re-established protection of cells against complement similar to that seen with NHS. A fH-derived peptide (pepAred), covering the N-terminal half of SCR 13 in fH, was able to enhance complement-mediated lysis of EhuA significantly. However, the oxidised form of this peptide (pepAox) had no effect. Biotinylated pepAred, but not pepAox, was able to directly bind to cells. Additionally, pepAred competed with direct fH-cell interaction which was observable only after treatment of purified fH with mercaptoethanol. Only pepAred increased the amount of C3 fragments and reduced levels of fH detectable on cells as shown by FACS analysis and radio-immuno assay. Furthermore, fH and factor I (fI)-mediated cleavage of agarose bound C3b into iC3b was decreased in the presence of pepAred. These data indicate that a fH-derived peptide can enhance complement-mediated lysis. We will continue to investigate whether the use of a fH peptide can be exploited for therapeutical purposes. PMID:11402501

  12. Anti-GD2 with an FC point mutation reduces complement fixation and decreases antibody-induced allodynia

    PubMed Central

    Sorkin, Linda S.; Otto, Mario; Baldwin, William M.; Vail, Emily; Gillies, Stephen D.; Handgretinger, Rupert; Barfield, Raymond C.; Yu, Hui Ming; Yu, Alice L.

    2013-01-01

    Monoclonal antibodies against GD2 ganglioside, such as ch14.18, the human–mouse chimeric antibody, have been shown to be effective for the treatment of neuroblastoma. However, treatment is associated with generalized, relatively opiate-resistant pain. We investigated if a point mutation in ch14.18 antibody (hu14.18K332A) to limit complement-dependent cytotoxicity (CDC) would ameliorate the pain behavior, while preserving antibody-dependent cellular cytotoxicity (ADCC). In vitro, CDC and ADCC were measured using europium-TDA assay. In vivo, allodynia was evaluated by measuring thresholds to von Frey filaments applied to the hindpaws after injection of either ch14.18 or hu14.18K332 into wild type rats or rats with deficient complement factor 6. Other rats were pretreated with complement factor C5a receptor antagonist and tested following ch14.18 injection. The mutation reduces the antibody’s ability to activate complement, while maintaining its ADCC capabilities. Injection of hu14.18K322 (1 or 3 mg/kg) produced faster resolving allodynia than that engendered by ch14.18 (1 mg/kg). Injection of ch14.18 (1 mg/kg) into rats with C6 complement deficiency further reduced antibody-induced allodynia, while pre-treatment with complement factor C5a receptor antagonist completely abolished ch14.18-induced allodynia. These findings showed that mutant hu14.18 K322 elicited less allodynia than ch14.18 and that ch14.18-elicited allodynia is due to activation of the complement cascade: in part, to formation of membrane attack complex, but more importantly to release of complement factor C5a. Development of immunotherapeutic agents with decreased complement-dependent lysis while maintaining cellular cytotoxicity may offer treatment options with reduced adverse side effects, thereby allowing dose escalation of therapeutic antibodies. PMID:20171010

  13. Complement Factor H Autoantibodies are Associated with Early Stage NSCLC

    PubMed Central

    Amornsiripanitch, Nita; Hong, Shaolin; Campa, Michael J.; Frank, Michael M.; Gottlin, Elizabeth B.; Patz, Edward F.

    2010-01-01

    Purpose In order to discover diagnostic biomarkers associated with early stage non-small cell lung cancer (NSCLC), we searched for autoantibodies preferentially present in stage I patients compared to patients with advanced stage disease. Here we describe an autoantibody against complement factor H (CFH) and its association with early stage NSCLC. Experimental Design Immunoblots were used to detect autoantibodies in the sera of stage I NSCLC patients. An autoantibody recognizing a 150 kDa protein was discovered and the protein was identified by mass spectrometry. The association of the autoantibody with early stage disease was suggested by the results of immunoblot analysis with sera from 28 stage I patients and 28 stage III/IV patients. This association was confirmed by protein microarray of sera from 125 NSCLC patients of all stages as well as 125 age, gender, and smoking history matched controls. Results The immunoreactive protein was identified as CFH. By immunoblot analysis, anti-CFH autoantibody was found in 50% of stage I NSCLC patients and 11% of late stage NSCLC patients (P=0.003). By protein microarray analysis, patients with stage I NSCLC had a significantly higher incidence of anti-CFH antibody than those with late stage NSCLC (P=0.0051). The percentage of sera with a positive level of CFH autoantibody was 30.4% in stage I, 21.1% in stage II, 12.5% in stage III, 7.4% in stage IV and 8.0% in the control group. Conclusions These findings suggest that in patients with NSCLC, CFH autoantibody is a molecular marker associated with early stage disease. PMID:20515868

  14. Pasteurella pneumotropica Evades the Human Complement System by Acquisition of the Complement Regulators Factor H and C4BP

    PubMed Central

    Sahagún-Ruiz, Alfredo; Granados Martinez, Adriana Patricia; Breda, Leandro Carvalho Dantas; Fraga, Tatiana Rodrigues; Castiblanco Valencia, Mónica Marcela; Barbosa, Angela Silva; Isaac, Lourdes

    2014-01-01

    Pasteurella pneumotropica is an opportunist Gram negative bacterium responsible for rodent pasteurellosis that affects upper respiratory, reproductive and digestive tracts of mammals. In animal care facilities the presence of P. pneumotropica causes severe to lethal infection in immunodeficient mice, being also a potential source for human contamination. Indeed, occupational exposure is one of the main causes of human infection by P. pneumotropica. The clinical presentation of the disease includes subcutaneous abscesses, respiratory tract colonization and systemic infections. Given the ability of P. pneumotropica to fully disseminate in the organism, it is quite relevant to study the role of the complement system to control the infection as well as the possible evasion mechanisms involved in bacterial survival. Here, we show for the first time that P. pneumotropica is able to survive the bactericidal activity of the human complement system. We observed that host regulatory complement C4BP and Factor H bind to the surface of P. pneumotropica, controlling the activation pathways regulating the formation and maintenance of C3-convertases. These results show that P. pneumotropica has evolved mechanisms to evade the human complement system that may increase the efficiency by which this pathogen is able to gain access to and colonize inner tissues where it may cause severe infections. PMID:25347183

  15. Differential Complement Activation Pathways Promote C3b Deposition on Native and Acetylated LDL thereby Inducing Lipoprotein Binding to the Complement Receptor 1

    PubMed Central

    Klop, Boudewijn; van der Pol, Pieter; van Bruggen, Robin; Wang, Yanan; de Vries, Marijke A.; van Santen, Selvetta; O'Flynn, Joseph; van de Geijn, Gert-Jan M.; Njo, Tjin L.; Janssen, Hans W.; de Man, Peter; Jukema, J. Wouter; Rabelink, Ton J.; Rensen, Patrick C. N.; van Kooten, Cees; Cabezas, Manuel Castro

    2014-01-01

    Lipoproteins can induce complement activation resulting in opsonization and binding of these complexes to complement receptors. We investigated the binding of opsonized native LDL and acetylated LDL (acLDL) to the complement receptor 1 (CR1). Binding of complement factors C3b, IgM, C1q, mannose-binding lectin (MBL), and properdin to LDL and acLDL were investigated by ELISA. Subsequent binding of opsonized LDL and acLDL to CR1 on CR1-transfected Chinese Hamster Ovarian cells (CHO-CR1) was tested by flow cytometry. Both native LDL and acLDL induced complement activation with subsequent C3b opsonization upon incubation with normal human serum. Opsonized LDL and acLDL bound to CR1. Binding to CHO-CR1 was reduced by EDTA, whereas MgEGTA only reduced the binding of opsonized LDL, but not of acLDL suggesting involvement of the alternative pathway in the binding of acLDL to CR1. In vitro incubations showed that LDL bound C1q, whereas acLDL bound to C1q, IgM, and properdin. MBL did neither bind to LDL nor to acLDL. The relevance of these findings was demonstrated by the fact that ex vivo up-regulation of CR1 on leukocytes was accompanied by a concomitant increased binding of apolipoprotein B-containing lipoproteins to leukocytes without changes in LDL-receptor expression. In conclusion, CR1 is able to bind opsonized native LDL and acLDL. Binding of LDL to CR1 is mediated via the classical pathway, whereas binding of acLDL is mediated via both the classical and alternative pathways. Binding of lipoproteins to CR1 may be of clinical relevance due to the ubiquitous cellular distribution of CR1. PMID:25349208

  16. Dynamics and reproductive effects of complement factors in the spontaneous abortion model of CBA/J×DBA/2 mice.

    PubMed

    Takeshita, Ai; Kusakabe, Ken Takeshi; Hiyama, Masato; Kuniyoshi, Nobue; Kondo, Tomohiro; Kano, Kiyoshi; Kiso, Yasuo; Okada, Toshiya

    2014-05-01

    The complement system is one component of innate immunity that could participate in fetal loss. We have already reported that adipsin, a complement activator in the alternative pathway, is stably expressed in the placenta and that an increase in this expression is related to spontaneous abortion. However, complement inhibitor Crry was concurrently expressed in the placenta, and the role of complement factors during pregnancy was not clear. In the present study, we examined the endogenous regulation of complement factors in placenta and serum by using another model mouse for spontaneous abortion and studied the effect of exogenous complement disruption on pregnancy. Compared to control mice, the CBA/J×DBA/2 model mice had higher expression levels of adipsin in the placenta and serum. Adipsin and complement C3 were localized in the metrial gland and labyrinth regions, and both positive reactive ranges were limited in the maternal blood current in normal implantation sites. These results suggest that extrauterine adipsin hematogenously reaches the placenta, activates complement C3, and promotes destruction of the feto-maternal barrier in aborted implantation sites. Crry was consistently expressed in the placenta and serum and reduced in the resorption sites of CBA/J×DBA/2 mice as compared to normal sites. Injection of recombinant adipsin increased the resorption rate and changed the expression of Th-type cytokines toward a Th1 bias. The present study indicates that adipsin could induce the fetal loss that accompanies the Th1 bias and may be a crucial cause of spontaneous abortion. In addition, the local expression of Crry prevents complement activation in placenta in response to a systemic increase of adipsin.

  17. Complement anaphylatoxin C3a is a potent inducer of embryonic chick retina regeneration

    PubMed Central

    Haynes, Tracy; Luz-Madrigal, Agustin; Reis, Edimara S.; Echeverri Ruiz, Nancy P.; Grajales-Esquivel, Erika; Tzekou, Apostolia; Tsonis, Panagiotis A.; Lambris, John D.; Del Rio-Tsonis, Katia

    2013-01-01

    Identifying the initiation signals for tissue regeneration in vertebrates is one of the major challenges in regenerative biology. Much of the research thus far has indicated that certain growth factors have key roles. Here we show that complement fragment C3a is sufficient to induce complete regeneration of the embryonic chick retina from stem/progenitor cells present in the eye, independent of fibroblast growth factor receptor signaling. Instead, C3a induces retina regeneration via STAT3 activation, which in turn activates the injury- and inflammation-responsive factors, IL-6, IL-8 and TNF-α. This activation sets forth regulation of Wnt2b, Six3 and Sox2, genes associated with retina stem and progenitor cells. Thus, our results establish a mechanism for retina regeneration based on injury and inflammation signals. Furthermore, our results indicate a unique function for complement anaphylatoxins that implicate these molecules in the induction and complete regeneration of the retina, opening new avenues of experimentation in the field. PMID:23942241

  18. Regulation of age-related macular degeneration-like pathology by complement factor H

    PubMed Central

    Toomey, Christopher B.; Kelly, Una; Saban, Daniel R.; Bowes Rickman, Catherine

    2015-01-01

    Complement factor H (CFH) is a major susceptibility gene for age-related macular degeneration (AMD); however, its impact on AMD pathobiology is unresolved. Here, the role of CFH in the development of AMD pathology in vivo was interrogated by analyzing aged Cfh+/− and Cfh−/− mice fed a high-fat, cholesterol-enriched diet. Strikingly, decreased levels of CFH led to increased sub-retinal pigmented epithelium (sub-RPE) deposit formation, specifically basal laminar deposits, following high-fat diet. Mechanistically, our data show that deposits are due to CFH competition for lipoprotein binding sites in Bruch’s membrane. Interestingly and despite sub-RPE deposit formation occurring in both Cfh+/− and Cfh−/− mice, RPE damage accompanied by loss of vision occurred only in old Cfh+/− mice. We demonstrate that such pathology is a function of excess complement activation in Cfh+/− mice versus complement deficiency in Cfh−/− animals. Due to the CFH-dependent increase in sub-RPE deposit height, we interrogated the potential of CFH as a previously unidentified regulator of Bruch’s membrane lipoprotein binding and show, using human Bruch’s membrane explants, that CFH removes endogenous human lipoproteins in aged donors. Thus, advanced age, high-fat diet, and decreased CFH induce sub-RPE deposit formation leading to complement activation, which contributes to RPE damage and visual function impairment. This new understanding of the complicated interactions of CFH in AMD-like pathology provides an improved foundation for the development of targeted therapies for AMD. PMID:25991857

  19. Elastase of Pseudomonas aeruginosa: Inactivation of Complement Components and Complement-Derived Chemotactic and Phagocytic Factors

    PubMed Central

    Schultz, Duane R.; Miller, Kent D.

    1974-01-01

    A purified elastase from Pseudomonas aeruginosa was highly destructive for fluid-phase and cell-bound C1 and C3 and fluid-phase C5, C8, and C9. Inactivation of C4, C2, C6, and C7 by the enzyme varied from 0 to 67%. Low concentrations of elastase generated, then inactivated, a chemotactic factor from human C5 but not from C3. Higher enzyme concentrations inactivated the C5 chemotactic activity at a faster rate. Elastase treatment of sensitized pseudomonads containing cell-bound C3 reduced the phagocytic indexes of polymorphonuclear leukocytes. The data support the proposed chemopathogenic role of the elastase in generation of the characteristic non-inflammatory Pseudomonas vasculitis. Images PMID:4210424

  20. Relationship between the complement system, risk factors and prediction models in age-related macular degeneration.

    PubMed

    Bora, Nalini S; Matta, Bharati; Lyzogubov, Valeriy V; Bora, Puran S

    2015-02-01

    Studies performed over the past decade in humans and experimental animals have been a major source of information and improved our understanding of how dysregulation of the complement system contributes to age-related macular degeneration (AMD) pathology. Drusen, the hall-mark of dry-type AMD are reported to be the by-product of complement mediated inflammatory processes. In wet AMD, unregulated complement activation results in increased production of angiogenic growth factors leading to choroidal neovascularization both in humans and in animal models. In this review article we have linked the complement system with modifiable and non-modifiable AMD risk factors as well as with prediction models of AMD. Understanding the association between the complement system, risk factors and prediction models will help improve our understanding of AMD pathology and management of this disease.

  1. Variants in Complement Factor H and Complement Factor H-Related Protein Genes, CFHR3 and CFHR1, Affect Complement Activation in IgA Nephropathy

    PubMed Central

    Zhu, Li; Zhai, Ya-Ling; Wang, Feng-Mei; Hou, Ping; Lv, Ji-Cheng; Xu, Da-Min; Shi, Su-Fang; Liu, Li-Jun; Yu, Feng; Zhao, Ming-Hui; Novak, Jan; Gharavi, Ali G.

    2015-01-01

    Complement activation is common in patients with IgA nephropathy (IgAN) and associated with disease severity. Our recent genome-wide association study of IgAN identified susceptibility loci on 1q32 containing the complement regulatory protein-encoding genes CFH and CFHR1–5, with rs6677604 in CFH as the top single-nucleotide polymorphism and CFHR3–1 deletion (CFHR3–1∆) as the top signal for copy number variation. In this study, to explore the clinical effects of variation in CFH, CFHR3, and CFHR1 on IgAN susceptibility and progression, we enrolled two populations. Group 1 included 1178 subjects with IgAN and available genome-wide association study data. Group 2 included 365 subjects with IgAN and available clinical follow-up data. In group 1, rs6677604 was associated with mesangial C3 deposition by genotype–phenotype correlation analysis. In group 2, we detected a linkage between the rs6677604-A allele and CFHR3–1∆ and found that the rs6677604-A allele was associated with higher serum levels of CFH and lower levels of the complement activation split product C3a. Furthermore, CFH levels were positively associated with circulating C3 levels and negatively associated with mesangial C3 deposition. Moreover, serum levels of the pathogenic galactose-deficient glycoform of IgA1 were also associated with the degree of mesangial C3 deposition in patients with IgAN. Our findings suggest that genetic variants in CFH, CFHR3, and CFHR1 affect complement activation and thereby, predispose patients to develop IgAN. PMID:25205734

  2. Effect of sulfide ions on complement factor C3.

    PubMed Central

    Granlund-Edstedt, M; Johansson, E; Claesson, R; Carlsson, J

    1991-01-01

    In infected sites such as the gingival pockets of patients with periodontal disease, sulfide levels up to 1 mmol/liter may be reached. There is little information, however, on how sulfide may interact with the host defense. In a previous study (R. Claesson, M. Granlund-Edstedt, S. Persson, and J. Carlsson, Infect. Immun. 57:2776-2781, 1989), it was shown that polymorphonuclear leukocytes were able to kill bacteria in the presence of 1 mM sulfide. However, sulfide seemed to interfere with the opsonization of the bacteria. It has been claimed that sulfide may be toxic by splitting disulfide bonds of proteins. In the present study, serum was exposed to 2 mM sulfide under anaerobic conditions, and the capacity of sulfide to split disulfide bonds of 10 serum proteins involved in opsonization was evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunodetection of the proteins after blotting. Sulfide had a low capacity to split the disulfide bonds of most proteins. Sulfide had, however, a pronounced effect on the complement component C3 in the form of C3bi. Sulfide released the C-terminal region of the alpha chain from C3bi. When C3 opsonizes bacteria, it is this region of C3bi which binds to complement receptor 3 (CR3) of the polymorphonuclear leukocytes. If sulfide has the same effect on C3bi deposited on the bacterial surface as it has on C3bi in solution, it will annihilate the very important contribution of C3bi to opsonization. Images PMID:1987085

  3. Inhibition of heparin/protamine complex-induced complement activation by Compstatin in baboons.

    PubMed

    Soulika, A M; Khan, M M; Hattori, T; Bowen, F W; Richardson, B A; Hack, C E; Sahu, A; Edmunds, L H; Lambris, J D

    2000-09-01

    Complement activation products are major components of the inflammatory response induced by cardiac surgery and cardiopulmonary bypass which contribute to postoperative organ dysfunction, fluid accumulation, and morbidity. Activation of the complement system occurs during extracorporeal circulation, during reperfusion of ischemic tissue, and after the formation of heparin-protamine complexes. In this study we examine the efficacy of Compstatin, a recently discovered peptide inhibitor of complement, in preventing heparin/protamine-induced complement activation in baboons. The study was performed in baboons because Compstatin binds to baboon C3 and is resistant to proteolytic cleavage in baboon blood (similar to humans); Compstatin inhibits only the activation of primates' complement system. After testing various doses and administration regimens, Compstatin produced complete inhibition at a total dose of 21 mg/kg when given as a combination of bolus injection and infusion. Compstatin completely inhibited in vivo heparin/protamine-induced complement activation without adverse effects on heart rate or systemic arterial, central venous, and pulmonary arterial pressures. This study indicates that Compstatin is a safe and effective complement inhibitor that has the potential to prevent complement activation during and after clinical cardiac surgery. Furthermore, Compstatin can serve as the prototype for designing an orally administrated drug.

  4. Translational Mini-Review Series on Complement Factor H: Therapies of renal diseases associated with complement factor H abnormalities: atypical haemolytic uraemic syndrome and membranoproliferative glomerulonephritis

    PubMed Central

    Noris, M; Remuzzi, G

    2008-01-01

    Genetic and acquired abnormalities in complement factor H (CFH) have been associated with two different human renal diseases: haemolytic uraemic syndrome and membrano proliferative glomerulonephritis. The new genetic and pathogenetic findings in these diseases and their clinical implications for the management and cure of patients are reviewed in this paper. PMID:18070148

  5. Gain-of-function mutations in complement factor B are associated with atypical hemolytic uremic syndrome

    PubMed Central

    de Jorge, Elena Goicoechea; Harris, Claire L.; Esparza-Gordillo, Jorge; Carreras, Luis; Arranz, Elena Aller; Garrido, Cynthia Abarrategui; López-Trascasa, Margarita; Sánchez-Corral, Pilar; Morgan, B. Paul; de Córdoba, Santiago Rodríguez

    2007-01-01

    Hemolytic uremic syndrome (HUS) is an important cause of acute renal failure in children. Mutations in one or more genes encoding complement-regulatory proteins have been reported in approximately one-third of nondiarrheal, atypical HUS (aHUS) patients, suggesting a defect in the protection of cell surfaces against complement activation in susceptible individuals. Here, we identified a subgroup of aHUS patients showing persistent activation of the complement alternative pathway and found within this subgroup two families with mutations in the gene encoding factor B (BF), a zymogen that carries the catalytic site of the complement alternative pathway convertase (C3bBb). Functional analyses demonstrated that F286L and K323E aHUS-associated BF mutations are gain-of-function mutations that result in enhanced formation of the C3bBb convertase or increased resistance to inactivation by complement regulators. These data expand our understanding of the genetic factors conferring predisposition to aHUS, demonstrate the critical role of the alternative complement pathway in the pathogenesis of aHUS, and provide support for the use of complement-inhibition therapies to prevent or reduce tissue damage caused by dysregulated complement activation. PMID:17182750

  6. Effects of two types of cobra venom factor on porcine complement activation and pulmonary artery pressure.

    PubMed Central

    Cheung, A K; Parker, C J; Wilcox, L

    1989-01-01

    Autologous porcine plasma that has been incubated with cuprophan haemodialysis membranes causes pulmonary hypertension and peripheral leucopenia following reinfusion into swine. These effects appear to be mediated by biologically active fragments of C3 and C5 that are generated as a consequence of ex vivo activation of complement. Putatively, C5a induces the leucopenia; however, the specific contributions of products of C3 and C5 activation to the pulmonary vasoconstriction have not been elucidated. In the present study, the effects of in vivo infusion of two different types of cobra venom factor (CVF) on peripheral leucocyte count and pulmonary artery pressure in the swine are reported. The CVF from Naja n. naja (CVF(TN)) was shown to activate both porcine C3 and C5, whereas the CVF from Naja h. haje (CVF(NH)) activated only C3. Both types of CVF produced pulmonary hypertension. Significant peripheral leucopenia, however, was observed only with CVF(TN). These results suggest that activation products of C3 contribute to the pulmonary hypertension but not to the peripheral leucopenia observed during haemodialysis using dialysis membranes that activate complement. PMID:12412765

  7. Complement control protein factor H: the good, the bad, and the inadequate

    PubMed Central

    Ferreira, Viviana P.; Pangburn, Michael K.; Cortés, Claudio

    2010-01-01

    The complement system is an essential component of the innate immune system that participates in elimination of pathogens and altered host cells and comprises an essential link between the innate and adaptive immune system. Soluble and membrane-bound complement regulators protect cells and tissues from unintended complement-mediated injury. Complement factor H is a soluble complement regulator essential for controlling the alternative pathway in blood and on cell surfaces. Normal recognition of self cell markers (i.e. polyanions) and C3b/C3d fragments is necessary for factor H function. Inadequate recognition of host cell surfaces by factor H due to mutations and polymorphisms have been associated with complement-mediated tissue damage and disease. On the other hand, unwanted recognition of pathogens and altered self cells (i.e. cancer) by factor H is used as an immune evasion strategy. This review will focus on the current knowledge related to these versatile recognition properties of factor H. PMID:20580090

  8. In vitro inactivation of complement by a serum factor present in Junin-virus infected guinea-pigs.

    PubMed Central

    Rimoldi, M T; de Bracco, M M

    1980-01-01

    A serum factor(s) of guinea-pigs infected with Junin virus, the etiological agent of Argentine haemorrhagic fever, is endowed with a potent anticomplementary activity. It is resistant to heat (56 degrees, 30 min) and elutes from a Sephadex G-200 column between albumin and haemoglobin. It is ineffective in the presence of EDTA or EGTA and does not sediment at 82,000 g. It has no direct effect on C4 unless functional Cl is present. However, it induces Cl activation that consumes C4 haemolytic activity in normal human and guinea-pig sera. The evidence presented in this report demonstrates that the complement activation observed in experimental Argentine haemorrhagic fever is at least in part due to a direct effect of this serum factor on the classical complement pathway. PMID:6247264

  9. Classical complement activation as a footprint for murine and human antiphospholipid antibody-induced fetal loss.

    PubMed

    Cohen, Danielle; Buurma, Aletta; Goemaere, Natascha N; Girardi, Guillermina; le Cessie, Saskia; Scherjon, Sicco; Bloemenkamp, Kitty W M; de Heer, Emile; Bruijn, Jan A; Bajema, Ingeborg M

    2011-12-01

    Recurrent miscarriage, fetal growth restriction and intrauterine fetal death are frequently occurring complications of pregnancy in patients with systemic lupus erythaematosus (SLE) and antiphospholipid syndrome (APS). Murine models show that complement activation plays a pivotal role in antiphospholipid antibody-mediated pregnancy morbidity, but the exact pathways of complement activation and their potential role in human pregnancy are insufficiently understood. We hypothesized that the classical pathway would play a major role in inducing fetal loss. Pregnant C57BL/6 mice and mice deficient in C1q and factor D were injected with antiphospholipid antibodies or normal human IgG. Mouse placentas were subsequently stained with an anti-C4 antibody and anti-normal human IgG to determine the presence of classical complement activation and IgG binding. Findings in mice were validated in 88 human placentae from 83 women (SLE and APS cases versus controls), which were immunohistochemically stained for C4d, C1q, properdin and MBL. Staining patterns were compared to pregnancy outcome. In murine placentae of mice pretreated with antiphospholipid antibodies, increased C4 deposition was observed, which was associated with adverse fetal outcome but not with IgG binding. In humans, diffuse C4d staining at the feto-maternal interface was present almost exclusively in patients with SLE and/or APS (p < 0.001) and was related to intrauterine fetal death (p = 0.03). Our data show that presence of C4d in murine and human placentae is strongly related to adverse fetal outcome in the setting of SLE and APS. The excessive deposition of C4d supports the concept of severe autoantibody-mediated injury at the fetal-maternal interface. We suggest C4d as a potential biomarker of autoantibody-mediated fetal loss in SLE and APS.

  10. Complement inhibition in biomaterial- and biosurface-induced thromboinflammation.

    PubMed

    Ekdahl, Kristina N; Huang, Shan; Nilsson, Bo; Teramura, Yuji

    2016-06-01

    Therapeutic medicine today includes a vast number of procedures involving the use of biomaterials, transplantation of therapeutic cells or cell clusters, as well as of solid organs. These treatment modalities are obviously of great benefit to the patient, but also present a great challenge to the innate immune system, since they involve direct exposure of non-biological materials, cells of non-hematological origin as well as endothelial cells, damaged by ischemia-perfusion in solid organs to proteins and cells in the blood. The result of such an exposure may be an inappropriate activation of the complement and contact/kallikrein systems, which produce mediators capable of triggering the platelets and PMNs and monocytes, which can ultimately result in thrombotic and inflammatory (i.e., a thrombo-inflammatory) response to the treatment modality. In this concept review, we give an overview of the mechanisms of recognition within the innate immunity system, with the aim to identify suitable points for intervention. Finally, we discuss emerging and promising techniques for surface modification of biomaterials and cells with specific inhibitors in order to diminish thromboinflammation and improve clinical outcome.

  11. Translational Mini-Review Series on Complement Factor H: Renal diseases associated with complement factor H: novel insights from humans and animals

    PubMed Central

    Pickering, M C; Cook, H T

    2008-01-01

    Factor H is the major regulatory protein of the alternative pathway of complement activation. Abnormalities in factor H have been associated with renal disease, namely glomerulonephritis with C3 deposition including membranoproliferative glomerulonephritis (MPGN) and the atypical haemolytic uraemic syndrome (aHUS). Furthermore, a common factor H polymorphism has been identified as a risk factor for the development of age-related macular degeneration. These associations suggest that alternative pathway dysregulation is a common feature in the pathogenesis of these conditions. However, with respect to factor H-associated renal disease, it is now clear that distinct molecular defects in the protein underlie the pathogenesis of glomerulonephritis and HUS. In this paper we review the associations between human factor H dysfunction and renal disease and explore how observations in both spontaneous and engineered animal models of factor H dysfunction have contributed to our understanding of the pathogenesis of factor H-related renal disease. PMID:18190458

  12. Quantitative differences between complement factor-B phenotypes.

    PubMed

    Mortensen, J P; Lamm, L U

    1981-04-01

    In a study of 365 unrelated blood donors the structural polymorphism was determined by high-voltage electrophoresis followed by immunofixation with monospecific anti-Bf serum as described by Alper, Boenish & Watson (1972), and serum levels were measured by rocket-immunoelectrophoresis as described by Sjöholm (1975). We found that the mean level of Factor B in relation to structural phenotypes varies significantly in the order BfF greater than BfFS greater than BfS, though great variation was observed within types. A similar difference was also found with a functional assay of Factor B in agarose plates, although this method is less accurate. Concerning the biological functions, such as opsonization and bacteriolysis, it might be that individuals with the BfF allele are more able to withstand infectious agents than BfS subjects. The Bf polymorphism may therefore be transient, the BfF allele being in a process of replacing BfS by natural selection.

  13. Anti-phospholipid antibodies restore mesenteric ischemia/reperfusion-induced injury in complement receptor 2/complement receptor 1-deficient mice.

    PubMed

    Fleming, Sherry D; Egan, Ryan P; Chai, Chunyan; Girardi, Guillermina; Holers, V Michael; Salmon, Jane; Monestier, Marc; Tsokos, George C

    2004-12-01

    Complement receptor 2-deficient (Cr2(-/-)) mice are resistant to mesenteric ischemia/reperfusion (I/R) injury because they lack a component of the natural Ab repertoire. Neither the nature of the Abs that are involved in I/R injury nor the composition of the target Ag, to which recognition is lacking in Cr2(-/-) mice, is known. Because anti-phospholipid Abs have been shown to mediate fetal growth retardation and loss when injected into pregnant mice, we performed experiments to determine whether anti-phospholipid Abs can also reconstitute I/R injury and, therefore, represent members of the injury-inducing repertoire that is missing in Cr2(-/-) mice. We demonstrate that both murine and human monoclonal and polyclonal Abs against negatively charged phospholipids can reconstitute mesenteric I/R-induced intestinal and lung tissue damage in Cr2(-/-) mice. In addition, Abs against beta2 glycoprotein I restore local and remote tissue damage in the Cr2(-/-) mice. Unlike Cr2(-/-) mice, reconstitution of I/R tissue damage in the injury-resistant Rag-1(-/-) mouse required the infusion of both anti-beta2-glycoprotein I and anti-phospholipid Ab. We conclude that anti-phospholipid Abs can bind to tissues subjected to I/R insult and mediate tissue damage. PMID:15557203

  14. Inhibition of biomaterial-induced complement activation attenuates the inflammatory host response to implantation

    PubMed Central

    Kourtzelis, Ioannis; Rafail, Stavros; DeAngelis, Robert A.; Foukas, Periklis G.; Ricklin, Daniel; Lambris, John D.

    2013-01-01

    Although complement is a known contributor to biomaterial-induced complications, pathological implications and therapeutic options remain to be explored. Here we investigated the involvement of complement in the inflammatory response to polypropylene meshes commonly used for hernia repair. In vitro assays revealed deposition of complement activation fragments on the mesh after incubation in plasma. Moreover, significant mesh-induced complement and granulocyte activation was observed in plasma and leukocyte preparations, respectively. Pretreatment of plasma with the complement inhibitor compstatin reduced opsonization >2-fold, and compstatin and a C5a receptor antagonist (C5aRa) impaired granulocyte activation by 50 and 67%, respectively. We established a clinically relevant mouse model of implantation and could confirm deposition of C3 activation fragments on mesh implants in vivo using immunofluorescence. In meshes extracted after subcutaneous or peritoneal implantation, the amount of immune cell infiltrate in mice deficient in key complement components (C3, C5aR), or treated with C5aRa, was approximately half of that observed in wild-type littermates or mice treated with inactive C5aRa, respectively. Our data suggest that implantation of a widely used surgical mesh triggers the formation of an inflammatory cell microenvironment at the implant site through complement activation, and indicates a path for the therapeutic modulation of implant-related complications.—Kourtzelis, I., Rafail, S., DeAngelis, R. A., Foukas, P. G., Ricklin, D., Lambris, J. D. Inhibition of biomaterial-induced complement activation attenuates the inflammatory host response to implantation. PMID:23558338

  15. Interaction of Shiga toxin 2 with complement regulators of the factor H protein family.

    PubMed

    Poolpol, Kulwara; Orth-Höller, Dorothea; Speth, Cornelia; Zipfel, Peter F; Skerka, Christine; de Córdoba, Santiago Rodriguez; Brockmeyer, Jens; Bielaszewska, Martina; Würzner, Reinhard

    2014-03-01

    Shiga toxin 2 (Stx2) is believed to be a major virulence factor of enterohemorrhagic Escherichia coli (EHEC) contributing to hemolytic uremic syndrome (HUS). The complement system has recently been found to be involved in the pathogenesis of EHEC-associated HUS. Stx2 was shown to activate complement via the alternative pathway, to bind factor H (FH) at short consensus repeats (SCRs) 6-8 and 18-20 and to delay and reduce FH cofactor activity on the cell surface. We now show that complement factor H-related protein 1 (FHR-1) and factor H-like protein 1 (FHL-1), proteins of the FH protein family that show amino acid sequence and regulatory function similarities with FH, also bind to Stx2. The FHR-1 binding site for Stx2 was located at SCRs 3-5 and the binding capacity of FHR-1*A allotype was higher than that of FHR-1*B. FHR-1 and FHL-1 competed with FH for Stx2 binding, and in the case of FHR-1 this competition resulted in a reduction of FH cofactor activity. FHL-1 retained its cofactor activity in the fluid phase when bound to Stx2. In conclusion, multiple interactions of key complement inhibitors FH, FHR-1 and FHL-1 with Stx2 corroborate our hypothesis of a direct role of complement in EHEC-associated HUS.

  16. Oncolytic and immunotherapeutic vaccinia induces antibody-mediated complement-dependent cancer cell lysis in humans.

    PubMed

    Kim, Mi Kyung; Breitbach, Caroline J; Moon, Anne; Heo, Jeong; Lee, Yu Kyoung; Cho, Mong; Lee, Jun Woo; Kim, Seong-Geun; Kang, Dae Hwan; Bell, John C; Park, Byeong Ho; Kirn, David H; Hwang, Tae-Ho

    2013-05-15

    Oncolytic viruses cause direct cytolysis and cancer-specific immunity in preclinical models. The goal of this study was to demonstrate induction of functional anticancer immunity that can lyse target cancer cells in humans. Pexa-Vec (pexastimogene devacirepvec; JX-594) is a targeted oncolytic and immunotherapeutic vaccinia virus engineered to express human granulocyte-macrophage colony-stimulating factor (GM-CSF). Pexa-Vec demonstrated replication, GM-CSF expression, and tumor responses in previous phase 1 trials. We now evaluated whether Pexa-Vec induced functional anticancer immunity both in the rabbit VX2 tumor model and in patients with diverse solid tumor types in phase 1. Antibody-mediated complement-dependent cancer cell cytotoxicity (CDC) was induced by intravenous Pexa-Vec in rabbits; transfer of serum from Pexa-Vec-treated animals to tumor-bearing animals resulted in tumor necrosis and improved survival. In patients with diverse tumor types treated on a phase 1 trial, CDC developed within 4 to 8 weeks in most patients; normal cells were resistant to the cytotoxic effects. T lymphocyte activation in patients was evidenced by antibody class switching. We determined that patients with the longest survival duration had the highest CDC activity, and identified candidate target tumor cell antigens. Thus, we demonstrated that Pexa-Vec induced polyclonal antibody-mediated CDC against multiple tumor antigens both in rabbits and in patients with diverse solid tumor types.

  17. An extended mini-complement factor H molecule ameliorates experimental C3 glomerulopathy

    PubMed Central

    Nichols, Eva-Maria; Barbour, Thomas D; Pappworth, Isabel Y; Wong, Edwin K S; Palmer, Jeremy M; Sheerin, Neil S; Pickering, Matthew C; Marchbank, Kevin J

    2015-01-01

    Abnormal regulation of the complement alternative pathway is associated with C3 glomerulopathy. Complement factor H is the main plasma regulator of the alternative pathway and consists of 20 short consensus repeat (SCR) domains. Although recombinant full-length factor H represents a logical treatment for C3 glomerulopathy, its production has proved challenging. We and others have designed recombinant mini-factor H proteins in which ‘non-essential' SCR domains have been removed. Here, we report the in vitro and in vivo effects of a mini-complement factor H protein, FH1–5^18–20, using the unique factor H–deficient (Cfh−/−) mouse model of C3 glomerulopathy. FH1–5^18–20 is comprised of the key complement regulatory domains (SCRs 1–5) linked to the surface recognition domains (SCRs 18–20). Intraperitoneal injection of FH1–5^18–20 in Cfh−/− mice reduced abnormal glomerular C3 deposition, similar to full-length factor H. Systemic effects on plasma alternative pathway control were comparatively modest, in association with a short half-life. Thus, FH1–5^18–20 is a potential therapeutic agent for C3 glomerulopathy and other renal conditions with alternative pathway-mediated tissue injury. PMID:26221753

  18. Protection of Nonself Surfaces from Complement Attack by Factor H-Binding Peptides: Implications for Therapeutic Medicine

    PubMed Central

    Wu, You-Qiang; Qu, Hongchang; Sfyroera, Georgia; Tzekou, Apostolia; Kay, Brian K.; Nilsson, Bo; Ekdahl, Kristina Nilsson; Ricklin, Daniel; Lambris, John D.

    2011-01-01

    Exposure of nonself surfaces such as those of biomaterials or transplanted cells and organs to host blood frequently triggers innate immune responses, thereby affecting both their functionality and tolerability. Activation of the alternative pathway of complement plays a decisive role in this unfavorable reaction. Whereas previous studies demonstrated that immobilization of physiological regulators of complement activation (RCA) can attenuate this foreign body-induced activation, simple and efficient approaches for coating artificial surfaces with intact RCA are still missing. The conjugation of small molecular entities that capture RCA with high affinity is an intriguing alternative, as this creates a surface with autoregulatory activity upon exposure to blood. We therefore screened two variable cysteine-constrained phage-displayed peptide libraries for factor H-binding peptides. We discovered three peptide classes that differed with respect to their main target binding areas. Peptides binding to the broad middle region of factor H (domains 5–18) were of particular interest, as they do not interfere with either regulatory or binding activities. One peptide in this group (5C6) was further characterized and showed high factor H-capturing activity while retaining its functional integrity. Most importantly, when 5C6 was coated to a model polystyrene surface and exposed to human lepirudin-anticoagulated plasma, the bound peptide captured factor H and substantially inhibited complement activation by the alternative pathway. Our study therefore provides a promising and novel approach to produce therapeutic materials with enhanced biocompatibility. PMID:21339361

  19. Alternative Complement Pathway Deficiency Ameliorates Chronic Smoke-Induced Functional and Morphological Ocular Injury

    PubMed Central

    Woodell, Alex; Coughlin, Beth; Kunchithapautham, Kannan; Casey, Sarah; Williamson, Tucker; Ferrell, W. Drew; Atkinson, Carl; Jones, Bryan W.; Rohrer, Bärbel

    2013-01-01

    Background Age-related macular degeneration (AMD), a complex disease involving genetic variants and environmental insults, is among the leading causes of blindness in Western populations. Genetic and histologic evidence implicate the complement system in AMD pathogenesis; and smoking is the major environmental risk factor associated with increased disease risk. Although previous studies have demonstrated that cigarette smoke exposure (CE) causes retinal pigment epithelium (RPE) defects in mice, and smoking leads to complement activation in patients, it is unknown whether complement activation is causative in the development of CE pathology; and if so, which complement pathway is required. Methods Mice were exposed to cigarette smoke or clean, filtered air for 6 months. The effects of CE were analyzed in wildtype (WT) mice or mice without a functional complement alternative pathway (AP; CFB−/−) using molecular, histological, electrophysiological, and behavioral outcomes. Results CE in WT mice exhibited a significant reduction in function of both rods and cones as determined by electroretinography and contrast sensitivity measurements, concomitant with a thinning of the nuclear layers as measured by SD-OCT imaging and histology. Gene expression analyses suggested that alterations in both photoreceptors and RPE/choroid might contribute to the observed loss of function, and visualization of complement C3d deposition implies the RPE/Bruch's membrane (BrM) complex as the target of AP activity. RPE/BrM alterations include an increase in mitochondrial size concomitant with an apical shift in mitochondrial distribution within the RPE and a thickening of BrM. CFB−/− mice were protected from developing these CE-mediated alterations. Conclusions Taken together, these findings provide clear evidence that ocular pathology generated in CE mice is dependent on complement activation and requires the AP. Identifying animal models with RPE/BrM damage and verifying which

  20. Competition between antagonistic complement factors for a single protein on N. meningitidis rules disease susceptibility

    PubMed Central

    Caesar, Joseph JE; Lavender, Hayley; Ward, Philip N; Exley, Rachel M; Eaton, Jack; Chittock, Emily; Malik, Talat H; Goiecoechea De Jorge, Elena; Pickering, Matthew C; Tang, Christoph M; Lea, Susan M

    2014-01-01

    Genome-wide association studies have found variation within the complement factor H gene family links to host susceptibility to meningococcal disease caused by infection with Neisseria meningitidis (Davila et al., 2010). Mechanistic insights have been challenging since variation within this locus is complex and biological roles of the factor H-related proteins, unlike factor H, are incompletely understood. N. meningitidis subverts immune responses by hijacking a host-immune regulator, complement factor H (CFH), to the bacterial surface (Schneider et al., 2006; Madico et al., 2007; Schneider et al., 2009). We demonstrate that complement factor-H related 3 (CFHR3) promotes immune activation by acting as an antagonist of CFH. Conserved sequences between CFH and CFHR3 mean that the bacterium cannot sufficiently distinguish between these two serum proteins to allow it to hijack the regulator alone. The level of protection from complement attack achieved by circulating N. meningitidis therefore depends on the relative levels of CFH and CFHR3 in serum. These data may explain the association between genetic variation in both CFH and CFHR3 and susceptibility to meningococcal disease. DOI: http://dx.doi.org/10.7554/eLife.04008.001 PMID:25534642

  1. Nerve Growth Factor Secretion From Pulp Fibroblasts is Modulated by Complement C5a Receptor and Implied in Neurite Outgrowth.

    PubMed

    Chmilewsky, Fanny; Ayaz, Warda; Appiah, James; About, Imad; Chung, Seung-Hyuk

    2016-01-01

    Given the importance of sensory innervation in tooth vitality, the identification of signals that control nerve regeneration and the cellular events they induce is essential. Previous studies demonstrated that the complement system, a major component of innate immunity and inflammation, is activated at the injured site of human carious teeth and plays an important role in dental-pulp regeneration via interaction of the active Complement C5a fragment with pulp progenitor cells. In this study, we further determined the role of the active fragment complement C5a receptor (C5aR) in dental nerve regeneration in regards to local secretion of nerve growth factor (NGF) upon carious injury. Using ELISA and AXIS co-culture systems, we demonstrate that C5aR is critically implicated in the modulation of NGF secretion by LTA-stimulated pulp fibroblasts. The NGF secretion by LTA-stimulated pulp fibroblasts, which is negatively regulated by C5aR activation, has a role in the control of the neurite outgrowth length in our axon regeneration analysis. Our data provide a scientific step forward that can guide development of future therapeutic tools for innovative and incipient interventions targeting the dentin-pulp regeneration process by linking the neurite outgrowth to human pulp fibroblast through complement system activation. PMID:27539194

  2. Nerve Growth Factor Secretion From Pulp Fibroblasts is Modulated by Complement C5a Receptor and Implied in Neurite Outgrowth

    PubMed Central

    Chmilewsky, Fanny; Ayaz, Warda; Appiah, James; About, Imad; Chung, Seung-Hyuk

    2016-01-01

    Given the importance of sensory innervation in tooth vitality, the identification of signals that control nerve regeneration and the cellular events they induce is essential. Previous studies demonstrated that the complement system, a major component of innate immunity and inflammation, is activated at the injured site of human carious teeth and plays an important role in dental-pulp regeneration via interaction of the active Complement C5a fragment with pulp progenitor cells. In this study, we further determined the role of the active fragment complement C5a receptor (C5aR) in dental nerve regeneration in regards to local secretion of nerve growth factor (NGF) upon carious injury. Using ELISA and AXIS co-culture systems, we demonstrate that C5aR is critically implicated in the modulation of NGF secretion by LTA-stimulated pulp fibroblasts. The NGF secretion by LTA-stimulated pulp fibroblasts, which is negatively regulated by C5aR activation, has a role in the control of the neurite outgrowth length in our axon regeneration analysis. Our data provide a scientific step forward that can guide development of future therapeutic tools for innovative and incipient interventions targeting the dentin-pulp regeneration process by linking the neurite outgrowth to human pulp fibroblast through complement system activation. PMID:27539194

  3. Systems Analysis of the Complement-Induced Priming Phase of Liver Regeneration.

    PubMed

    Min, Jun S; DeAngelis, Robert A; Reis, Edimara S; Gupta, Shakti; Maurya, Mano R; Evans, Charles; Das, Arun; Burant, Charles; Lambris, John D; Subramaniam, Shankar

    2016-09-15

    Liver regeneration is a well-orchestrated process in the liver that allows mature hepatocytes to reenter the cell cycle to proliferate and replace lost or damaged cells. This process is often impaired in fatty or diseased livers, leading to cirrhosis and other deleterious phenotypes. Prior research has established the role of the complement system and its effector proteins in the progression of liver regeneration; however, a detailed mechanistic understanding of the involvement of complement in regeneration is yet to be established. In this study, we have examined the role of the complement system during the priming phase of liver regeneration through a systems level analysis using a combination of transcriptomic and metabolomic measurements. More specifically, we have performed partial hepatectomy on mice with genetic deficiency in C3, the major component of the complement cascade, and collected their livers at various time points. Based on our analysis, we show that the C3 cascade activates c-fos and promotes the TNF-α signaling pathway, which then activates acute-phase genes such as serum amyloid proteins and orosomucoids. The complement activation also regulates the efflux and the metabolism of cholesterol, an important metabolite for cell cycle and proliferation. Based on our systems level analysis, we provide an integrated model for the complement-induced priming phase of liver regeneration. PMID:27511733

  4. The Staphylococcus aureus Protein Sbi Acts as a Complement Inhibitor and Forms a Tripartite Complex with Host Complement Factor H and C3b

    PubMed Central

    van den Elsen, Jean; Burman, Julia; Hälbich, Steffi; Richter, Julia; Skerka, Christine; Zipfel, Peter F.

    2008-01-01

    The Gram-positive bacterium Staphylococcus aureus, similar to other pathogens, binds human complement regulators Factor H and Factor H related protein 1 (FHR-1) from human serum. Here we identify the secreted protein Sbi (Staphylococcus aureus binder of IgG) as a ligand that interacts with Factor H by a—to our knowledge—new type of interaction. Factor H binds to Sbi in combination with C3b or C3d, and forms tripartite Sbi∶C3∶Factor H complexes. Apparently, the type of C3 influences the stability of the complex; surface plasmon resonance studies revealed a higher stability of C3d complexed to Sbi, as compared to C3b or C3. As part of this tripartite complex, Factor H is functionally active and displays complement regulatory activity. Sbi, by recruiting Factor H and C3b, acts as a potent complement inhibitor, and inhibits alternative pathway-mediated lyses of rabbit erythrocytes by human serum and sera of other species. Thus, Sbi is a multifunctional bacterial protein, which binds host complement components Factor H and C3 as well as IgG and β2-glycoprotein I and interferes with innate immune recognition. PMID:19112495

  5. Antibodies That Efficiently Form Hexamers upon Antigen Binding Can Induce Complement-Dependent Cytotoxicity under Complement-Limiting Conditions

    PubMed Central

    Cook, Erika M.; Lindorfer, Margaret A.; van der Horst, Hilma; Oostindie, Simone; Beurskens, Frank J.; Schuurman, Janine; Zent, Clive S.; Burack, Richard; Parren, Paul W. H. I.

    2016-01-01

    Recently, we demonstrated that IgG Abs can organize into ordered hexamers after binding their cognate Ags expressed on cell surfaces. This process is dependent on Fc:Fc interactions, which promote C1q binding, the first step in classical pathway complement activation. We went on to engineer point mutations that stimulated IgG hexamer formation and complement-dependent cytotoxicity (CDC). The hexamer formation–enhanced (HexaBody) CD20 and CD38 mAbs support faster, more robust CDC than their wild-type counterparts. To further investigate the CDC potential of these mAbs, we used flow cytometry, high-resolution digital imaging, and four-color confocal microscopy to examine their activity against B cell lines and primary chronic lymphocytic leukemia cells in sera depleted of single complement components. We also examined the CDC activity of alemtuzumab (anti-CD52) and mAb W6/32 (anti-HLA), which bind at high density to cells and promote substantial complement activation. Although we observed little CDC for mAb-opsonized cells reacted with sera depleted of early complement components, we were surprised to discover that the Hexabody mAbs, as well as ALM and W6/32, were all quite effective at promoting CDC in sera depleted of individual complement components C6 to C9. However, neutralization studies conducted with an anti-C9 mAb verified that C9 is required for CDC activity against cell lines. These highly effective complement-activating mAbs efficiently focus activated complement components on the cell, including C3b and C9, and promote CDC with a very low threshold of MAC binding, thus providing additional insight into their enhanced efficacy in promoting CDC. PMID:27474078

  6. Substrate recognition by complement convertases revealed in the C5–cobra venom factor complex

    PubMed Central

    Laursen, Nick S; Andersen, Kasper R; Braren, Ingke; Spillner, Edzard; Sottrup-Jensen, Lars; Andersen, Gregers R

    2011-01-01

    Complement acts as a danger-sensing system in the innate immune system, and its activation initiates a strong inflammatory response and cleavage of the proteins C3 and C5 by proteolytic enzymes, the convertases. These contain a non-catalytic substrate contacting subunit (C3b or C4b) in complex with a protease subunit (Bb or C2a). We determined the crystal structures of the C3b homologue cobra venom factor (CVF) in complex with C5, and in complex with C5 and the inhibitor SSL7 at 4.3 Å resolution. The structures reveal a parallel two-point attachment between C5 and CVF, where the presence of SSL7 only slightly affects the C5–CVF interface, explaining the IgA dependence for SSL7-mediated inhibition of C5 cleavage. CVF functions as a relatively rigid binding scaffold inducing a conformational change in C5, which positions its cleavage site in proximity to the serine protease Bb. A general model for substrate recognition by the convertases is presented based on the C5–CVF and C3b–Bb–SCIN structures. Prior knowledge concerning interactions between the endogenous convertases and their substrates is rationalized by this model. PMID:21217642

  7. Species-specific interaction of Streptococcus pneumoniae with human complement factor H

    PubMed Central

    Lu, Ling; Ma, Zhuo; Jokiranta, T. Sakari; Whitney, Adeline R.; DeLeo, Frank R.; Zhang, Jing-Ren

    2008-01-01

    Streptococcus pneumoniae naturally colonizes the nasopharynx as a commensal organism and sometimes causes infections in remote tissue sites. This bacterium is highly capable of resisting host innate immunity during nasopharyngeal colonization and disseminating infections. The ability to recruit complement factor H (FH) by S. pneumoniae has been implicated as a bacterial immune evasion mechanism against complement-mediated bacterial clearance because FH is a complement alternative pathway inhibitor. S. pneumoniae recruits FH through a previously defined FH-binding domain of choline-binding protein A (CbpA), a major surface protein of S. pneumoniae. In this study, we show that CbpA binds to human FH but not to the FH proteins of mouse and other animal species tested thus far. Accordingly, deleting the FH-binding domain of CbpA in strain D39 did not result in obvious change in the levels of pneumococcal bacteremia or virulence in a bacteremia mouse model. Furthermore, this species-specific pneumococcal interaction with FH was shown to occur in multiple pneumococcal isolates from the blood and cerebrospinal fluid (CSF). Finally, our phagocytosis experiments with human- and mouse phagocytes and complement systems provide additional evidence to support our hypothesis that CbpA acts as a bacterial determinant for pneumococcal resistance to complement-mediated host defense in humans. PMID:18981135

  8. Aged complement factor H knockout mice kept in a clean barriered environment have reduced retinal pathology.

    PubMed

    Hoh Kam, Jaimie; Morgan, James E; Jeffery, Glen

    2016-08-01

    Age-related macular degeneration (AMD) is the largest cause of visual loss in those over 60 years in the West and is a condition increasing in prevalence. Many diseases result from genetic/environmental interactions and 50% of AMD cases have an association with polymorphisms of the complement system including complement factor H. Here we explore interactions between genetic predisposition and environmental conditions in triggering retinal pathology in two groups of aged complement factor H knock out (Cfh(-/-)) mice. Mice were maintained over 9 months in either a conventional open environment or a barriered pathogen free environment. Open environment Cfh(-/-) mice had significant increases in subretinal macrophage numbers, inflammatory and stress responses and reduced photoreceptor numbers over mice kept in a pathogen free environment. Hence, environmental factors can drive retinal disease in these mice when linked to complement deficits impairing immune function. Both groups of mice had similar levels of retinal amyloid beta accumulation. Consequently there is no direct link between this and inflammation in Cfh(-/-) mice.

  9. Complement is a central mediator of radiotherapy-induced tumor-specific immunity and clinical response.

    PubMed

    Surace, Laura; Lysenko, Veronika; Fontana, Andrea Orlando; Cecconi, Virginia; Janssen, Hans; Bicvic, Antonela; Okoniewski, Michal; Pruschy, Martin; Dummer, Reinhard; Neefjes, Jacques; Knuth, Alexander; Gupta, Anurag; van den Broek, Maries

    2015-04-21

    Radiotherapy induces DNA damage and cell death, but recent data suggest that concomitant immune stimulation is an integral part of the therapeutic action of ionizing radiation. It is poorly understood how radiotherapy supports tumor-specific immunity. Here we report that radiotherapy induced tumor cell death and transiently activated complement both in murine and human tumors. The local production of pro-inflammatory anaphylatoxins C3a and C5a was crucial to the tumor response to radiotherapy and concomitant stimulation of tumor-specific immunity. Dexamethasone, a drug frequently given during radiotherapy, limited complement activation and the anti-tumor effects of the immune system. Overall, our findings indicate that anaphylatoxins are key players in radiotherapy-induced tumor-specific immunity and the ensuing clinical responses.

  10. Complement-fixing properties of antinuclear antibodies distinguish drug-induced lupus from systemic lupus erythematosus.

    PubMed

    Rubin, R L; Teodorescu, M; Beutner, E H; Plunkett, R W

    2004-01-01

    The immunofluorescence antinuclear antibody (ANA) test has been widely used to monitor autoimmune disease, but its value for diagnostic purposes is compromised by low specificity and high prevalence in disease-free individuals. The capacity of autoantibodies to fix serum complement proteins when bound to antigen is an important effector function because this property is associated with acute and chronic inflammatory processes. The current study evaluates the complement-fixing properties of antinuclear antibodies (CANA) in three well-defined and clinically-related patient groups: systemic lupus erythematosus (SLE), drug-induced lupus (DIL) and drug-induced autoimmunity (DIA). Of 20 patients diagnosed with SLE, 90% displayed complement-fixing ANA while this feature was present in only two of 18 patients with DIL and no patients with DIA without associated disease even though the mean ANA titres were similar among these patient groups. CANA was significantly correlated with anti-Sm activity. Because SLE but not DIL or DIA can be a life-threatening disease associated with complement consumption in vivo, these results demonstrate that measurement of CANA is a diagnostically useful tool and may have immunopathologic implications.

  11. A complement-dependent model of thrombotic thrombocytopenic purpura induced by antibodies reactive with endothelial cells.

    PubMed

    Ren, Guohui; Hack, Bradley K; Minto, Andrew W; Cunningham, Patrick N; Alexander, Jessy J; Haas, Mark; Quigg, Richard J

    2002-04-01

    Thrombotic thrombocytopenic purpura (TTP) is an immunologically mediated disease characterized by thrombocytopenia, hemolytic anemia, and pathologic changes in various organs, including the kidney, which are secondary to widespread thromboses. Central to TTP is platelet activation, which may occur from a variety of mechanisms, including endothelial cell activation or injury. In this study, injection of K6/1, a monoclonal antibody with widespread reactivity toward endothelia, led to dose-dependent thrombocytopenia in rats. This was magnified if animals were preimmunized with mouse IgG, thereby resulting in an accelerated autologous phase of injury. In this setting, significant anemia also resulted. Rats injected with K6/1 developed renal injury, consisting of tubular damage and glomerular thrombi. Thrombocytopenia and renal morphological abnormalities were eliminated if animals were complement depleted with cobra venom factor prior to K6/1 injection and worsened when the activity of the ubiquitous complement regulator Crry was inhibited with function-neutralizing antibodies. Therefore, we have developed a complement-dependent model of TTP in rats by injecting monoclonal antibodies reactive with endothelial cells. Antibody-directed complement activation leads to stimulation of platelets, through direct interactions with complement fragments and/or indirectly through endothelial cell activation or injury, with the subsequent development of TTP.

  12. Efficacy of plasma therapy in atypical hemolytic uremic syndrome with complement factor H mutations.

    PubMed

    Lapeyraque, Anne-Laure; Wagner, Eric; Phan, Véronique; Clermont, Marie-José; Merouani, Aïcha; Frémeaux-Bacchi, Véronique; Goodship, Timothy H J; Robitaille, Pierre

    2008-08-01

    Atypical hemolytic uremic syndrome (aHUS) frequently results in end-stage renal failure and can be lethal. Several studies have established an association between quantitative or qualitative abnormalities in complement factor H and aHUS. Although plasma infusion and exchange are often advocated, guidelines have yet to be established. Long-term outcome for patients under treatment is still unknown. We describe a patient who, at 7 months of age, presented with aHUS associated with combined de novo complement factor H mutations (S1191L and V1197A) on the same allele. Laboratory investigations showed normal levels of complements C4, C3 and factor H. Plasma exchanges and large-dose infusion therapy resulted in a resolution of hemolysis and recovery of renal function. Three recurrences were successfully treated by intensification of the plasma infusion treatment to intervals of 2 or 3 days. This patient showed good response to large doses of plasma infusions and her condition remained stable for 30 months with weekly plasma infusions (30 ml/kg). Long-term tolerance and efficacy of such intensive plasma therapy are still unknown. Reported secondary failure of plasma therapy in factor H deficiency warrants the search for alternative therapeutic approaches. PMID:18425537

  13. Complement C1q-induced activation of β-catenin signalling causes hypertensive arterial remodelling

    PubMed Central

    Sumida, Tomokazu; Naito, Atsuhiko T.; Nomura, Seitaro; Nakagawa, Akito; Higo, Tomoaki; Hashimoto, Akihito; Okada, Katsuki; Sakai, Taku; Ito, Masamichi; Yamaguchi, Toshihiro; Oka, Toru; Akazawa, Hiroshi; Lee, Jong-Kook; Minamino, Tohru; Offermanns, Stefan; Noda, Tetsuo; Botto, Marina; Kobayashi, Yoshio; Morita, Hiroyuki; Manabe, Ichiro; Nagai, Toshio; Shiojima, Ichiro; Komuro, Issei

    2015-01-01

    Hypertension induces structural remodelling of arteries, which leads to arteriosclerosis and end-organ damage. Hyperplasia of vascular smooth muscle cells (VSMCs) and infiltration of immune cells are the hallmark of hypertensive arterial remodelling. However, the precise molecular mechanisms of arterial remodelling remain elusive. We have recently reported that complement C1q activates β-catenin signalling independent of Wnts. Here, we show a critical role of complement C1-induced activation of β-catenin signalling in hypertensive arterial remodelling. Activation of β-catenin and proliferation of VSMCs were observed after blood-pressure elevation, which were prevented by genetic and chemical inhibition of β-catenin signalling. Macrophage depletion and C1qa gene deletion attenuated the hypertension-induced β-catenin signalling, proliferation of VSMCs and pathological arterial remodelling. Our findings unveil the link between complement C1 and arterial remodelling and suggest that C1-induced activation of β-catenin signalling becomes a novel therapeutic target to prevent arteriosclerosis in patients with hypertension. PMID:25716000

  14. The complement receptor 2/factor H fusion protein TT30 protects paroxysmal nocturnal hemoglobinuria erythrocytes from complement-mediated hemolysis and C3 fragment.

    PubMed

    Risitano, Antonio M; Notaro, Rosario; Pascariello, Caterina; Sica, Michela; del Vecchio, Luigi; Horvath, Christopher J; Fridkis-Hareli, Masha; Selleri, Carmine; Lindorfer, Margaret A; Taylor, Ronald P; Luzzatto, Lucio; Holers, V Michael

    2012-06-28

    Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by complement-mediated intravascular hemolysis because of the lack from erythrocyte surface of the complement regulators CD55 and CD59, with subsequent uncontrolled continuous spontaneous activation of the complement alternative pathway (CAP), and at times of the complement classic pathway. Here we investigate in an in vitro model the effect on PNH erythrocytes of a novel therapeutic strategy for membrane-targeted delivery of a CAP inhibitor. TT30 is a 65 kDa recombinant human fusion protein consisting of the iC3b/C3d-binding region of complement receptor 2 (CR2) and the inhibitory domain of the CAP regulator factor H (fH). TT30 completely inhibits in a dose-dependent manner hemolysis of PNH erythrocytes in a modified extended acidified serum assay, and also prevents C3 fragment deposition on surviving PNH erythrocytes. The efficacy of TT30 derives from its direct binding to PNH erythrocytes; if binding to the erythrocytes is disrupted, only partial inhibition of hemolysis is mediated by TT30 in solution, which is similar to that produced by the fH moiety of TT30 alone, or by intact human fH. TT30 is a membrane-targeted selective CAP inhibitor that may prevent both intravascular and C3-mediated extravascular hemolysis of PNH erythrocytes and warrants consideration for the treatment of PNH patients.

  15. Contribution of complement-stimulated hepatic macrophages and neutrophils to endotoxin-induced liver injury in rats.

    PubMed

    Jaeschke, H; Farhood, A; Smith, C W

    1994-04-01

    The role of complement as potential activator for tissue macrophages and neutrophils was investigated in an experimental model of endotoxin-induced liver injury in male Fischer rats. Injection of Salmonella enteritidis endotoxin (1 mg/kg) into Corynebacterium parvum-pretreated animals (7 mg/kg; single dose 6 days before endotoxin) resulted in severe oxidant stress, as indicated by a 37-fold increase of plasma levels of glutathione disulfide (basal concentration, 0.36 +/- 14 mumol/L), accumulation of neutrophils in the liver (600 +/- 31 neutrophils/50 high-power fields) and liver injury (plasma ALT, 1184 +/- 185 U/l; necrosis; 19% +/- 3%) 10 hr after endotoxin. The oxidant stress induced by 1 mg/kg endotoxin in the C. parvum-treated animals was always significantly higher than that in control animals receiving the same dose of endotoxin. Inhibition of complement activation with the soluble complement receptor type 1 attenuated the oxidant stress and liver injury by 50% to 65% but had no effect on hepatic neutrophil accumulation or plasma tumor necrosis factor-alpha levels. Treatment with a monoclonal antibody directed against the alpha-chain of CD11b/CD18 adhesion proteins (clone 17), which was highly effective in attenuating ischemia-reperfusion injury in the liver by reducing the number of neutrophils and functionally inactivating these cells, neither protected against parenchymal cell injury nor affected hepatic neutrophil infiltration in the C. parvum model. We conclude that reactive oxygen derived from complement-stimulated macrophages is critical for the development of liver injury in the C. parvum/endotoxin model. PMID:8138272

  16. Expression and Subcellular Targeting of Human Complement Factor C5a in Nicotiana species

    PubMed Central

    Nausch, Henrik; Mischofsky, Heike; Koslowski, Roswitha; Meyer, Udo; Broer, Inge; Huckauf, Jana

    2012-01-01

    We evaluated transgenic tobacco plants as an alternative to Escherichia coli for the production of recombinant human complement factor 5a (C5a). C5a has not been expressed in plants before and is highly unstable in vivo in its native form, so it was necessary to establish the most suitable subcellular targeting strategy. We used the strong and constitutive CaMV 35S promoter to drive transgene expression and compared three different subcellular compartments. The yields of C5a in the T0 transgenic plants were low in terms of the proportion of total soluble protein (TSP) when targeted to the apoplast (0.0002% TSP) or endoplasmic reticulum (0.0003% TSP) but was one order of magnitude higher when targeted to the vacuole (0.001% TSP). The yields could be increased by conventional breeding (up to 0.014% TSP in the T2 generation). C5a accumulated to the same level in seeds and leaves when targeted to the apoplast but was up to 1.7-fold more abundant in the seeds when targeted to the ER or vacuole, although this difference was less striking in the better-performing lines. When yields were calculated as an amount per gram fresh weight of transgenic plant tissue, the vacuole targeting strategy was clearly more efficient in seeds, reaching 35.8 µg C5a per gram of fresh seed weight compared to 10.62 µg C5a per gram fresh weight of leaves. Transient expression of C5aER and C5aVac in N. benthamiana, using MagnICON vectors, reached up to 0.2% and 0.7% of TSP, respectively, but was accompanied by cytotoxic effects and induced leaf senescence. Western blot of the plant extracts revealed a band matching the corresponding glycosylated native protein and the bioassay demonstrated that recombinant C5a was biologically active. PMID:23285250

  17. Complement factor H modulates the activation of human neutrophil granulocytes and the generation of neutrophil extracellular traps.

    PubMed

    Schneider, Andrea E; Sándor, Noémi; Kárpáti, Éva; Józsi, Mihály

    2016-04-01

    Factor H (FH) is a major inhibitor of the alternative pathway of complement activation in plasma and on certain host surfaces. In addition to being a complement regulator, FH can bind to various cells via specific receptors, including binding to neutrophil granulocytes through complement receptor type 3 (CR3; CD11b/CD18), and modulate their function. The cellular roles of FH are, however, poorly understood. Because neutrophils are important innate immune cells in inflammatory processes and the host defense against pathogens, we aimed at studying the effects of FH on various neutrophil functions, including the generation of extracellular traps. FH co-localized with CD11b on the surface of neutrophils isolated from peripheral blood of healthy individuals, and cell-bound FH retained its cofactor activity and enhanced C3b degradation. Soluble FH supported neutrophil migration and immobilized FH induced cell spreading. In addition, immobilized but not soluble FH enhanced IL-8 release from neutrophils. FH alone did not trigger the cells to produce neutrophil extracellular traps (NETs), but NET formation induced by PMA and by fibronectin plus fungal β-glucan were inhibited by immobilized, but not by soluble, FH. Moreover, in parallel with NET formation, immobilized FH also inhibited the production of reactive oxygen species induced by PMA and by fibronectin plus β-glucan. Altogether, these data indicate that FH has multiple regulatory roles on neutrophil functions. While it can support the recruitment of neutrophils, FH may also exert anti-inflammatory effects and influence local inflammatory and antimicrobial reactions, and reduce tissue damage by modulating NET formation. PMID:26938503

  18. A complement-microglial axis drives synapse loss during virus-induced memory impairment.

    PubMed

    Vasek, Michael J; Garber, Charise; Dorsey, Denise; Durrant, Douglas M; Bollman, Bryan; Soung, Allison; Yu, Jinsheng; Perez-Torres, Carlos; Frouin, Arnaud; Wilton, Daniel K; Funk, Kristen; DeMasters, Bette K; Jiang, Xiaoping; Bowen, James R; Mennerick, Steven; Robinson, John K; Garbow, Joel R; Tyler, Kenneth L; Suthar, Mehul S; Schmidt, Robert E; Stevens, Beth; Klein, Robyn S

    2016-06-23

    Over 50% of patients who survive neuroinvasive infection with West Nile virus (WNV) exhibit chronic cognitive sequelae. Although thousands of cases of WNV-mediated memory dysfunction accrue annually, the mechanisms responsible for these impairments are unknown. The classical complement cascade, a key component of innate immune pathogen defence, mediates synaptic pruning by microglia during early postnatal development. Here we show that viral infection of adult hippocampal neurons induces complement-mediated elimination of presynaptic terminals in a murine WNV neuroinvasive disease model. Inoculation of WNV-NS5-E218A, a WNV with a mutant NS5(E218A) protein leads to survival rates and cognitive dysfunction that mirror human WNV neuroinvasive disease. WNV-NS5-E218A-recovered mice (recovery defined as survival after acute infection) display impaired spatial learning and persistence of phagocytic microglia without loss of hippocampal neurons or volume. Hippocampi from WNV-NS5-E218A-recovered mice with poor spatial learning show increased expression of genes that drive synaptic remodelling by microglia via complement. C1QA was upregulated and localized to microglia, infected neurons and presynaptic terminals during WNV neuroinvasive disease. Murine and human WNV neuroinvasive disease post-mortem samples exhibit loss of hippocampal CA3 presynaptic terminals, and murine studies revealed microglial engulfment of presynaptic terminals during acute infection and after recovery. Mice with fewer microglia (Il34(-/-) mice with a deficiency in IL-34 production) or deficiency in complement C3 or C3a receptor were protected from WNV-induced synaptic terminal loss. Our study provides a new murine model of WNV-induced spatial memory impairment, and identifies a potential mechanism underlying neurocognitive impairment in patients recovering from WNV neuroinvasive disease. PMID:27337340

  19. A complement-microglial axis drives synapse loss during virus-induced memory impairment.

    PubMed

    Vasek, Michael J; Garber, Charise; Dorsey, Denise; Durrant, Douglas M; Bollman, Bryan; Soung, Allison; Yu, Jinsheng; Perez-Torres, Carlos; Frouin, Arnaud; Wilton, Daniel K; Funk, Kristen; DeMasters, Bette K; Jiang, Xiaoping; Bowen, James R; Mennerick, Steven; Robinson, John K; Garbow, Joel R; Tyler, Kenneth L; Suthar, Mehul S; Schmidt, Robert E; Stevens, Beth; Klein, Robyn S

    2016-06-22

    Over 50% of patients who survive neuroinvasive infection with West Nile virus (WNV) exhibit chronic cognitive sequelae. Although thousands of cases of WNV-mediated memory dysfunction accrue annually, the mechanisms responsible for these impairments are unknown. The classical complement cascade, a key component of innate immune pathogen defence, mediates synaptic pruning by microglia during early postnatal development. Here we show that viral infection of adult hippocampal neurons induces complement-mediated elimination of presynaptic terminals in a murine WNV neuroinvasive disease model. Inoculation of WNV-NS5-E218A, a WNV with a mutant NS5(E218A) protein leads to survival rates and cognitive dysfunction that mirror human WNV neuroinvasive disease. WNV-NS5-E218A-recovered mice (recovery defined as survival after acute infection) display impaired spatial learning and persistence of phagocytic microglia without loss of hippocampal neurons or volume. Hippocampi from WNV-NS5-E218A-recovered mice with poor spatial learning show increased expression of genes that drive synaptic remodelling by microglia via complement. C1QA was upregulated and localized to microglia, infected neurons and presynaptic terminals during WNV neuroinvasive disease. Murine and human WNV neuroinvasive disease post-mortem samples exhibit loss of hippocampal CA3 presynaptic terminals, and murine studies revealed microglial engulfment of presynaptic terminals during acute infection and after recovery. Mice with fewer microglia (Il34(-/-) mice with a deficiency in IL-34 production) or deficiency in complement C3 or C3a receptor were protected from WNV-induced synaptic terminal loss. Our study provides a new murine model of WNV-induced spatial memory impairment, and identifies a potential mechanism underlying neurocognitive impairment in patients recovering from WNV neuroinvasive disease.

  20. Complement factor H deficiency in aged mice causes retinal abnormalities and visual dysfunction.

    PubMed

    Coffey, Peter J; Gias, Carlos; McDermott, Caroline J; Lundh, Peter; Pickering, Matthew C; Sethi, Charanjit; Bird, Alan; Fitzke, Fred W; Maass, Annelie; Chen, Li Li; Holder, Graham E; Luthert, Philip J; Salt, Thomas E; Moss, Stephen E; Greenwood, John

    2007-10-16

    Age-related macular degeneration is the most common form of legal blindness in westernized societies, and polymorphisms in the gene encoding complement factor H (CFH) are associated with susceptibility to age-related macular degeneration in more than half of affected individuals. To investigate the relationship between complement factor H (CFH) and retinal disease, we performed functional and anatomical analysis in 2-year-old CFH-deficient (cfh(-/-)) mice. cfh(-/-) animals exhibited significantly reduced visual acuity and rod response amplitudes on electroretinography compared with age-matched controls. Retinal imaging by confocal scanning laser ophthalmoscopy revealed an increase in autofluorescent subretinal deposits in the cfh(-/-) mice, whereas the fundus and vasculature appeared normal. Examination of tissue sections showed an accumulation of complement C3 in the neural retina of the cfh(-/-) mice, together with a decrease in electron-dense material, thinning of Bruch's membrane, changes in the cellular distribution of retinal pigment epithelial cell organelles, and disorganization of rod photoreceptor outer segments. Collectively, these data show that, in the absence of any specific exogenous challenge to the innate immune system, CFH is critically required for the long-term functional health of the retina.

  1. An Anti-C1s Monoclonal, TNT003, Inhibits Complement Activation Induced by Antibodies Against HLA.

    PubMed

    Thomas, K A; Valenzuela, N M; Gjertson, D; Mulder, A; Fishbein, M C; Parry, G C; Panicker, S; Reed, E F

    2015-08-01

    Antibody-mediated rejection (AMR) of solid organ transplants (SOT) is characterized by damage triggered by donor-specific antibodies (DSA) binding donor Class I and II HLA (HLA-I and HLA-II) expressed on endothelial cells. While F(ab')2 portions of DSA cause cellular activation and proliferation, Fc regions activate the classical complement cascade, resulting in complement deposition and leukocyte recruitment, both hallmark features of AMR. We characterized the ability of an anti-C1s monoclonal antibody, TNT003, to inhibit HLA antibody (HLA-Ab)-induced complement activation. Complement deposition induced by HLA-Ab was evaluated using novel cell- and bead-based assays. Human aortic endothelial cells (HAEC) were cultured with HLA-Ab and human complement; production of activated complement proteins was measured by flow cytometry. Additionally, C3d deposition was measured on single antigen beads (SAB) mixed with HLA-Ab and human complement. TNT003 inhibited HLA-Ab mediated complement deposition on HAEC in a concentration-dependent manner; C3a, C4a and C5a anaphylatoxin production was also diminished by TNT003. Finally, TNT003 blocked C3d deposition induced by Class I (HLAI-Ab)- and Class II (HLAII-Ab)-specific antibodies on SAB. These data suggest TNT003 may be useful for modulating the effects of DSA, as TNT003 inhibits complement deposition and split product formation generated by HLA-I/II-Ab in vitro. PMID:25904443

  2. An Anti-C1s Monoclonal, TNT003, Inhibits Complement Activation Induced by Antibodies Against HLA

    PubMed Central

    Thomas, K A; Valenzuela, N M; Gjertson, D; Mulder, A; Fishbein, M C; Parry, G C; Panicker, S; Reed, E F

    2015-01-01

    Antibody-mediated rejection (AMR) of solid organ transplants (SOT) is characterized by damage triggered by donor-specific antibodies (DSA) binding donor Class I and II HLA (HLA-I and HLA-II) expressed on endothelial cells. While F(ab′)2 portions of DSA cause cellular activation and proliferation, Fc regions activate the classical complement cascade, resulting in complement deposition and leukocyte recruitment, both hallmark features of AMR. We characterized the ability of an anti-C1s monoclonal antibody, TNT003, to inhibit HLA antibody (HLA-Ab)-induced complement activation. Complement deposition induced by HLA-Ab was evaluated using novel cell- and bead-based assays. Human aortic endothelial cells (HAEC) were cultured with HLA-Ab and human complement; production of activated complement proteins was measured by flow cytometry. Additionally, C3d deposition was measured on single antigen beads (SAB) mixed with HLA-Ab and human complement. TNT003 inhibited HLA-Ab mediated complement deposition on HAEC in a concentration-dependent manner; C3a, C4a and C5a anaphylatoxin production was also diminished by TNT003. Finally, TNT003 blocked C3d deposition induced by Class I (HLAI-Ab)- and Class II (HLAII-Ab)-specific antibodies on SAB. These data suggest TNT003 may be useful for modulating the effects of DSA, as TNT003 inhibits complement deposition and split product formation generated by HLA-I/II-Ab in vitro. PMID:25904443

  3. Cysteine proteinase from Streptococcus pyogenes enables evasion of innate immunity via degradation of complement factors.

    PubMed

    Honda-Ogawa, Mariko; Ogawa, Taiji; Terao, Yutaka; Sumitomo, Tomoko; Nakata, Masanobu; Ikebe, Kazunori; Maeda, Yoshinobu; Kawabata, Shigetada

    2013-05-31

    Streptococcus pyogenes is an important human pathogen that causes invasive diseases such as necrotizing fasciitis, sepsis, and streptococcal toxic shock syndrome. We investigated the function of a major cysteine protease from S. pyogenes that affects the amount of C1-esterase inhibitor (C1-INH) and other complement factors and aimed to elucidate the mechanism involved in occurrence of streptococcal toxic shock syndrome from the aspect of the complement system. First, we revealed that culture supernatant of a given S. pyogenes strain and recombinant SpeB degraded the C1-INH. Then, we determined the N-terminal sequence of the C1-INH fragment degraded by recombinant SpeB. Interestingly, the region containing one of the identified cleavage sites is not present in patients with C1-INH deficiency. Scanning electron microscopy of the speB mutant incubated in human serum showed the abnormal superficial architecture and irregular oval structure. Furthermore, unlike the wild-type strain, that mutant strain showed lower survival capacity than normal as compared with heat-inactivated serum, whereas it had a significantly higher survival rate in serum without the C1-INH than in normal serum. Also, SpeB degraded multiple complement factors and the membrane attack complex. Flow cytometric analyses revealed deposition of C9, one of the components of membrane the attack complex, in greater amounts on the surface of the speB mutant, whereas lower amounts of C9 were bound to the wild-type strain surface. These results suggest that SpeB can interrupt the human complement system via degrading the C1-INH, thus enabling S. pyogenes to evade eradication in a hostile environment.

  4. Complement factor I from flatfish half-smooth tongue (Cynoglossus semilaevis) exhibited anti-microbial activities.

    PubMed

    Xiang, Jinsong; Li, Xihong; Chen, Yadong; Lu, Yang; Yu, Mengjun; Chen, Xuejie; Zhang, Wenting; Zeng, Yan; Sun, Luming; Chen, Songlin; Sha, Zhenxia

    2015-11-01

    Complement factor I (Cfi) is a soluble serine protease which plays a crucial role in the modulation of complement cascades. In the presence of substrate modulating cofactors (such as complement factor H, C4bp, CR1, etc), Cfi cleaves and inactivates C3b and C4b, thereby controlling the complement-mediated processes. In this study, we sequenced and characterized Cfi gene from Cynoglossus Semilaevis (designated as CsCfi) for the first time. The full-length cDNA of CsCfi was 2230 bp in length, including a 98 bp 5'-untranslated region (UTR), a 164 bp 3'-UTR and a 1968 bp open reading frame (ORF). It encoded a polypeptide of 656 amino acids, with a molecular mass of 72.28 kDa and an isoelectric point of 7.71. A signal peptide was defined at N-terminus, resulting in a 626-residue mature protein. Multiple sequence alignment revealed that Cfi proteins were well conserved with the typical modular architecture and identical active sites throughout the vertebrates, which suggested the conserved function of Cfi. Phylogenetic analysis indicated that CsCfi and the homologous Cfi sequences from teleosts clustered into a clade, separating from another clade from the cartilaginous fish and other vertebrates. Tissue expression profile analysis by quantitative real-time PCR (qRT-PCR) showed that CsCfi mRNA constitutively expressed in all tested tissues, with the predominant expression in liver and the lowest in stomach. Temporal expression levels of CsCfi after challenging with Vibrio anguillarum showed different expression patterns in intestine, spleen, skin, blood, head kidney and liver. The recombinant CsCfi (rCsCfi) protein showed broad-spectrum antimicrobial activities against the Gram-positive bacteria Staphylococcus aureus and the Gram-negative bacteria Escherichia coli, Pseudomonas aeruginosa and Shewanella putrefaciens. The research revealed that CsCfi plays an important role in C. Semilaevis immunity.

  5. Complement complex C5b-8 induces PGI/sub 2/ formation in culture endothelial cells

    SciTech Connect

    Suttorp, N.; Seeger, W.; Zinsky, S.; Bhakdi, S.

    1987-07-01

    The effects of the terminal complement sequence on prostacyclin (PGI/sub 2/) generation in antibody-sensitized pulmonary arterial endothelial cells were examined. Whereas C5b-7 complement complexes induced no PGI/sub 2/ formation, addition of purified complement component C8 resulted in a time- and dose-dependent burst of PGI/sub 2/ release in the absence of overt cell damage. Formation of the complete terminal complement complex C5b-9 enhanced PGI/sub 2/ release but was accompanied by cytolysis. Extracellular Ca/sup 2 +/ was required for C5b-8-dependent PGI/sub 2/ formation. Three different blockers of physiological calcium channels failed to suppress the observed stimulatory effect. In contrast, W7 (N-(6-amino-hexyl)-5-chloro-1-napththalene sulfonamide) and trifluoperazine, inhibitors of calmodulin activity, all reduced the C5b-8-dependent PGI/sub 2/ generation. None of the inhibitors used impaired Ca/sup 2 +/ flux into the cells. One minute after addition of C8 to endothelial cells carrying C5b-7 complexes, a six- to seven-fold enhanced passive influx of /sup 45/Ca/sup 2 +/ into the cells was noted. An enhanced passive influx was also observed for /sup 51/CrO/sub 4//sup 2 -/, (/sup H/) aminobutyric acid, and (/sup 3/H) sucrose, but not for (/sup 3/H)inulin and (/sup 3/H)dextran. These data together suggest that complement C5b-8 complexes may serve as Ca/sup 2 +/bypass gates in endothelial cells, the ensuring influx of Ca/sup 2 +/ leading to subsequent activation of the arachiodonic acid pathway.

  6. Complement regulator CD59 protects against angiotensin II-induced abdominal aortic aneurysms in mice

    PubMed Central

    Wu, Gongxiong; Chen, Ting; Shahsafaei, Aliakbar; Hu, Weiguo; Bronson, Rod T.; Shi, Guo-Ping; Halperin, Jose A; Aktas, Huseyin; Qin, Xuebin

    2010-01-01

    Background Complement system, an innate immunity, has been well documented to play a critical role in many inflammatory diseases. However, the role of complement in pathogenesis of abdominal aortic aneurysm (AAA), which is considered as an immune and inflammatory disease, remains obscure. Methods and Results Here, we evaluated the pathogenic roles of complement membrane attack complex (MAC) and CD59, a key regulator that inhibits MAC, in the development of AAA. We demonstrated that in the angiotensin II-induced AAA model, deficiency of MAC regulator CD59 in ApoE-null mice (mCd59ab−/−/ApoE−/−) accelerated the disease development, while transgenic over-expression of human CD59 (hCD59ICAM-2+/−/ApoE−/−) in this model attenuated progression of AAA. The severity of aneurysm among these three groups positively correlates with C9 deposition, and/or the activities of MMP2 and MMP9, and/or the levels of phosphor (p)-c-Jun, p-c-Fos, p-IKK-α/β, and p-65. Furthermore, we demonstrated that MAC directly induced gene expression of MMP2 and MMP9 in vitro, which required activation of AP-1 and NF-κB signaling pathways. Conclusions Together, these results defined the protective role of CD59 and shed light on the important pathogenic role of MAC in AAA. PMID:20212283

  7. Recruitment of Factor H as a Novel Complement Evasion Strategy for Blood-Stage Plasmodium falciparum Infection.

    PubMed

    Kennedy, Alexander T; Schmidt, Christoph Q; Thompson, Jennifer K; Weiss, Greta E; Taechalertpaisarn, Tana; Gilson, Paul R; Barlow, Paul N; Crabb, Brendan S; Cowman, Alan F; Tham, Wai-Hong

    2016-02-01

    The human complement system is the frontline defense mechanism against invading pathogens. The coexistence of humans and microbes throughout evolution has produced ingenious molecular mechanisms by which microorganisms escape complement attack. A common evasion strategy used by diverse pathogens is the hijacking of soluble human complement regulators to their surfaces to afford protection from complement activation. One such host regulator is factor H (FH), which acts as a negative regulator of complement to protect host tissues from aberrant complement activation. In this report, we show that Plasmodium falciparum merozoites, the invasive form of the malaria parasites, actively recruit FH and its alternative spliced form FH-like protein 1 when exposed to human serum. We have mapped the binding site in FH that recognizes merozoites and identified Pf92, a member of the six-cysteine family of Plasmodium surface proteins, as its direct interaction partner. When bound to merozoites, FH retains cofactor activity, a key function that allows it to downregulate the alternative pathway of complement. In P. falciparum parasites that lack Pf92, we observed changes in the pattern of C3b cleavage that are consistent with decreased regulation of complement activation. These results also show that recruitment of FH affords P. falciparum merozoites protection from complement-mediated lysis. Our study provides new insights on mechanisms of immune evasion of malaria parasites and highlights the important function of surface coat proteins in the interplay between complement regulation and successful infection of the host.

  8. Complement factor D homolog involved in the alternative complement pathway of rock bream (Oplegnathus fasciatus): Molecular and functional characterization and immune responsive mRNA expression analysis.

    PubMed

    Godahewa, G I; Perera, N C N; Bathige, S D N K; Nam, Bo-Hye; Noh, Jae Koo; Lee, Jehee

    2016-08-01

    The complement system serves conventional role in the innate defense against common invading pathogens. Complement factor D (CfD) is vital to alternative complement pathway activation in cleaving complement factor B. This catalytic reaction forms the alternative C3 convertase that is crucial for complement-mediated pathogenesis. In this study, rock bream (Oplegnathus fasciatus) CfD (OfCfD) was characterized and OfCfD mRNA expression was investigated. OfCfD encodes 277 amino acids (aa) for a 30-kDa polypeptide. A domain analysis of the deduced OfCfD aa sequence showed a single serine protease trypsin superfamily domain, a serine active region, three active sites, and three substrate-binding sites. Pairwise sequence comparisons indicated that OfCfD has the highest identity (84.5%) with Oreochromis niloticus CfD. The phylogenetic tree revealed a common ancestral origin of CfD members, with fish CfD distinct from other vertebrate orthologs. The structural arrangement of the OfCfD gene (2451 bp) contained five exons interrupted by four introns. A spatial transcriptional analysis indicated that OfCfD transcripts constitutively expressed in all of the examined rock bream tissues, and that they were highest in the spleen and liver. In addition, OfCfD transcripts were immunologically upregulated by lipopolysaccharide (LPS) (12 h p.i.), Streptococcus iniae (12 h p.i.), rock bream iridovirus (RBIV) (6-12 h p.i.), and poly I:C (6 h p.i.) in spleen tissue. OfCfD is a trypsin protease and its recombinant protein showed strong protease activity similar to that of trypsin, indicating its catalytic function in the alternative pathway. Together, our findings suggest that OfCfD might be involved in immune responses in rock bream.

  9. Complement factor D homolog involved in the alternative complement pathway of rock bream (Oplegnathus fasciatus): Molecular and functional characterization and immune responsive mRNA expression analysis.

    PubMed

    Godahewa, G I; Perera, N C N; Bathige, S D N K; Nam, Bo-Hye; Noh, Jae Koo; Lee, Jehee

    2016-08-01

    The complement system serves conventional role in the innate defense against common invading pathogens. Complement factor D (CfD) is vital to alternative complement pathway activation in cleaving complement factor B. This catalytic reaction forms the alternative C3 convertase that is crucial for complement-mediated pathogenesis. In this study, rock bream (Oplegnathus fasciatus) CfD (OfCfD) was characterized and OfCfD mRNA expression was investigated. OfCfD encodes 277 amino acids (aa) for a 30-kDa polypeptide. A domain analysis of the deduced OfCfD aa sequence showed a single serine protease trypsin superfamily domain, a serine active region, three active sites, and three substrate-binding sites. Pairwise sequence comparisons indicated that OfCfD has the highest identity (84.5%) with Oreochromis niloticus CfD. The phylogenetic tree revealed a common ancestral origin of CfD members, with fish CfD distinct from other vertebrate orthologs. The structural arrangement of the OfCfD gene (2451 bp) contained five exons interrupted by four introns. A spatial transcriptional analysis indicated that OfCfD transcripts constitutively expressed in all of the examined rock bream tissues, and that they were highest in the spleen and liver. In addition, OfCfD transcripts were immunologically upregulated by lipopolysaccharide (LPS) (12 h p.i.), Streptococcus iniae (12 h p.i.), rock bream iridovirus (RBIV) (6-12 h p.i.), and poly I:C (6 h p.i.) in spleen tissue. OfCfD is a trypsin protease and its recombinant protein showed strong protease activity similar to that of trypsin, indicating its catalytic function in the alternative pathway. Together, our findings suggest that OfCfD might be involved in immune responses in rock bream. PMID:27311435

  10. NMO sera down-regulate AQP4 in human astrocyte and induce cytotoxicity independent of complement.

    PubMed

    Haruki, Hiroyo; Sano, Yasuteru; Shimizu, Fumitaka; Omoto, Masatoshi; Tasaki, Ayako; Oishi, Mariko; Koga, Michiaki; Saito, Kazuyuki; Takahashi, Toshiyuki; Nakada, Tsutomu; Kanda, Takashi

    2013-08-15

    Autoantibodies against astrocyte water channel aquaporin-4 (AQP4) are highly specific for neuromyelitis optica (NMO). However, the molecular mechanism of NMO still remains unclear. The purpose of this study was to identify the possible humoral mechanisms responsible for the occurrence of astrocytic damage. Human primary astrocytes (AST) were immortalized by retroviral vectors harboring temperature-sensitive SV40 T antigen gene and AQP4 cDNA (M23), designated as hAST-AQP4. The effects of NMO sera on the content and localization of AQP4, including cytotoxicity and astrocytic morphology, were evaluated. In addition, this study examined whether the amount and localization of AQP4 protein in astrocytes were influenced by direct contact with the immortalized human brain microvascular endothelial cell line, TY09. NMO sera alone induced cytotoxicity and addition of complement had a more harmful effect on hAST-AQP4. NMO sera also decreased AQP4 mRNA and protein. NMO sera alone up-regulated TNFα and IL-6 in astrocytes and co-incubation with anti-TNFα and anti-IL-6 neutralizing antibodies blocked both the cytotoxicity and reduction of AQP4 in astrocytes. In the experiment using the in vitro BBB models, AQP4 protein mainly localized at the astrocytic membrane after co-culture with TY09, in contact with TY09. The future elucidation of factors that up-regulate AQP4 in astrocytes presumably released by blood brain barrier forming endothelial cells and that block the production of inflammatory cytokines may therefore lead to the development of a novel therapeutic strategy.

  11. Direct binding of C1q to apoptotic cells and cell blebs induces complement activation.

    PubMed

    Nauta, Alma J; Trouw, Leendert A; Daha, Mohamed R; Tijsma, Odette; Nieuwland, Rienk; Schwaeble, Wilhelm J; Gingras, Alexandre R; Mantovani, Alberto; Hack, Erik C; Roos, Anja

    2002-06-01

    Deficiency of early components of the classical pathway of complement, particularly C1q, predisposes to the development of systemic lupus erythematosus. Several studies have suggested an association between the classical complement pathway and the clearance of apoptotic cells. Mice with a targeted deletion of the C1q gene develop a lupus-like renal disease, which is associated with the presence of multiple apoptotic bodies in the kidney. In the present study we demonstrate that highly purified C1q binds to apoptotic cells and isolated blebs derived from these apoptotic cells. Binding of C1q to apoptotic cells occurs via the globular heads of C1q and induces activation of the classical complement pathway, as shown by the deposition of C4 and C3 on the surface of these cells and on cell-derived blebs. In addition, for the first time, we demonstrate that surface-bound C1q is present on a subpopulation of microparticles isolated from human plasma. Taken together, these observations demonstrate that C1q binds directly to apoptotic cells and blebs derived therefrom and support a role for C1q, possibly in concert with C4 and C3, in the clearance of apoptotic cells and blebs by the phagocytic system.

  12. A complement C3 inhibitor specifically targeted to sites of complement activation effectively ameliorates collagen-induced arthritis in DBA/1J mice.

    PubMed

    Song, Hongbin; Qiao, Fei; Atkinson, Carl; Holers, V Michael; Tomlinson, Stephen

    2007-12-01

    Collagen-induced arthritis (CIA) represents an animal model of autoimmune polyarthritis with similarities to human rheumatoid arthritis, and therapy with various systemic complement-inhibitory proteins has been investigated in this model with varying results. We investigated the use of complement receptor 2 (CR2)-Crry, a complement inhibitor with the ability to target C3 breakdown products deposited in a rheumatic joint. Following induction of CIA in DBA/1J mice, animals were treated with either PBS or CR2-Crry (every other day, every 4 days, or with a single injection). The severity of clinical disease was significantly reduced in all CR2-Crry-treated groups compared with controls. Joints from mice receiving multiple doses of CR2-Crry showed significantly decreased inflammatory cell infiltrate, cartilage damage, pannus formation, and bone damage. CR2-Crry treatment also significantly decreased production of anti-collagen IgG and the inflammatory cytokines TNF-alpha and IL-1beta. IL-10 and IL-1Ra levels were increased in CR2-Crry-treated mice. CR2-Crry localized preferentially in the joints of mice with CIA. Analysis of IgG and C3 deposition in the joints of treated animals indicated that both complement regulation and the modulation of anti-collagen Ab production contributed to the protective effects of CR2-Crry. Of interest, a previous study reported that Crry-Ig, an untargeted counterpart of CR2-Crry, had minimal effect on disease, even when administered at a sufficiently high dose to maintain chronic complement inhibition. PMID:18025232

  13. Molecular cloning and characterization of a complement-depleting factor from king cobra, Ophiophagus hannah.

    PubMed

    Zeng, Lin; Sun, Qian-Yun; Jin, Yang; Zhang, Yong; Lee, Wen-Hui; Zhang, Yun

    2012-09-01

    Cobra venom factor (CVF) is an anti-complement factor existing in cobra venom. CVF proteins have been purified from the venoms of Naja haje, Naja siamensis, Naja atra, Naja kaouthia, Naja naja, Naja melanoleuca and Austrelaps superbus, but only three full-length cDNA sequences of CVF are available. In the present work, a cobra venom factor termed OVF was purified from the crude venom of Ophiophagus hannah by successive gel filtration, ion-exchange and heparin affinity chromatography steps. The purified OVF was homogenous on the SDS-PAGE gel with an apparent molecular weight of 140 kDa under non-reducing conditions. Under reducing conditions, OVF was divided into three bands with apparent molecular weight of 72 kDa (α chain), 45 kDa (β chain) and 32 kDa (γ chain), respectively. OVF consumed complement components with anti-complement activity of 154 units per mg. By using Reverse transcription-PCR and 5'-RACE assay, the open reading frame of OVF was obtained. MALDI-TOF and protein sequencing assays confirmed the cloned cDNA coding for OVF protein. The cDNA sequence of OVF is conservative when aligned with that of other CVFs. Phylogenetic analysis revealed OVF is closer to CVF from N. kaouthia than to AVF-1 and AVF-2 from A. superbus. Our results demonstrated that OVF has its unique features as following: 1) The N-terminal amino acid sequence of OVF γ chain is different from that of other known CVFs, suggesting that the OVF γ chain might be further processed; 2) Unlike N. kaouthia CVF and A. superbus AVF-1, which have potential N-linked glycosylation sites located in both α and β chain, OVF only has N-linked glycosylation site in its α chain as revealed by Schiff's reagent staining and protein sequence analysis; 3) In addition to the 27 well conserved cysteine residues in all known CVFs, OVF have an additional cysteine residue in its γ chain. Understanding the importance of above mentioned specific characteristics might provide useful information on structure

  14. Molecular cloning and characterization of a complement-depleting factor from king cobra, Ophiophagus hannah.

    PubMed

    Zeng, Lin; Sun, Qian-Yun; Jin, Yang; Zhang, Yong; Lee, Wen-Hui; Zhang, Yun

    2012-09-01

    Cobra venom factor (CVF) is an anti-complement factor existing in cobra venom. CVF proteins have been purified from the venoms of Naja haje, Naja siamensis, Naja atra, Naja kaouthia, Naja naja, Naja melanoleuca and Austrelaps superbus, but only three full-length cDNA sequences of CVF are available. In the present work, a cobra venom factor termed OVF was purified from the crude venom of Ophiophagus hannah by successive gel filtration, ion-exchange and heparin affinity chromatography steps. The purified OVF was homogenous on the SDS-PAGE gel with an apparent molecular weight of 140 kDa under non-reducing conditions. Under reducing conditions, OVF was divided into three bands with apparent molecular weight of 72 kDa (α chain), 45 kDa (β chain) and 32 kDa (γ chain), respectively. OVF consumed complement components with anti-complement activity of 154 units per mg. By using Reverse transcription-PCR and 5'-RACE assay, the open reading frame of OVF was obtained. MALDI-TOF and protein sequencing assays confirmed the cloned cDNA coding for OVF protein. The cDNA sequence of OVF is conservative when aligned with that of other CVFs. Phylogenetic analysis revealed OVF is closer to CVF from N. kaouthia than to AVF-1 and AVF-2 from A. superbus. Our results demonstrated that OVF has its unique features as following: 1) The N-terminal amino acid sequence of OVF γ chain is different from that of other known CVFs, suggesting that the OVF γ chain might be further processed; 2) Unlike N. kaouthia CVF and A. superbus AVF-1, which have potential N-linked glycosylation sites located in both α and β chain, OVF only has N-linked glycosylation site in its α chain as revealed by Schiff's reagent staining and protein sequence analysis; 3) In addition to the 27 well conserved cysteine residues in all known CVFs, OVF have an additional cysteine residue in its γ chain. Understanding the importance of above mentioned specific characteristics might provide useful information on structure

  15. Maternal Haploids Are Preferentially Induced by CENH3-tailswap Transgenic Complementation in Maize

    PubMed Central

    Kelliher, Timothy; Starr, Dakota; Wang, Wenling; McCuiston, Jamie; Zhong, Heng; Nuccio, Michael L.; Martin, Barry

    2016-01-01

    Doubled haploid plants are invaluable breeding tools but many crop species are recalcitrant to available haploid induction techniques. To test if haploid inducer lines can be engineered into crops, CENH3−∕− and CENH3:RNAi lines were complemented by AcGREEN-tailswap-CENH3 or AcGREEN-CENH3 transgenes. Haploid induction rates were determined following testcrosses to wild-type plants after independently controlling for inducer parent sex and transgene zygosity. CENH3 fusion proteins were localized to centromeres and did not cause vegetative defects or male sterility. CENH3:RNAi lines did not demonstrate consistent knockdown and rarely produced haploids. In contrast, many of the complemented CENH3−∕− lines produced haploids at low frequencies. The rate of gynogenic haploid induction reached a maximum of 3.6% in several hemizygous individuals when backcrossed as males. These results demonstrate that CENH3-tailswap transgenes can be used to engineer in vivo haploid induction systems into maize plants. PMID:27066050

  16. Complement component 6 deficiency increases susceptibility to dextran sulfate sodium-induced murine colitis.

    PubMed

    Ding, Peipei; Li, Ling; Huang, Tianbao; Yang, Chaoqun; Xu, Enjie; Wang, Na; Zhang, Long; Gu, Hongyu; Yao, Xudong; Zhou, Xuhui; Hu, Weiguo

    2016-11-01

    As a potent effector of innate immunity, the complement system has been shown to be involved in the pathogenesis of inflammatory bowel disease (IBD). However, the role of the membrane attack complex (MAC) in the development of IBD is still largely unknown. Here, we used C6-deficient mice in which MAC formation was blocked due to the absence of C6 to develop an acute colitis model by the administration of dextran sulfate sodium (DSS). The results showed that DSS-induced colitis was aggravated in C6-deficient mice compared with wild-type (WT) mice, as represented by the markedly greater weight loss, higher disease activity index (DAI), shortened colon length, more severe histological injury with increased epithelial ulcerations, and massively increased infiltration of leukocytes accompanied by much higher myeloperoxidase (MPO) levels in local inflammatory colonic sites. In addition, the DSS-induced colitis in C6-deficient mice could be significantly ameliorated by the exogenous C6 from WT sera. Furthermore, the significantly enhanced production of pro-inflammatory mediators, including IL-1β, IL-6, CXCL-1, CCL-3, TGF-β1 and IL-17F, was also observed in C6-deficient mice. Unexpectedly, the aggravated colitis in C6-deficient mice may be not due to the increase of lipopolysaccharide (LPS) levels in serum. Overall, we demonstrated that MAC exerts a protective role in acute colitis, strongly highlighting the host defense function of the complement system. PMID:27316715

  17. Regulation of Cre recombinase by ligand-induced complementation of inactive fragments.

    PubMed

    Jullien, Nicolas; Sampieri, François; Enjalbert, Alain; Herman, Jean-Paul

    2003-11-01

    Cre recombinase is extensively used to engineer the genome of experimental animals. However, its usefulness is still limited by the lack of an efficient temporal control over its activity. To overcome this, we have developed DiCre, a regulatable fragment complementation system for Cre. The enzyme was split into two moieties that were fused to FKBP12 (FK506-binding protein) and FRB (binding domain of the FKBP12-rapamycin-associated protein), respectively. These can be efficiently heterodimerized by rapamycin. Several variants, based on splitting Cre at different sites and using different linker peptides, were tested in an indicator cell line. The fusion proteins, taken separately, had no recombinase activity. Stable transformants, co-expressing complementing fragments based on splitting Cre between Asn59 and Asn60, displayed low background activity affecting 0.05-0.4% of the cells. Rapamycin induced a rapid recombination, reaching 100% by 48-72 h, with an EC50 of 0.02 nM. Thus, ligand-induced dimerization can efficiently regulate Cre, and should be useful to achieve a tight temporal control of its activity, such as in the case of the creation of conditional knock-out animals.

  18. Regulation of Cre recombinase by ligand-induced complementation of inactive fragments

    PubMed Central

    Jullien, Nicolas; Sampieri, François; Enjalbert, Alain; Herman, Jean-Paul

    2003-01-01

    Cre recombinase is extensively used to engineer the genome of experimental animals. However, its usefulness is still limited by the lack of an efficient temporal control over its activity. To overcome this, we have developed DiCre, a regulatable fragment complementation system for Cre. The enzyme was split into two moieties that were fused to FKBP12 (FK506-binding protein) and FRB (binding domain of the FKBP12–rapamycin-associated protein), respectively. These can be efficiently heterodimerized by rapamycin. Several variants, based on splitting Cre at different sites and using different linker peptides, were tested in an indicator cell line. The fusion proteins, taken separately, had no recombinase activity. Stable transformants, co-expressing complementing fragments based on splitting Cre between Asn59 and Asn60, displayed low background activity affecting 0.05–0.4% of the cells. Rapamycin induced a rapid recombination, reaching 100% by 48–72 h, with an EC50 of 0.02 nM. Thus, ligand-induced dimerization can efficiently regulate Cre, and should be useful to achieve a tight temporal control of its activity, such as in the case of the creation of conditional knock-out animals. PMID:14576331

  19. Function of the factor I modules (FIMS) of human complement component C6.

    PubMed

    DiScipio, R G; Linton, S M; Rushmere, N K

    1999-11-01

    In order to elucidate the function of complement component C6, truncated C6 molecules were expressed recombinantly. These were either deleted of the factor I modules (FIMs) (C6des-748-913) or both complement control protein (CCP) modules and FIMs (C6des-611-913). C6des-748-913 exhibited approximately 60-70% of the hemolytic activity of full-length C6 when assayed for Alternative Pathway activity, but when measured for the Classical Pathway, C6des-748-914 was only 4-6% as effective as C6. The activity difference between C6 and C6des-748-913 for the two complement pathways can be explained by a greater stability of newly formed metastable C5b* when produced by the Alternative Pathway compared with that made by the Classical Pathway. The half-lives of metastable C5b* and the decay of (125)I-C5b measured from cells used to activate the Alternative Pathway were found to be about 5-12-fold longer than those same parameters derived from cells that had activated the Classical Pathway. (125)I-C5 binds reversibly to C6 in an ionic strength-dependent fashion, but (125)I-C5 binds only weakly to C6des-FIMs and not at all to C6des-CCP/FIMs. Therefore, although the FIMs are not required absolutely for C6 activity, these modules promote interaction of C6 with C5 enabling a more efficient bimolecular coupling ultimately leading to the formation of the C5b-6 complex. PMID:10542204

  20. The Complement Anaphylatoxin C5a Induces Apoptosis in Adrenomedullary Cells during Experimental Sepsis

    PubMed Central

    Flierl, Michael A.; Rittirsch, Daniel; Chen, Anthony J.; Nadeau, Brian A.; Day, Danielle E.; Sarma, J. Vidya; Huber-Lang, Markus S.; Ward, Peter A.

    2008-01-01

    Sepsis remains a poorly understood, enigmatic disease. One of the cascades crucially involved in its pathogenesis is the complement system. Especially the anaphylatoxin C5a has been shown to have numerous harmful effects during sepsis. We have investigated the impact of high levels of C5a on the adrenal medulla following cecal ligation and puncture (CLP)-induced sepsis in rats as well as the role of C5a on catecholamine production from pheochromocytoma-derived PC12 cells. There was significant apoptosis of adrenal medulla cells in rats 24 hrs after CLP, as assessed by the TUNEL technique. These effects could be reversed by dual-blockade of the C5a receptors, C5aR and C5L2. When rats were subjected to CLP, levels of C5a and norepinephrine were found to be antipodal as a function of time. PC12 cell production of norepinephrine and dopamine was significantly blunted following exposure to recombinant rat C5a in a time-dependent and dose-dependent manner. This impaired production could be related to C5a-induced initiation of apoptosis as defined by binding of Annexin V and Propidium Iodine to PC12 cells. Collectively, we describe a C5a-dependent induction of apoptotic events in cells of adrenal medulla in vivo and pheochromocytoma PC12 cells in vitro. These data suggest that experimental sepsis induces apoptosis of adrenomedullary cells, which are responsible for the bulk of endogenous catecholamines. Septic shock may be linked to these events. Since blockade of both C5a receptors virtually abolished adrenomedullary apoptosis in vivo, C5aR and C5L2 become promising targets with implications on future complement-blocking strategies in the clinical setting of sepsis. PMID:18648551

  1. Activated signal transducers and activators of transcription 3 signaling induces CD46 expression and protects human cancer cells from complement-dependent cytotoxicity.

    PubMed

    Buettner, Ralf; Huang, Mei; Gritsko, Tanya; Karras, Jim; Enkemann, Steve; Mesa, Tania; Nam, Sangkil; Yu, Hua; Jove, Richard

    2007-08-01

    CD46 is one of the complement-regulatory proteins expressed on the surface of normal and tumor cells for protection against complement-dependent cytotoxicity. Cancer cells need to access the blood circulation for continued growth and metastasis, thus exposing themselves to destruction by complement system components. Previous studies have established that the signal transducers and activators of transcription 3 (STAT3) transcription factor is persistently activated in a wide variety of human cancer cells and primary tumor tissues compared with their normal counterparts. Using microarray gene expression profiling, we identified the CD46 gene as a target for activated STAT3 signaling in human breast and prostate cancer cells. The CD46 promoter contains two binding sites for activated STAT3 and mutations introduced into the major site abolished STAT3 binding. Chromatin immunoprecipitation confirms binding of STAT3 to the CD46 promoter. CD46 promoter activity is induced by activation of STAT3 and blocked by a dominant-negative form of STAT3 in luciferase reporter assays. CD46 mRNA expression is induced by interleukin-6 and by transient transfection of normal human epithelial cells with a persistently active mutant construct of STAT3, STAT3C. Furthermore, we show that inhibition of STAT3-mediated CD46 cell surface expression sensitizes DU145 prostate cancer cells to cytotoxicity in an in vitro complement lysis assay using rabbit anti-DU145 antiserum and rabbit complement. These results show that activated STAT3 signaling induces the CD46 promoter and protects human cancer cells from complement-dependent cytotoxicity, suggesting a potential mechanism whereby oncogenic signaling contributes to tumor cell evasion of antibody-mediated immunity.

  2. Sickness: From the focus on cytokines, prostaglandins, and complement factors to the perspectives of neurons.

    PubMed

    Poon, David Chun-Hei; Ho, Yuen-Shan; Chiu, Kin; Wong, Hoi-Lam; Chang, Raymond Chuen-Chung

    2015-10-01

    Systemic inflammation leads to a variety of physiological (e.g. fever) and behavioral (e.g. anorexia, immobility, social withdrawal, depressed mood, disturbed sleep) responses that are collectively known as sickness. While these phenomena have been studied for the past few decades, the neurobiological mechanisms by which sickness occurs remain unclear. In this review, we first revisit how the body senses and responds to infections and injuries by eliciting systemic inflammation. Next, we focus on how peripheral inflammatory molecules such as cytokines, prostaglandins, and activated complement factors communicate with the brain to trigger neuroinflammation and sickness. Since depression also involves inflammation, we further elaborate on the interrelationship between sickness and depression. Finally, we discuss how immune activation can modulate neurons in the brain, and suggest future perspectives to help unravel how changes in neuronal functions relate to sickness responses. PMID:26363665

  3. Characterization of a Factor H Mutation That Perturbs the Alternative Pathway of Complement in a Family with Membranoproliferative GN

    PubMed Central

    Wong, Edwin K.S.; Anderson, Holly E.; Herbert, Andrew P.; Challis, Rachel C.; Brown, Paul; Reis, Geisilaine S.; Tellez, James O.; Strain, Lisa; Fluck, Nicholas; Humphrey, Ann; Macleod, Alison; Richards, Anna; Ahlert, Daniel; Santibanez-Koref, Mauro; Barlow, Paul N.; Marchbank, Kevin J.; Harris, Claire L.; Goodship, Timothy H.J.

    2014-01-01

    Complement C3 activation is a characteristic finding in membranoproliferative GN (MPGN). This activation can be caused by immune complex deposition or an acquired or inherited defect in complement regulation. Deficiency of complement factor H has long been associated with MPGN. More recently, heterozygous genetic variants have been reported in sporadic cases of MPGN, although their functional significance has not been assessed. We describe a family with MPGN and acquired partial lipodystrophy. Although C3 nephritic factor was shown in family members with acquired partial lipodystrophy, it did not segregate with the renal phenotype. Genetic analysis revealed a novel heterozygous mutation in complement factor H (R83S) in addition to known risk polymorphisms carried by individuals with MPGN. Patients with MPGN had normal levels of factor H, and structural analysis of the mutant revealed only subtle alterations. However, functional analysis revealed profoundly reduced C3b binding, cofactor activity, and decay accelerating activity leading to loss of regulation of the alternative pathway. In summary, this family showed a confluence of common and rare functionally significant genetic risk factors causing disease. Data from our analysis of these factors highlight the role of the alternative pathway of complement in MPGN. PMID:24722444

  4. Sodium butyrate enhances complement-mediated cell injury via down-regulation of decay-accelerating factor expression in colonic cancer cells.

    PubMed

    Andoh, Akira; Shimada, Mitsue; Araki, Yoshio; Fujiyama, Yoshihide; Bamba, Tadao

    2002-02-01

    Decay-accelerating factor (DAF) expressed on the surface of colonic cancer cells presents a barrier to complement-mediated clearance by contributing to the ineffectiveness of the humoral immune response. In this study, to investigate the mechanisms responsible for the anti-tumor effects of butyrate, we evaluated how butyrate modulates DAF expression in colonic cancer cells. Three colonic cancer cell lines (HT-29, Caco-2, and T84 cells) were studied. DAF protein expression was assessed by western blot, and DAF mRNA expression was evaluated by northern blot. Complement C3 deposition on the surface of colonic cancer cells was determined by enzyme-linked immunosorbent assay (ELISA). The promoter activity of the DAF gene was assessed by a reporter gene-luciferase assay. Butyrate reduced the basal and interleukin-4 (IL-4)- and tumor necrosis factor-alpha (TNF-alpha)-induced expression of DAF protein and mRNA in HT-29 cells. It increased the susceptibility to complement attack and enhanced C3 deposition on HT-29 cells. The inhibitory effect of butyrate on DAF mRNA expression was also observed in T84 and Caco-2 cells. Butyrate decreased basal DAF expression at both transcriptional and post-transcriptional levels. The inhibitory effect of butyrate on IL-4-induced DAF expression was closely associated with a blockade of IL-4-induced DAF mRNA stability. TNF-alpha-induced transcriptional activation and the increased stability of the DAF gene were also blocked by butyrate. Similar but weak effects were induced by trichostatin A, a potent histone deacetylase inhibitor, suggesting that histone acetylation might participate in butyrate activity. These observations indicate that both a down-regulation of DAF expression and the induction of susceptibility to complement attack contribute to the anti-tumor effects of butyrate in colonic cancer.

  5. A Targeted Inhibitor of the Alternative Complement Pathway Accelerates Recovery From Smoke-Induced Ocular Injury

    PubMed Central

    Woodell, Alex; Jones, Bryan W.; Williamson, Tucker; Schnabolk, Gloriane; Tomlinson, Stephen; Atkinson, Carl; Rohrer, Bärbel

    2016-01-01

    Purpose Morphologic and genetic evidence exists that an overactive complement system driven by the complement alternative pathway (AP) is involved in pathogenesis of age-related macular degeneration (AMD). Smoking is the only modifiable risk factor for AMD. As we have shown that smoke-related ocular pathology can be prevented in mice that lack an essential activator of AP, we ask here whether this pathology can be reversed by increasing inhibition in AP. Methods Mice were exposed to either cigarette smoke (CS) or filtered air (6 hours/day, 5 days/week, 6 months). Smoke-exposed animals were then treated with the AP inhibitor (CR2-fH) or vehicle control (PBS) for the following 3 months. Spatial frequency and contrast sensitivity were assessed by optokinetic response paradigms at 6 and 9 months; additional readouts included assessment of retinal morphology by electron microscopy (EM) and gene expression analysis by quantitative RT-PCR. Results The CS mice treated with CR2-fH showed significant improvement in contrast threshold compared to PBS-treated mice, whereas spatial frequency was unaffected by CS or pharmacologic intervention. Treatment with CR2-fH in CS animals reversed thinning of the retina observed in PBS-treated mice as analyzed by spectral-domain optical coherence tomography, and reversed most morphologic changes in RPE and Bruch's membrane seen in CS animals by EM. Conclusions Taken together, these findings suggest that AP inhibitors not only prevent, but have the potential to accelerate the clearance of complement-mediated ocular injury. Improving our understanding of the regulation of the AP is paramount to developing novel treatment approaches for AMD. PMID:27064393

  6. Detection of Initiator Caspase Induced Proximity in Single Cells by Caspase Bimolecular Fluorescence Complementation.

    PubMed

    Parsons, Melissa J; Fassio, Sara R; Bouchier-Hayes, Lisa

    2016-01-01

    The caspase family of proteases includes key regulators of apoptosis and inflammation. The caspases can be divided into two groups, the initiator caspases and the executioner caspases. Initiator caspases include caspase-2, caspase-8, and caspase-9 and are activated by proximity-induced dimerization upon recruitment to large molecular weight protein complexes called activation platforms. This protocol describes an imaging-based technique called caspase Bimolecular Fluorescence Complementation (BiFC) that measures induced proximity of initiator caspases. This method uses nonfluorescent fragments of the fluorescent protein Venus fused to initiator caspase monomers. When the caspase is recruited to its activation platform, the resulting induced proximity of the caspase monomers facilitates refolding of the Venus fragments into the full molecule, reconstituting its fluorescence. Thus, the assembly of initiator caspase activation platforms can be followed in single cells in real time. Induced proximity is the most apical step in the activation of initiator caspases, and therefore, caspase BiFC is a robust and specific method to measure initiator caspase activation. PMID:27108430

  7. Development of a Sterne-Based Complement Fixation Test to Monitor the Humoral Response Induced by Anthrax Vaccines

    PubMed Central

    Adone, Rosanna; Sali, Michela; Francia, Massimiliano; Iatarola, Michela; Donatiello, Adelia; Fasanella, Antonio

    2016-01-01

    Anthrax is a zoonotic disease caused by Bacillus anthracis spore-forming bacterium. Since it is primarily a disease of animals, the control in animals, and humans depend on the prevention in livestock, principally cattle, sheep, and goats. Most veterinary vaccines utilize the toxigenic, uncapsulated (pXO1+/pXO2–) B. anthracis strain 34F2 which affords protection through the production of neutralizing antibodies directed to the toxin components Protective Antigen (PA), Lethal Factor (LF), and Edema Factor (EF). The titration of specific antibodies in sera of vaccinated animals is crucial to evaluate the efficacy of the vaccination and to obtain epidemiological information for an effective anthrax surveillance. In this study, we developed a Sterne-based Complement Fixation Test (CFT) to detect specific antibodies induced in animals vaccinated with Sterne 34F2. We assessed its efficacy in laboratory animals and under field conditions by monitoring the humoral response induced by vaccination in cattle. The results indicated that the Sterne-based CFT is able to correctly identify vaccinated animals. It proved to be a very sensitive and specific test. Moreover, the Sterne-based CFT offers many benefits with regard to costs, standardization and reproducibility of the assay procedure. PMID:26858700

  8. Association of Genetic Variants in Complement Factor H and Factor H-Related Genes with Systemic Lupus Erythematosus Susceptibility

    PubMed Central

    Zhao, Jian; Wu, Hui; Khosravi, Melanie; Cui, Huijuan; Qian, Xiaoxia; Kelly, Jennifer A.; Kaufman, Kenneth M.; Langefeld, Carl D.; Williams, Adrienne H.; Comeau, Mary E.; Ziegler, Julie T.; Marion, Miranda C.; Adler, Adam; Glenn, Stuart B.; Alarcón-Riquelme, Marta E.; Pons-Estel, Bernardo A.; Harley, John B.; Bae, Sang-Cheol; Bang, So-Young; Cho, Soo-Kyung; Jacob, Chaim O.; Vyse, Timothy J.; Niewold, Timothy B.; Gaffney, Patrick M.; Moser, Kathy L.; Kimberly, Robert P.; Edberg, Jeffrey C.; Brown, Elizabeth E.; Alarcon, Graciela S.; Petri, Michelle A.; Ramsey-Goldman, Rosalind; Vilá, Luis M.; Reveille, John D.; James, Judith A.; Gilkeson, Gary S.; Kamen, Diane L.; Freedman, Barry I.; Anaya, Juan-Manuel; Merrill, Joan T.; Criswell, Lindsey A.; Scofield, R. Hal; Stevens, Anne M.; Guthridge, Joel M.; Chang, Deh-Ming; Song, Yeong Wook; Park, Ji Ah; Lee, Eun Young; Boackle, Susan A.; Grossman, Jennifer M.; Hahn, Bevra H.; Goodship, Timothy H. J.; Cantor, Rita M.; Yu, Chack-Yung; Shen, Nan; Tsao, Betty P.

    2011-01-01

    Systemic lupus erythematosus (SLE), a complex polygenic autoimmune disease, is associated with increased complement activation. Variants of genes encoding complement regulator factor H (CFH) and five CFH-related proteins (CFHR1-CFHR5) within the chromosome 1q32 locus linked to SLE, have been associated with multiple human diseases and may contribute to dysregulated complement activation predisposing to SLE. We assessed 60 SNPs covering the CFH-CFHRs region for association with SLE in 15,864 case-control subjects derived from four ethnic groups. Significant allelic associations with SLE were detected in European Americans (EA) and African Americans (AA), which could be attributed to an intronic CFH SNP (rs6677604, in intron 11, Pmeta = 6.6×10−8, OR = 1.18) and an intergenic SNP between CFHR1 and CFHR4 (rs16840639, Pmeta = 2.9×10−7, OR = 1.17) rather than to previously identified disease-associated CFH exonic SNPs, including I62V, Y402H, A474A, and D936E. In addition, allelic association of rs6677604 with SLE was subsequently confirmed in Asians (AS). Haplotype analysis revealed that the underlying causal variant, tagged by rs6677604 and rs16840639, was localized to a ∼146 kb block extending from intron 9 of CFH to downstream of CFHR1. Within this block, the deletion of CFHR3 and CFHR1 (CFHR3-1Δ), a likely causal variant measured using multiplex ligation-dependent probe amplification, was tagged by rs6677604 in EA and AS and rs16840639 in AA, respectively. Deduced from genotypic associations of tag SNPs in EA, AA, and AS, homozygous deletion of CFHR3-1Δ (Pmeta = 3.2×10−7, OR = 1.47) conferred a higher risk of SLE than heterozygous deletion (Pmeta = 3.5×10−4, OR = 1.14). These results suggested that the CFHR3-1Δ deletion within the SLE-associated block, but not the previously described exonic SNPs of CFH, might contribute to the development of SLE in EA, AA, and AS, providing new insights into the role of complement

  9. Alkylglycerols reduce serum complement and plasma vascular endothelial growth factor in obese individuals.

    PubMed

    Parri, A; Fitó, Montserrat; Torres, C F; Muñoz-Aguayo, D; Schröder, H; Cano, J F; Vázquez, L; Reglero, G; Covas, María-Isabel

    2016-06-01

    Alkylglycerols (AKGs), isolated or present in shark liver oil have anti-inflammatory properties. Complement 3 (C3) and 4 (C4) participate in lipid metabolism and in obesity, contributing to the metabolic syndrome and to the low-grade inflammation associated with obesity. In a randomized, controlled, crossover study, 26 non-diabetes obese individuals were assigned two preparations with low (LAC, 10 mg AKGs) and high (HAC, 20 mg AKGs) AKG content. Intervention periods were of 3 weeks preceded by 2-week washout periods in which shark liver oil was avoided. Cholesterol, C3, C4, and vascular endothelial growth factor (VEGF) decreased in a linear trend (P < 0.01) from baseline (control) to LAC and HAC. Values after HAC were significantly lower (P < 0.05) versus both baseline and after LAC. No adverse effects were observed or reported. Data from this pilot study open a promising field for the study of the beneficial effects of AKGs on cardiovascular risk factors in obese individuals. PMID:27188987

  10. Alkylglycerols reduce serum complement and plasma vascular endothelial growth factor in obese individuals.

    PubMed

    Parri, A; Fitó, Montserrat; Torres, C F; Muñoz-Aguayo, D; Schröder, H; Cano, J F; Vázquez, L; Reglero, G; Covas, María-Isabel

    2016-06-01

    Alkylglycerols (AKGs), isolated or present in shark liver oil have anti-inflammatory properties. Complement 3 (C3) and 4 (C4) participate in lipid metabolism and in obesity, contributing to the metabolic syndrome and to the low-grade inflammation associated with obesity. In a randomized, controlled, crossover study, 26 non-diabetes obese individuals were assigned two preparations with low (LAC, 10 mg AKGs) and high (HAC, 20 mg AKGs) AKG content. Intervention periods were of 3 weeks preceded by 2-week washout periods in which shark liver oil was avoided. Cholesterol, C3, C4, and vascular endothelial growth factor (VEGF) decreased in a linear trend (P < 0.01) from baseline (control) to LAC and HAC. Values after HAC were significantly lower (P < 0.05) versus both baseline and after LAC. No adverse effects were observed or reported. Data from this pilot study open a promising field for the study of the beneficial effects of AKGs on cardiovascular risk factors in obese individuals.

  11. Role of Complement C5 in Experimental Blunt Chest Trauma-Induced Septic Acute Lung Injury (ALI)

    PubMed Central

    Karbach, Michael; Braumueller, Sonja; Kellermann, Philipp; Gebhard, Florian; Huber-Lang, Markus; Perl, Mario

    2016-01-01

    Background Severe blunt chest trauma is associated with high mortality. Sepsis represents a serious risk factor for mortality in acute respiratory distress syndrome (ARDS). In septic patients with ARDS complement activation products were found to be elevated in the plasma. In single models like LPS or trauma complement has been studied to some degree, however in clinically highly relevant double hit models such as the one used here little data is available. Here, we hypothesized that absence of C5 is correlated with a decreased inflammatory response in trauma induced septic acute lung injury. Methods 12 hrs after DH in mice the local and systemic cytokines and chemokines were quantified by multiplex bead array or ELISA, activated caspase-3 by western blot. Data were analyzed using one-way ANOVA followed by post-hoc Sidak’s multiple comparison test (significance, p≤ 0.05). Results In lung tissue interleukin (IL)-6, monocyte chemo attractant protein-1 (MCP-1) and granulocyte-colony stimulating factor (G-CSF) was elevated in both C5-/- mice and wildtype littermates (wt), whereas caspase-3 was reduced in lungs after DH in C5-/- mice. Systemically, reduced keratinocyte-derived chemokine (KC) levels were observed after DH in C5-/- compared to wt mice. Locally, lung myeloperoxidase (MPO), protein, IL-6, MCP-1 and G-CSF in brochoalveolar lavage fluid (BALF) were elevated after DH in C5-/- compared to wt. Conclusions In the complex but clinically relevant DH model the local and systemic inflammatory immune response features both, C5-dependent and C5-independent characteristics. Activation of caspase-3 in lung tissue after DH was C5-dependent whereas local inflammation in lung tissue was C5-independent. PMID:27437704

  12. Complement sensitivity and factor H binding of European Francisella tularensis ssp. holarctica strains in selected animal species.

    PubMed

    Kreizinger, Zsuzsa; Bhide, Mangesh; Bencurova, Elena; Dolinska, Saskia; Gyuranecz, Miklós

    2015-09-01

    Francisella tularensis is a Gram-negative bacterium, the causative agent of the zoonotic disease tularaemia. The bacterium has developed several extracellular and intracellular strategies to evade the hosts' innate and adaptive immune responses. The aims of the study were to examine complement sensitivity of wild and attenuated F. tularensis ssp. holarctica strains in animal hosts of distinct sensitivity to the bacterium, to compare the complement-evading ability of wild strains of different phylogeographic background, and to examine the role of factor H in the host-pathogen interactions. Complement sensitivity assays were carried out on various F. tularensis ssp. holarctica wild strains and on the attenuated live vaccine strain (LVS) with sera of the highly sensitive house mouse (Mus musculus), the moderately sensitive European brown hare (Lepus europaeus) and the relatively resistant cattle (Bos taurus). Specific binding of complement regulator factor H to bacterial membrane proteins was examined by Western blot assays. All wild strains interacted with the hosts' complement system and showed no significant differences in their survivability. The attenuated LVS was resistant to serum killing in mouse, but was lysed in the sera of hare and cattle. Direct binding of factor H to F. tularensis membrane proteins was not detected.

  13. Complement Factor B Mutations in Atypical Hemolytic Uremic Syndrome—Disease-Relevant or Benign?

    PubMed Central

    Marinozzi, Maria Chiara; Vergoz, Laura; Rybkine, Tania; Ngo, Stephanie; Bettoni, Serena; Pashov, Anastas; Cayla, Mathieu; Tabarin, Fanny; Jablonski, Mathieu; Hue, Christophe; Smith, Richard J.; Noris, Marina; Halbwachs-Mecarelli, Lise; Donadelli, Roberta; Fremeaux-Bacchi, Veronique

    2014-01-01

    Atypical hemolytic uremic syndrome (aHUS) is a genetic ultrarare renal disease associated with overactivation of the alternative pathway of complement. Four gain-of-function mutations that form a hyperactive or deregulated C3 convertase have been identified in Factor B (FB) ligand binding sites. Here, we studied the functional consequences of 10 FB genetic changes recently identified from different aHUS cohorts. Using several tests for alternative C3 and C5 convertase formation and regulation, we identified two gain-of-function and potentially disease-relevant mutations that formed either an overactive convertase (M433I) or a convertase resistant to decay by FH (K298Q). One mutation (R178Q) produced a partially cleaved protein with no ligand binding or functional activity. Seven genetic changes led to near-normal or only slightly reduced ligand binding and functional activity compared with the most common polymorphism at position 7, R7. Notably, none of the algorithms used to predict the disease relevance of FB mutations agreed completely with the experimental data, suggesting that in silico approaches should be undertaken with caution. These data, combined with previously published results, suggest that 9 of 15 FB genetic changes identified in patients with aHUS are unrelated to disease pathogenesis. This study highlights that functional assessment of identified nucleotide changes in FB is mandatory to confirm disease association. PMID:24652797

  14. Ensemble refinement shows conformational flexibility in crystal structures of human complement factor D

    SciTech Connect

    Forneris, Federico; Burnley, B. Tom; Gros, Piet

    2014-03-01

    Ensemble-refinement analysis of native and mutant factor D (FD) crystal structures indicates a dynamical transition in FD from a self-inhibited inactive conformation to a substrate-bound active conformation that is reminiscent of the allostery in thrombin. Comparison with previously observed dynamics in thrombin using NMR data supports the crystallographic ensembles. Human factor D (FD) is a self-inhibited thrombin-like serine proteinase that is critical for amplification of the complement immune response. FD is activated by its substrate through interactions outside the active site. The substrate-binding, or ‘exosite’, region displays a well defined and rigid conformation in FD. In contrast, remarkable flexibility is observed in thrombin and related proteinases, in which Na{sup +} and ligand binding is implied in allosteric regulation of enzymatic activity through protein dynamics. Here, ensemble refinement (ER) of FD and thrombin crystal structures is used to evaluate structure and dynamics simultaneously. A comparison with previously published NMR data for thrombin supports the ER analysis. The R202A FD variant has enhanced activity towards artificial peptides and simultaneously displays active and inactive conformations of the active site. ER revealed pronounced disorder in the exosite loops for this FD variant, reminiscent of thrombin in the absence of the stabilizing Na{sup +} ion. These data indicate that FD exhibits conformational dynamics like thrombin, but unlike in thrombin a mechanism has evolved in FD that locks the unbound native state into an ordered inactive conformation via the self-inhibitory loop. Thus, ensemble refinement of X-ray crystal structures may represent an approach alternative to spectroscopy to explore protein dynamics in atomic detail.

  15. Expression, purification, cocrystallization and preliminary crystallographic analysis of sucrose octasulfate/human complement regulator factor H SCRs 6–8

    SciTech Connect

    Prosser, Beverly E.; Johnson, Steven; Roversi, Pietro; Clark, Simon J.; Tarelli, Edward; Sim, Robert B.; Day, Antony J.; Lea, Susan M.

    2007-06-01

    The crystallization of human complement regulator FH-678{sub 402H} with a glycosaminoglycan analogue is described. Human plasma protein complement factor H (FH) is an inhibitor of the spontaneously activated alternative complement pathway. An allotypic variant of FH, 402His, has been associated with age-related macular degeneration, the leading cause of blindness in the elderly. Crystals of FH domains 6–8 (FH678) containing 402His have been grown in the presence of a polyanionic sucrose octasulfate ligand (an analogue of the natural glycosaminoglycan ligands of FH) using both native and selenomethionine-derivatized protein. Native data sets diffracting to 2.3 Å and SeMet data sets of up to 2.8 Å resolution have been collected. An anomalous difference Patterson map reveals self- and cross-peaks from two incorporated Se atoms. The corresponding selenium substructure has been solved.

  16. Visualizing K48 Ubiquitination during Presynaptic Formation By Ubiquitination-Induced Fluorescence Complementation (UiFC)

    PubMed Central

    Pinto, Maria J.; Pedro, Joana R.; Costa, Rui O.; Almeida, Ramiro D.

    2016-01-01

    In recent years, signaling through ubiquitin has been shown to be of great importance for normal brain development. Indeed, fluctuations in ubiquitin levels and spontaneous mutations in (de)ubiquitination enzymes greatly perturb synapse formation and neuronal transmission. In the brain, expression of lysine (K) 48-linked ubiquitin chains is higher at a developmental stage coincident with synaptogenesis. Nevertheless, no studies have so far delved into the involvement of this type of polyubiquitin chains in synapse formation. We have recently proposed a role for polyubiquitinated conjugates as triggering signals for presynaptic assembly. Herein, we aimed at characterizing the axonal distribution of K48 polyubiquitin and its dynamics throughout the course of presynaptic formation. To accomplish so, we used an ubiquitination-induced fluorescence complementation (UiFC) strategy for the visualization of K48 polyubiquitin in live hippocampal neurons. We first validated its use in neurons by analyzing changing levels of polyubiquitin. UiFC signal is diffusely distributed with distinct aggregates in somas, dendrites and axons, which perfectly colocalize with staining for a K48-specific antibody. Axonal UiFC aggregates are relatively stable and new aggregates are formed as an axon grows. Approximately 65% of UiFC aggregates colocalize with synaptic vesicle clusters and they preferentially appear in the axonal domains of axo-somatodendritic synapses when compared to isolated axons. We then evaluated axonal accumulation of K48 ubiquitinated signals in bead-induced synapses. We observed rapid accumulation of UiFC signal and endogenous K48 ubiquitin at the sites of newly formed presynapses. Lastly, we show by means of a microfluidic platform, for the isolation of axons, that presynaptic clustering on beads is dependent on E1-mediated ubiquitination at the axonal level. Altogether, these results indicate that enrichment of K48 polyubiquitin at the site of nascent presynaptic

  17. Bacillus anthracis Spore Surface Protein BclA Mediates Complement Factor H Binding to Spores and Promotes Spore Persistence.

    PubMed

    Wang, Yanyu; Jenkins, Sarah A; Gu, Chunfang; Shree, Ankita; Martinez-Moczygemba, Margarita; Herold, Jennifer; Botto, Marina; Wetsel, Rick A; Xu, Yi

    2016-06-01

    Spores of Bacillus anthracis, the causative agent of anthrax, are known to persist in the host lungs for prolonged periods of time, however the underlying mechanism is poorly understood. In this study, we demonstrated that BclA, a major surface protein of B. anthracis spores, mediated direct binding of complement factor H (CFH) to spores. The surface bound CFH retained its regulatory cofactor activity resulting in C3 degradation and inhibition of downstream complement activation. By comparing results from wild type C57BL/6 mice and complement deficient mice, we further showed that BclA significantly contributed to spore persistence in the mouse lungs and dampened antibody responses to spores in a complement C3-dependent manner. In addition, prior exposure to BclA deletion spores (ΔbclA) provided significant protection against lethal challenges by B. anthracis, whereas the isogenic parent spores did not, indicating that BclA may also impair protective immunity. These results describe for the first time an immune inhibition mechanism of B. anthracis mediated by BclA and CFH that promotes spore persistence in vivo. The findings also suggested an important role of complement in persistent infections and thus have broad implications. PMID:27304426

  18. Deficiency of the Complement Component 3 but Not Factor B Aggravates Staphylococcus aureus Septic Arthritis in Mice.

    PubMed

    Na, Manli; Jarneborn, Anders; Ali, Abukar; Welin, Amanda; Magnusson, Malin; Stokowska, Anna; Pekna, Marcela; Jin, Tao

    2016-04-01

    The complement system plays an essential role in the innate immune response and protection against bacterial infections. However, detailed knowledge regarding the role of complement in Staphylococcus aureus septic arthritis is still largely missing. In this study, we elucidated the roles of selected complement proteins in S. aureus septic arthritis. Mice lacking the complement component 3 (C3(-/-)), complement factor B (fB(-/-)), and receptor for C3-derived anaphylatoxin C3a (C3aR(-/-)) and wild-type (WT) control mice were intravenously or intra-articularly inoculated with S. aureus strain Newman. The clinical course of septic arthritis, as well as histopathological and radiological changes in joints, was assessed. After intravenous inoculation, arthritis severity and frequency were significantly higher in C3(-/-)mice than in WT controls, whereas fB(-/-)mice displayed intermediate arthritis severity and frequency. This was in accordance with both histopathological and radiological findings. C3, but not fB, deficiency was associated with greater weight loss, more frequent kidney abscesses, and higher bacterial burden in kidneys. S. aureus opsonized with C3(-/-)sera displayed decreased uptake by mouse peritoneal macrophages compared with bacteria opsonized with WT or fB(-/-)sera. C3aR deficiency had no effect on the course of hematogenous S. aureus septic arthritis. We conclude that C3 deficiency increases susceptibility to hematogenous S. aureus septic arthritis and impairs host bacterial clearance, conceivably due to diminished opsonization and phagocytosis of S. aureus.

  19. Bacillus anthracis Spore Surface Protein BclA Mediates Complement Factor H Binding to Spores and Promotes Spore Persistence

    PubMed Central

    Gu, Chunfang; Martinez-Moczygemba, Margarita; Herold, Jennifer; Botto, Marina; Wetsel, Rick A.; Xu, Yi

    2016-01-01

    Spores of Bacillus anthracis, the causative agent of anthrax, are known to persist in the host lungs for prolonged periods of time, however the underlying mechanism is poorly understood. In this study, we demonstrated that BclA, a major surface protein of B. anthracis spores, mediated direct binding of complement factor H (CFH) to spores. The surface bound CFH retained its regulatory cofactor activity resulting in C3 degradation and inhibition of downstream complement activation. By comparing results from wild type C57BL/6 mice and complement deficient mice, we further showed that BclA significantly contributed to spore persistence in the mouse lungs and dampened antibody responses to spores in a complement C3-dependent manner. In addition, prior exposure to BclA deletion spores (ΔbclA) provided significant protection against lethal challenges by B. anthracis, whereas the isogenic parent spores did not, indicating that BclA may also impair protective immunity. These results describe for the first time an immune inhibition mechanism of B. anthracis mediated by BclA and CFH that promotes spore persistence in vivo. The findings also suggested an important role of complement in persistent infections and thus have broad implications. PMID:27304426

  20. Complement factor H interferes with Mycobacterium bovis BCG entry into macrophages and modulates the pro-inflammatory cytokine response.

    PubMed

    Abdul-Aziz, Munirah; Tsolaki, Anthony G; Kouser, Lubna; Carroll, Maria V; Al-Ahdal, Mohammed N; Sim, Robert B; Kishore, Uday

    2016-09-01

    Mycobacterium tuberculosis is an accomplished intracellular pathogen, particularly within the macrophage and this is of the utmost importance in the host-pathogen stand-off observed in the granuloma during latent tuberculosis. Contact with innate immune molecules is one of the primary interactions that can occur with the pathogen M. tuberculosis once inhaled. Complement proteins may play a role in facilitating M. tuberculosis interactions with macrophages. Here, we demonstrate that factor H, a complement regulatory protein that down-regulates complement alternative pathway activation, binds directly to the model organism M. bovis BCG. Binding of factor H reaches saturation at 5-10μg of factor H/ml, well below the plasma level. C4 binding protein (C4BP) competed with factor H for binding to mycobacteria. Factor H was also found to inhibit uptake of M. bovis BCG by THP-1 macrophage cells in a dose-dependent manner. Real-time qPCR analysis showed stark differential responses of pro- and anti-inflammatory cytokines during the early stages of phagocytosis, as evident from elevated levels of TNF-α, IL-1β and IL-6, and a concomitant decrease in IL-10, TGF-β and IL-12 levels, when THP-1:BCG interaction took place in the presence of factor H. Our results suggest that factor H can interfere with mycobacterial entry into macrophages and modulate inflammatory cytokine responses, particularly during the initial stages of infection, thus affecting the extracellular survival of the pathogen. Our results offer novel insights into complement activation-independent functions of factor H during the host-pathogen interaction in tuberculosis.

  1. Polydom: a secreted protein with pentraxin, complement control protein, epidermal growth factor and von Willebrand factor A domains.

    PubMed Central

    Gilgès, D; Vinit, M A; Callebaut, I; Coulombel, L; Cacheux, V; Romeo, P H; Vigon, I

    2000-01-01

    To identify extracellular proteins with epidermal growth factor (EGF) domains that are potentially involved in the control of haemopoiesis, we performed degenerate reverse-transcriptase-mediated PCR on the murine bone-marrow stromal cell line MS-5 and isolated a new partial cDNA encoding EGF-like domains related to those in the Notch proteins. Cloning and sequencing of the full-length cDNA showed that it encoded a new extracellular multi-domain protein that we named polydom. This 387 kDa mosaic protein contained a signal peptide followed by a new association of eight different protein domains, including a pentraxin domain and a von Willebrand factor type A domain, ten EGF domains, and 34 complement control protein modules. The human polydom mRNA is strongly expressed in placenta, its expression in the other tissues being weak or undetectable. The particular multidomain structure of the encoded protein suggests an important biological role in cellular adhesion and/or in the immune system. PMID:11062057

  2. Rare Complement Factor H Variant Associated With Age-Related Macular Degeneration in the Amish

    PubMed Central

    Hoffman, Joshua D.; CookeBailey, Jessica N.; D'Aoust, Laura; Cade, William; Ayala-Haedo, Juan; Fuzzell, Denise; Laux, Renee; Adams, Larry D.; Reinhart-Mercer, Lori; Caywood, Laura; Whitehead-Gay, Patrice; Agarwal, Anita; Wang, Gaofeng; Scott, William K.; Pericak-Vance, Margaret A.; Haines, Jonathan L.

    2014-01-01

    Purpose. Age-related macular degeneration is the leading cause of blindness among the adult population in the developed world. To further the understanding of this disease, we have studied the genetically isolated Amish population of Ohio and Indiana. Methods. Cumulative genetic risk scores were calculated using the 19 known allelic associations. Exome sequencing was performed in three members of a small Amish family with AMD who lacked the common risk alleles in complement factor H (CFH) and ARMS2/HTRA1. Follow-up genotyping and association analysis was performed in a cohort of 973 Amish individuals, including 95 with self-reported AMD. Results. The cumulative genetic risk score analysis generated a mean genetic risk score of 1.12 (95% confidence interval [CI]: 1.10, 1.13) in the Amish controls and 1.18 (95% CI: 1.13, 1.22) in the Amish cases. This mean difference in genetic risk scores is statistically significant (P = 0.0042). Exome sequencing identified a rare variant (P503A) in CFH. Association analysis in the remainder of the Amish sample revealed that the P503A variant is significantly associated with AMD (P = 9.27 × 10−13). Variant P503A was absent when evaluated in a cohort of 791 elderly non-Amish controls, and 1456 non-Amish cases. Conclusions. Data from the cumulative genetic risk score analysis suggests that the variants reported by the AMDGene consortium account for a smaller genetic burden of disease in the Amish compared with the non-Amish Caucasian population. Using exome sequencing data, we identified a novel missense mutation that is shared among a densely affected nuclear Amish family and located in a gene that has been previously implicated in AMD risk. PMID:24906858

  3. Localization of complement factor H gene expression and protein distribution in the mouse outer retina

    PubMed Central

    Smit-McBride, Zeljka; Oltjen, Sharon L.; Radu, Roxana A.; Estep, Jason; Nguyen, Anthony T.; Gong, Qizhi

    2015-01-01

    Purpose To determine the localization of complement factor H (Cfh) mRNA and its protein in the mouse outer retina. Methods Quantitative real-time PCR (qPCR) was used to determine the expression of Cfh and Cfh-related (Cfhr) transcripts in the RPE/choroid. In situ hybridization (ISH) was performed using the novel RNAscope 2.0 FFPE assay to localize the expression of Cfh mRNA in the mouse outer retina. Immunohistochemistry (IHC) was used to localize Cfh protein expression, and western blots were used to characterize CFH antibodies used for IHC. Results Cfh and Cfhr2 transcripts were detected in the mouse RPE/choroid using qPCR, while Cfhr1, Cfhr3, and Cfhrc (Gm4788) were not detected. ISH showed abundant Cfh mRNA in the RPE of all mouse strains (C57BL/6, BALB/c, 129/Sv) tested, with the exception of the Cfh−/− eye. Surprisingly, the Cfh protein was detected by immunohistochemistry in photoreceptors rather than in RPE cells. The specificity of the CFH antibodies was tested by western blotting. Our CFH antibodies recognized purified mouse Cfh protein, serum Cfh protein in wild-type C57BL/6, BALB/c, and 129/Sv, and showed an absence of the Cfh protein in the serum of Cfh−/− mice. Greatly reduced Cfh protein immunohistological signals in the Cfh−/− eyes also supported the specificity of the Cfh protein distribution results. Conclusions Only Cfh and Cfhr2 genes are expressed in the mouse outer retina. Only Cfh mRNA was detected in the RPE, but no protein. We hypothesize that the steady-state concentration of Cfh protein is low in the cells due to secretion, and therefore is below the detection level for IHC. PMID:25684976

  4. Genomic analysis of the F subtypes of human complement factor B.

    PubMed

    Jahn, I; Mejía, J E; Thomas, M; Darke, C; Schröder, H; Geserick, G; Hauptmann, G

    1994-12-01

    Factor B of human complement is encoded within the Major Histocompatibility Complex (MHC) and is polymorphic, with up to 30 alleles defined by electrophoretic mobility. One of the most common alleles, BF*F, is subdivided into the FA and FB subtypes, which differ at the gene level by non-synonymous base substitutions in the seventh codon. We have found at this position a new restriction site polymorphism, as a Bsl I site absent from the FB allele. Using this restriction polymorphism, we have developed a method for BF F subtype determination, based on amplification by polymerase chain reaction of the 5' end of the BF gene, and digestion with Bsl I. This new method has been applied to a panel of 29 selected BF F individuals. A single strand DNA conformation analysis of the same region of the gene allowed us to confirm the above DNA-based BF F subtyping. During this study, two BF*F1 alleles showed discrepancies between protein and DNA typing, which were confirmed by our sequencing data. These were identical, in the 5' region, to BF*S and BF*FB genes, respectively. In a comparison with two protein subtyping methods, identical results were found for only one third of the selected samples. The conflicting results may arise, in part, from previously undescribed molecular heterogeneity within BF F subtypes, or from the presence of a null allele. Our new method allows BF*F subtyping to be used with confidence in the definition of disease-associated MHC haplotypes.

  5. Complement factor H gene associations with end-stage kidney disease in African Americans

    PubMed Central

    Bonomo, Jason A.; Palmer, Nicholette D.; Hicks, Pamela J.; Lea, Janice P.; Okusa, Mark D.; Langefeld, Carl D.; Bowden, Donald W.; Freedman, Barry I.

    2014-01-01

    Background Mutations in the complement factor H gene (CFH) region associate with renal-limited mesangial proliferative forms of glomerulonephritis including IgA nephropathy (IgAN), dense deposit disease (DDD) and C3 glomerulonephritis (C3GN). Lack of kidney biopsies could lead to under diagnosis of CFH-associated end-stage kidney disease (ESKD) in African Americans (AAs), with incorrect attribution to other causes. A prior genome-wide association study in AAs with non-diabetic ESKD implicated an intronic CFH single nucleotide polymorphism (SNP). Methods Thirteen CFH SNPs (8 exonic, 2 synonymous, 2 3′UTR, and the previously associated intronic variant rs379489) were tested for association with common forms of non-diabetic and type 2 diabetes-associated (T2D) ESKD in 3770 AAs (1705 with non-diabetic ESKD, 1305 with T2D-ESKD, 760 controls). Most cases lacked kidney biopsies; those with known IgAN, DDD or C3GN were excluded. Results Adjusting for age, gender, ancestry and apolipoprotein L1 gene risk variants, single SNP analyses detected 6 CFH SNPs (5 exonic and the intronic variant) as significantly associated with non-diabetic ESKD (P = 0.002–0.01), three of these SNPs were also associated with T2D-ESKD. Weighted CFH locus-wide Sequence Kernel Association Testing (SKAT) in non-diabetic ESKD (P = 0.00053) and T2D-ESKD (P = 0.047) confirmed significant evidence of association. Conclusions CFH was associated with commonly reported etiologies of ESKD in the AA population. These results suggest that a subset of cases with ESKD clinically ascribed to the effects of hypertension or glomerulosclerosis actually have CFH-related forms of mesangial proliferative glomerulonephritis. Genetic testing may prove useful to identify the causes of renal-limited kidney disease in patients with ESKD who lack renal biopsies. PMID:24586071

  6. Hepatitis B virus X protein up-regulates C4b-binding protein α through activating transcription factor Sp1 in protection of hepatoma cells from complement attack

    PubMed Central

    Feng, Guoxing; Li, Jiong; Zheng, Minying; Yang, Zhe; Liu, Yunxia; Zhang, Shuqin; Ye, Lihong; Zhang, Weiying; Zhang, Xiaodong

    2016-01-01

    Hepatitis B virus X protein (HBx) plays crucial roles in the development of hepatocellular carcinoma (HCC). We previously showed that HBx protected hepatoma cells from complement attack by activation of CD59. Moreover, in this study we found that HBx protected hepatoma cells from complement attack by activation of C4b-binding protein α (C4BPα), a potent inhibitor of complement system. We observed that HBx were positively correlated with those of C4BPα in clinical HCC tissues. Mechanistically, HBx activated the promoter core region of C4BPα, located at −1199/−803nt, through binding to transcription factor Sp1. In addition, chromatin immunoprecipitation (ChIP) assays showed that HBx was able to bind to the promoter of C4BPα, which could be blocked by Sp1 silencing. Functionally, knockdown of C4BPα obviously increased the deposition of C5b-9, a complex of complement membrane attack, and remarkably abolished the HBx-induced resistance of hepatoma cells from complement attack in vitro and in vivo. Thus, we conclude that HBx up-regulates C4BPα through activating transcription factor Sp1 in protection of liver cancer cells from complement attack. Our finding provides new insights into the mechanism by which HBx enhances protection of hepatoma cells from complement attack. PMID:27050367

  7. The relationship between complement factor C3, APOE ε4, amyloid and tau in Alzheimer's disease.

    PubMed

    Bonham, Luke W; Desikan, Rahul S; Yokoyama, Jennifer S

    2016-01-01

    Inflammation is becoming increasingly recognized as an important contributor to Alzheimer's disease (AD) pathogenesis. As a part of the innate immune system, the complement cascade enhances the body's ability to destroy and remove pathogens and has recently been shown to influence Alzheimer's associated amyloid and tau pathology. However, little is known in humans about the effects of the complement system and genetic modifiers of AD risk like the ε4 allele of apolioprotein E (APOE ε4) on AD pathobiology. We evaluated cerebrospinal fluid (CSF) protein levels from 267 individuals clinically diagnosed as cognitively normal, mild cognitive impairment, and AD. Using linear models, we assessed the relationship between APOE ε4 genotype, CSF Complement 3 (C3), CSF amyloid-β (amyloid) and CSF hyperphosphorylated tau (ptau). We found a significant interaction between APOE ε4 and CSF C3 on both CSF amyloid and CSF ptau. We also found that CSF C3 is only associated with CSF ptau after accounting for CSF amyloid. Our results support a conceptual model of the AD pathogenic cascade where a synergistic relationship between the complement cascade (C3) and APOE ε4 results in elevated Alzheimer's neurodegeneration and in turn, amyloid further regulates the effect of the complement cascade on downstream tau pathology. PMID:27357286

  8. Factor H-related protein 5 interacts with pentraxin 3 and the extracellular matrix and modulates complement activation.

    PubMed

    Csincsi, Ádám I; Kopp, Anne; Zöldi, Miklós; Bánlaki, Zsófia; Uzonyi, Barbara; Hebecker, Mario; Caesar, Joseph J E; Pickering, Matthew C; Daigo, Kenji; Hamakubo, Takao; Lea, Susan M; Goicoechea de Jorge, Elena; Józsi, Mihály

    2015-05-15

    The physiological roles of the factor H (FH)-related proteins are controversial and poorly understood. Based on genetic studies, FH-related protein 5 (CFHR5) is implicated in glomerular diseases, such as atypical hemolytic uremic syndrome, dense deposit disease, and CFHR5 nephropathy. CFHR5 was also identified in glomerular immune deposits at the protein level. For CFHR5, weak complement regulatory activity and competition for C3b binding with the plasma complement inhibitor FH have been reported, but its function remains elusive. In this study, we identify pentraxin 3 (PTX3) as a novel ligand of CFHR5. Binding of native CFHR5 to PTX3 was detected in human plasma and the interaction was characterized using recombinant proteins. The binding of PTX3 to CFHR5 is of ∼2-fold higher affinity compared with that of FH. CFHR5 dose-dependently inhibited FH binding to PTX3 and also to the monomeric, denatured form of the short pentraxin C-reactive protein. Binding of PTX3 to CFHR5 resulted in increased C1q binding. Additionally, CFHR5 bound to extracellular matrix in vitro in a dose-dependent manner and competed with FH for binding. Altogether, CFHR5 reduced FH binding and its cofactor activity on pentraxins and the extracellular matrix, while at the same time allowed for enhanced C1q binding. Furthermore, CFHR5 allowed formation of the alternative pathway C3 convertase and supported complement activation. Thus, CFHR5 may locally enhance complement activation via interference with the complement-inhibiting function of FH, by enhancement of C1q binding, and by activating complement, thereby contributing to glomerular disease.

  9. The effect of substrate molecular mobility on surface induced immune complement activation and blood plasma coagulation.

    PubMed

    Berglin, Mattias; Andersson, Marcus; Sellborn, Anders; Elwing, Hans

    2004-08-01

    Changing the length of the alkyl ester side chain in poly(alkyl methacrylates) provides a unique opportunity to systematically vary the mobility of the polymer chains, or in other words vary the glass transition temperature (T(g)), without greatly affect the solid surface energy (gamma(s)) of the polymer. A series of poly(alkyl methacrylate) coatings was therefore analysed with regard to the human immune complement (IC) activation and the surface associated blood plasma coagulation cascade (CC) properties. For the IC and CC measurements we used a quartz crystal microbalance (QCM) where we modified the chemistry of the sensor surface by applying 10-30 nm thick poly(alkyl methacrylate) coatings. The surface energy was calculated from water contact angles and small differences between the coatings were observed. The surface chemistry of the coatings, as determined with X-ray photoelectron spectroscopy (XPS), showed no deviation from expected compositions. Tapping mode atomic force microscopy (TM-AFM) measurements revealed that all coatings displayed similar morphology and the roughness was in the range of 0.7-0.9 nm. Increased polymer mobility correlated with a decrease in IC activation, measured as a decreased C3c deposition at the surface. The surface induced CC, measured as fibrin clot formation at the surface, was different between the different coatings but no correlation with molecular mobility was observed. Thus, the molecular mobility of the polymer chains had a major effect on both the IC and the CC and it seems that different aspects of the chemistry of the solid surface regulate activation of the IC and the CC.

  10. Sundanese Complementation

    ERIC Educational Resources Information Center

    Kurniawan, Eri

    2013-01-01

    The focus of this thesis is the description and analysis of clausal complementation in Sundanese, an Austronesian language spoken in Indonesia. The thesis examined a range of clausal complement types in Sundanese, which consists of (i) "yen/(wi)rehna" "that" complements, (ii) "pikeun" "for" complements,…

  11. Complement-dependent cytotoxicity in rats bearing human adenovirus type 12-induced primary retinoblastoma-like tumor in the eye.

    PubMed

    Nishida, T; Mukai, N; Solish, S P

    1981-01-01

    Using an animal model of retinoblastoma in inbred rats and cultured human adenovirus type 12-induced retinoblastoma-like tumor cells (RAO 188), complement-dependent cytotoxicity was determined by measuring release of 3H-uridine labelled RNA. Sera from rats in which tumors did not grow after adenovirus type 12 inoculation had higher cytotoxicity against RAO 188 cells than sera from rats bearing primary adenovirus type 12-induced retinoblastoma-like tumor. These results showed that the rat which could raise antibodies against adenovirus type 12-induced retinoblastoma-like tumor cells did not allow the tumor growth in the eye after virus inoculation.

  12. [Modification of complement factors and their inhibitors during meningococcal sepsis (author's transl)].

    PubMed

    Blanco, A; Solís, P; Alvarez Guisasola, F J; Valbuena, C; Gómez, C; Prieto, P; Ruíz, C

    1980-08-01

    Various complement components (C'1s, C'3, C'4, C'5, C'8, C'9, C'3 act., C'1 inh., C'3b inact.) and seric immunocomplexes (by polyethylene glycol, PEG) were evaluated in 43 children with meningococcal sepsis. 28 patients had disseminated intravascular coagulation (DIC), group I, and 15 did not show it, group II; 14 patients died in group I and none of group II. In 21 cases studies were repeated 24 hours later. In group I all complement components were decreased, specially C'3 (x: 67 mg./100 ml., p < 0.01) and C'5 (x: 8 mg./100 ml., p < 0.01) and they were lower 24 h. later. Results of group II were normal, except a decrease of C'5. Catabolic products of C'3 were founded in 11/14 cases of group I and two/nine of group II and products of C'3 act. in four/14 and one/10. PEG precipitation was positive in 10/14 cases of group I and 10/12 of group II and IgG, IgM, C'3 and C'4 were found in precipitations. This complement components were more frequently present in sepsis without DIC and after 24 h. of evolution. C'1 and C'3b inhibitors decreased after evolution in group I and by contrast increased in group II. This fall enhances complement activation.

  13. Complement factor I deficiency: a not so rare immune defect. Characterization of new mutations and the first large gene deletion

    PubMed Central

    2012-01-01

    Background Complement Factor I (CFI) is a serine protease with an important role in complement alternative pathway regulation. Complete factor I deficiency is strongly associated with severe infections. Approximately 30 families with this deficiency have been described worldwide. Patients and methods We have studied five new Spanish families suffering from CFI deficiency. From 19 screened people, 7 homozygous, 10 heterozygous and 2 healthy subjects were identified. Clinical, biochemical and genetic descriptions are included. Results Molecular studies demonstrated 4 novel mutations in the screened individuals; amongst them, we describe here the first great gene deletion reported in the CFI locus, which includes full exon 2 and part of the large intron 1. Conclusion CFI deficiency is possibly an underestimated defect and the eventual existence of this deficiency should be tested in those patients exhibiting low C3 and recurrent bacterial infections. We propose a simple diagnostic flowchart to help clinicians in the identification and correct diagnosis of such patients. PMID:22710145

  14. Dual interaction of factor H with C3d and glycosaminoglycans in host-nonhost discrimination by complement.

    PubMed

    Kajander, Tommi; Lehtinen, Markus J; Hyvärinen, Satu; Bhattacharjee, Arnab; Leung, Elisa; Isenman, David E; Meri, Seppo; Goldman, Adrian; Jokiranta, T Sakari

    2011-02-15

    The alternative pathway of complement is important in innate immunity, attacking not only microbes but all unprotected biological surfaces through powerful amplification. It is unresolved how host and nonhost surfaces are distinguished at the molecular level, but key components are domains 19-20 of the complement regulator factor H (FH), which interact with host (i.e., nonactivator surface glycosaminoglycans or sialic acids) and the C3d part of C3b. Our structure of the FH19-20:C3d complex at 2.3-Å resolution shows that FH19-20 has two distinct binding sites, FH19 and FH20, for C3b. We show simultaneous binding of FH19 to C3b and FH20 to nonactivator surface glycosaminoglycans, and we show that both of these interactions are necessary for full binding of FH to C3b on nonactivator surfaces (i.e., for target discrimination). We also show that C3d could replace glycosaminoglycan binding to FH20, thus providing a feedback control for preventing excess C3b deposition and complement amplification. This explains the molecular basis of atypical hemolytic uremic syndrome, where mutations on the binding interfaces between FH19-20 and C3d or between FH20 and glycosaminoglycans lead to complement attack against host surfaces.

  15. Microwaves induce an increase in the frequency of complement receptor-bearing lymphoid spleen cells in mice.

    PubMed

    Wiktor-Jedrzejczak, W; Ahmed, A; Sell, K W; Czerski, P; Leach, W M

    1977-04-01

    A single 30-min exposure of mice to 2450 MHz microwaves (12 to 15 mW/g body weight) in an environmentally controlled waveguide facility induced a significant increase in the proportion of complement-receptor positive lymphoid cells in the spleen. This effect was further enhanced by repeated (three times) exposures, which in addition produced a significant increase in the proportion of Ig+ cells. The proportion of theta-positive cells and the total number of spleen cells remained unchanged.

  16. The complement system in human cardiometabolic disease.

    PubMed

    Hertle, E; Stehouwer, C D A; van Greevenbroek, M M J

    2014-10-01

    The complement system has been implicated in obesity, fatty liver, diabetes and cardiovascular disease (CVD). Complement factors are produced in adipose tissue and appear to be involved in adipose tissue metabolism and local inflammation. Thereby complement links adipose tissue inflammation to systemic metabolic derangements, such as low-grade inflammation, insulin resistance and dyslipidaemia. Furthermore, complement has been implicated in pathophysiological mechanisms of diet- and alcohol induced liver damage, hyperglycaemia, endothelial dysfunction, atherosclerosis and fibrinolysis. In this review, we summarize current evidence on the role of the complement system in several processes of human cardiometabolic disease. C3 is the central component in complement activation, and has most widely been studied in humans. C3 concentrations are associated with insulin resistance, liver dysfunction, risk of the metabolic syndrome, type 2 diabetes and CVD. C3 can be activated by the classical, the lectin and the alternative pathway of complement activation; and downstream activation of C3 activates the terminal pathway. Complement may also be activated via extrinsic proteases of the coagulation, fibrinolysis and the kinin systems. Studies on the different complement activation pathways in human cardiometabolic disease are limited, but available evidence suggests that they may have distinct roles in processes underlying cardiometabolic disease. The lectin pathway appeared beneficial in some studies on type 2 diabetes and CVD, while factors of the classical and the alternative pathway were related to unfavourable cardiometabolic traits. The terminal complement pathway was also implicated in insulin resistance and liver disease, and appears to have a prominent role in acute and advanced CVD. The available human data suggest a complex and potentially causal role for the complement system in human cardiometabolic disease. Further, preferably longitudinal studies are needed to

  17. Decay-accelerating factor induction by tumour necrosis factor-alpha, through a phosphatidylinositol-3 kinase and protein kinase C-dependent pathway, protects murine vascular endothelial cells against complement deposition.

    PubMed

    Ahmad, Saifur R; Lidington, Elaine A; Ohta, Rieko; Okada, Noriko; Robson, Michael G; Davies, Kevin A; Leitges, Michael; Harris, Claire L; Haskard, Dorian O; Mason, Justin C

    2003-10-01

    We have shown that human endothelial cells (EC) are protected against complement-mediated injury by the inducible expression of decay-accelerating factor (DAF). To understand further the importance of DAF regulation, we characterized EC DAF expression on murine EC in vitro and in vivo using a model of glomerulonephritis. Flow cytometry using the monoclonal antibody (mAb) Riko-3 [binds transmembrane- and glycosylphosphatidylinositol (GPI)-anchored DAF], mAb Riko-4 (binds GPI-anchored DAF) and reverse transcription-polymerase chain reaction (RT-PCR), demonstrated that murine EC DAF is GPI-anchored. Tumour necrosis factor-alpha (TNF-alpha) increased EC DAF expression, detectable at 6 hr and maximal at 24-48 hr poststimulation. DAF upregulation required increased steady-state DAF mRNA and protein synthesis. In contrast, no increased expression of the murine complement receptor-related protein-Y (Crry) was seen with TNF-alpha. DAF upregulation was mediated via a protein kinase C (PKC)alpha, phosphoinositide-3 kinase (PI-3 kinase), p38 mitogen-activated protein kinase (MAPK) and nuclear factor-kappaB (NF-kappaB)-dependent pathway. The increased DAF was functionally relevant, resulting in a marked reduction in C3 deposition following complement activation. In a nephrotoxic nephritis model, DAF expression on glomerular capillaries was significantly increased 2 hr after the induction of disease. The demonstration of DAF upregulation above constitutive levels suggests that this may be important in the maintenance of vascular integrity during inflammation, when the risk of complement-mediated injury is increased. The mouse represents a suitable model for the study of novel therapeutic approaches by which vascular endothelium may be conditioned against complement-mediated injury.

  18. Heparin-protamine complexes and C-reactive protein induce activation of the classical complement pathway: studies in patients undergoing cardiac surgery and in vitro.

    PubMed

    Bruins, P; te Velthuis, H; Eerenberg-Belmer, A J; Yazdanbakhsh, A P; de Beaumont, E M; Eijsman, L; Trouwborst, A; Hack, C E

    2000-08-01

    The administration of protamine to patients undergoing cardiopulmonary bypass (CPB) to neutralize heparin and to reduce the risk of bleeding, induces activation of the classical complement pathway mainly by heparin-protamine complexes. We investigated whether C-reactive protein (CRP) contributes to protamine-induced complement activation. In 24 patients during myocardial revascularization, we measured complement, CRP, and complement-CRP complexes, reflecting CRP-mediated complement activation in vivo. We also incubated plasma from healthy volunteers and patients with heparin and protamine in vitro to study CRP-mediated complement activation. During CPB, CRP levels remained unchanged while C3 activation products increased. C4 activation occurred after protamine administration. CRP-complement complexes increased at the end of CPB and upon protamine administration. Incubation of plasma with heparin and protamine in vitro generated complement-CRP complexes, which was blocked by phosphorylcholine and stimulated by exogenous CRP. C4d-CRP complex formation after protamine administration correlated clinically with the incidence of postoperative arrhythmia. Protamine administration during cardiac surgery induces complement activation which in part is CRP-dependent, and correlates with postoperative arrhythmia.

  19. Complement Modulation of Anti-Aging Factor Klotho in Ischemia/Reperfusion Injury and Delayed Graft Function.

    PubMed

    Castellano, G; Intini, A; Stasi, A; Divella, C; Gigante, M; Pontrelli, P; Franzin, R; Accetturo, M; Zito, A; Fiorentino, M; Montinaro, V; Lucarelli, G; Ditonno, P; Battaglia, M; Crovace, A; Staffieri, F; Oortwijn, B; van Amersfoort, E; Pertosa, G; Grandaliano, G; Gesualdo, L

    2016-01-01

    Klotho is an anti-aging factor mainly produced by renal tubular epithelial cells (TEC) with pleiotropic functions. Klotho is down-regulated in acute kidney injury in native kidney; however, the modulation of Klotho in kidney transplantation has not been investigated. In a swine model of ischemia/reperfusion injury (IRI), we observed a remarkable reduction of renal Klotho by 24 h from IRI. Complement inhibition by C1-inhibitor preserved Klotho expression in vivo by abrogating nuclear factor kappa B (NF-kB) signaling. In accordance, complement anaphylotoxin C5a led to a significant down-regulation of Klotho in TEC in vitro that was NF-kB mediated. Analysis of Klotho in kidneys from cadaveric donors demonstrated a significant expression of Klotho in pre-implantation biopsies; however, patients affected by delayed graft function (DGF) showed a profound down-regulation of Klotho compared with patients with early graft function. Quantification of serum Klotho after 2 years from transplantation demonstrated significant lower levels in DGF patients. Our data demonstrated that complement might be pivotal in the down-regulation of Klotho in IRI leading to a permanent deficiency after years from transplantation. Considering the anti-senescence and anti-fibrotic effects of Klotho at renal levels, we hypothesize that this acquired deficiency of Klotho might contribute to DGF-associated chronic allograft dysfunction.

  20. Complement Modulation of Anti-Aging Factor Klotho in Ischemia/Reperfusion Injury and Delayed Graft Function.

    PubMed

    Castellano, G; Intini, A; Stasi, A; Divella, C; Gigante, M; Pontrelli, P; Franzin, R; Accetturo, M; Zito, A; Fiorentino, M; Montinaro, V; Lucarelli, G; Ditonno, P; Battaglia, M; Crovace, A; Staffieri, F; Oortwijn, B; van Amersfoort, E; Pertosa, G; Grandaliano, G; Gesualdo, L

    2016-01-01

    Klotho is an anti-aging factor mainly produced by renal tubular epithelial cells (TEC) with pleiotropic functions. Klotho is down-regulated in acute kidney injury in native kidney; however, the modulation of Klotho in kidney transplantation has not been investigated. In a swine model of ischemia/reperfusion injury (IRI), we observed a remarkable reduction of renal Klotho by 24 h from IRI. Complement inhibition by C1-inhibitor preserved Klotho expression in vivo by abrogating nuclear factor kappa B (NF-kB) signaling. In accordance, complement anaphylotoxin C5a led to a significant down-regulation of Klotho in TEC in vitro that was NF-kB mediated. Analysis of Klotho in kidneys from cadaveric donors demonstrated a significant expression of Klotho in pre-implantation biopsies; however, patients affected by delayed graft function (DGF) showed a profound down-regulation of Klotho compared with patients with early graft function. Quantification of serum Klotho after 2 years from transplantation demonstrated significant lower levels in DGF patients. Our data demonstrated that complement might be pivotal in the down-regulation of Klotho in IRI leading to a permanent deficiency after years from transplantation. Considering the anti-senescence and anti-fibrotic effects of Klotho at renal levels, we hypothesize that this acquired deficiency of Klotho might contribute to DGF-associated chronic allograft dysfunction. PMID:26280899

  1. Expression, purification, cocrystallization and preliminary crystallographic analysis of sucrose octasulfate/human complement regulator factor H SCRs 6-8.

    PubMed

    Prosser, Beverly E; Johnson, Steven; Roversi, Pietro; Clark, Simon J; Tarelli, Edward; Sim, Robert B; Day, Antony J; Lea, Susan M

    2007-06-01

    Human plasma protein complement factor H (FH) is an inhibitor of the spontaneously activated alternative complement pathway. An allotypic variant of FH, 402His, has been associated with age-related macular degeneration, the leading cause of blindness in the elderly. Crystals of FH domains 6-8 (FH678) containing 402His have been grown in the presence of a polyanionic sucrose octasulfate ligand (an analogue of the natural glycosaminoglycan ligands of FH) using both native and selenomethionine-derivatized protein. Native data sets diffracting to 2.3 A and SeMet data sets of up to 2.8 A resolution have been collected. An anomalous difference Patterson map reveals self- and cross-peaks from two incorporated Se atoms. The corresponding selenium substructure has been solved. PMID:17554167

  2. Antimicrobial Peptides and Complement in Neonatal Hypoxia-Ischemia Induced Brain Damage

    PubMed Central

    Rocha-Ferreira, Eridan; Hristova, Mariya

    2015-01-01

    Hypoxic-ischemic encephalopathy (HIE) is a clinical condition in the neonate, resulting from oxygen deprivation around the time of birth. HIE affects 1–5/1000 live births worldwide and is associated with the development of neurological deficits, including cerebral palsy, epilepsy, and cognitive disabilities. Even though the brain is considered as an immune-privileged site, it has innate and adaptive immune response and can produce complement (C) components and antimicrobial peptides (AMPs). Dysregulation of cerebral expression of AMPs and C can exacerbate or ameliorate the inflammatory response within the brain. Brain ischemia triggers a prolonged inflammatory response affecting the progression of injury and secondary energy failure and involves both innate and adaptive immune systems, including immune-competent and non-competent cells. Following injury to the central nervous system (CNS), including neonatal hypoxia-ischemia (HI), resident microglia, and astroglia are the main cells providing immune defense to the brain in a stimulus-dependent manner. They can express and secrete pro-inflammatory cytokines and therefore trigger prolonged inflammation, resulting in neurodegeneration. Microglial cells express and release a wide range of inflammation-associated molecules including several components of the complement system. Complement activation following neonatal HI injury has been reported to contribute to neurodegeneration. Astrocytes can significantly affect the immune response of the CNS under pathological conditions through production and release of pro-inflammatory cytokines and immunomodulatory AMPs. Astrocytes express β-defensins, which can chemoattract and promote maturation of dendritic cells (DC), and can also limit inflammation by controlling the viability of these same DC. This review will focus on the balance of complement components and AMPs within the CNS following neonatal HI injury and the effect of that balance on the subsequent brain damage

  3. Complement associated pathogenic mechanisms in myasthenia gravis.

    PubMed

    Tüzün, Erdem; Christadoss, Premkumar

    2013-07-01

    The complement system is profoundly involved in the pathogenesis of acetylcholine receptor (AChR) antibody (Ab) related myasthenia gravis (MG) and its animal model experimental autoimmune myasthenia gravis (EAMG). The most characteristic finding of muscle pathology in both MG and EAMG is the abundance of IgG and complement deposits at the nerve-muscle junction (NMJ), suggesting that AChR-Ab induces muscle weakness by complement pathway activation and consequent membrane attack complex (MAC) formation. This assumption has been supported with EAMG resistance of complement factor C3 knockout (KO), C4 KO and C5 deficient mice and amelioration of EAMG symptoms following treatment with complement inhibitors such as cobra venom factor, soluble complement receptor 1, anti-C1q, anti-C5 and anti-C6 Abs. Moreover, the complement inhibitor decay accelerating factor (DAF) KO mice exhibit increased susceptibility to EAMG. These findings have brought forward improvisation of novel therapy methods based on inhibition of classical and common complement pathways in MG treatment.

  4. Creating functional sophistication from simple protein building blocks, exemplified by factor H and the regulators of complement activation.

    PubMed

    Makou, Elisavet; Herbert, Andrew P; Barlow, Paul N

    2015-10-01

    Complement control protein modules (CCPs) occur in numerous functionally diverse extracellular proteins. Also known as short consensus repeats (SCRs) or sushi domains each CCP contains approximately 60 amino acid residues, including four consensus cysteines participating in two disulfide bonds. Varying in length and sequence, CCPs adopt a β-sandwich type fold and have an overall prolate spheroidal shape with N- and C-termini lying close to opposite poles of the long axis. CCP-containing proteins are important as cytokine receptors and in neurotransmission, cell adhesion, blood clotting, extracellular matrix formation, haemoglobin metabolism and development, but CCPs are particularly well represented in the vertebrate complement system. For example, factor H (FH), a key soluble regulator of the alternative pathway of complement activation, is made up entirely from a chain of 20 CCPs joined by short linkers. Collectively, therefore, the 20 CCPs of FH must mediate all its functional capabilities. This is achieved via collaboration and division of labour among these modules. Structural studies have illuminated the dynamic architectures that allow FH and other CCP-rich proteins to perform their biological functions. These are largely the products of a highly varied set of intramolecular interactions between CCPs. The CCP can act as building block, spacer, highly versatile recognition site or dimerization mediator. Tandem CCPs may form composite binding sites or contribute to flexible, rigid or conformationally 'switchable' segments of the parent proteins.

  5. Creating functional sophistication from simple protein building blocks, exemplified by factor H and the regulators of complement activation.

    PubMed

    Makou, Elisavet; Herbert, Andrew P; Barlow, Paul N

    2015-10-01

    Complement control protein modules (CCPs) occur in numerous functionally diverse extracellular proteins. Also known as short consensus repeats (SCRs) or sushi domains each CCP contains approximately 60 amino acid residues, including four consensus cysteines participating in two disulfide bonds. Varying in length and sequence, CCPs adopt a β-sandwich type fold and have an overall prolate spheroidal shape with N- and C-termini lying close to opposite poles of the long axis. CCP-containing proteins are important as cytokine receptors and in neurotransmission, cell adhesion, blood clotting, extracellular matrix formation, haemoglobin metabolism and development, but CCPs are particularly well represented in the vertebrate complement system. For example, factor H (FH), a key soluble regulator of the alternative pathway of complement activation, is made up entirely from a chain of 20 CCPs joined by short linkers. Collectively, therefore, the 20 CCPs of FH must mediate all its functional capabilities. This is achieved via collaboration and division of labour among these modules. Structural studies have illuminated the dynamic architectures that allow FH and other CCP-rich proteins to perform their biological functions. These are largely the products of a highly varied set of intramolecular interactions between CCPs. The CCP can act as building block, spacer, highly versatile recognition site or dimerization mediator. Tandem CCPs may form composite binding sites or contribute to flexible, rigid or conformationally 'switchable' segments of the parent proteins. PMID:26517887

  6. Acquisition of complement inhibitor serine protease factor I and its cofactors C4b-binding protein and factor H by Prevotella intermedia.

    PubMed

    Malm, Sven; Jusko, Monika; Eick, Sigrun; Potempa, Jan; Riesbeck, Kristian; Blom, Anna M

    2012-01-01

    Infection with the Gram-negative pathogen Prevotella intermedia gives rise to periodontitis and a growing number of studies implies an association of P. intermedia with rheumatoid arthritis. The serine protease Factor I (FI) is the central inhibitor of complement degrading complement components C3b and C4b in the presence of cofactors such as C4b-binding protein (C4BP) and Factor H (FH). Yet, the significance of complement inhibitor acquisition in P. intermedia infection and FI binding by Gram-negative pathogens has not been addressed. Here we show that P. intermedia isolates bound purified FI as well as FI directly from heat-inactivated human serum. FI bound to bacteria retained its serine protease activity as shown in degradation experiments with (125)I-labeled C4b. Since FI requires cofactors for its activity we also investigated the binding of purified cofactors C4BP and FH and found acquisition of both proteins, which retained their activity in FI mediated degradation of C3b and C4b. We propose that FI binding by P. intermedia represents a new mechanism contributing to complement evasion by a Gram-negative bacterial pathogen associated with chronic diseases.

  7. The Structure of the Cell-Wall Protease from Streptococci that Inactivates the Human Complement Factor 5A

    SciTech Connect

    Brown,C.; Gu, Z.; Matsuka, Y.; Olmsted, S.; Cleary, P.; Ohlendorf, D.; Earhart, C.

    2006-01-01

    The structure of a 949-residue fragment of complement factor 5a peptidase (SCP) was determined to 1.9 Angstroms resolution. The molecule is made of five distinct domains in an elongated head-stalk structure. The structure suggests that activity of SCP can be modulated through binding of integrins to 2 RGD sequences. This structure is the first of an enzyme that is covalently attached to the cell wall of a Gram-positive bacteria. SCP is also the first functional protease containing a protease-associated domain to have its structure elucidated.

  8. Complement Factor H-Related Protein 3 Serum Levels Are Low Compared to Factor H and Mainly Determined by Gene Copy Number Variation in CFHR3

    PubMed Central

    Pouw, Richard B.; Brouwer, Mieke C.; Geissler, Judy; van Herpen, Laurens V.; Zeerleder, Sacha S.; Wuillemin, Walter A.; Wouters, Diana; Kuijpers, Taco W.

    2016-01-01

    The major human complement regulator in blood, complement factor H (FH), has several closely related proteins, called FH-related (FHR) proteins. As all FHRs lack relevant complement regulatory activity, their physiological role is not well understood. FHR protein 3 (FHR-3) has been suggested to compete with FH for binding to Neisseria meningitidis, thereby affecting complement-mediated clearance. Clearly, the in vivo outcome of such competition greatly depends on the FH and FHR-3 concentrations. While FH levels have been established, accurate FHR-3 levels were never unequivocally reported to date. Moreover, CFHR3 gene copy numbers commonly vary, which may impact the FHR-3 concentration. Hence, we generated five anti-FHR-3 mAbs to specifically measure FHR-3 in human healthy donors of which we determined the gene copy number variation at the CFH/CFHR locus. Finally, we examined the acute-phase response characteristics of FHR-3 in a small sepsis cohort. We determined FHR-3 levels to have a mean of 19 nM and that under normal conditions the copy number of CFHR3 correlates to a very large extent with the FHR-3 serum levels. On average, FHR-3 was 132-fold lower compared to the FH concentration in the same serum samples and FHR-3 did not behave as a major acute phase response protein. PMID:27007437

  9. Complement Factor H-Related Protein 3 Serum Levels Are Low Compared to Factor H and Mainly Determined by Gene Copy Number Variation in CFHR3.

    PubMed

    Pouw, Richard B; Brouwer, Mieke C; Geissler, Judy; van Herpen, Laurens V; Zeerleder, Sacha S; Wuillemin, Walter A; Wouters, Diana; Kuijpers, Taco W

    2016-01-01

    The major human complement regulator in blood, complement factor H (FH), has several closely related proteins, called FH-related (FHR) proteins. As all FHRs lack relevant complement regulatory activity, their physiological role is not well understood. FHR protein 3 (FHR-3) has been suggested to compete with FH for binding to Neisseria meningitidis, thereby affecting complement-mediated clearance. Clearly, the in vivo outcome of such competition greatly depends on the FH and FHR-3 concentrations. While FH levels have been established, accurate FHR-3 levels were never unequivocally reported to date. Moreover, CFHR3 gene copy numbers commonly vary, which may impact the FHR-3 concentration. Hence, we generated five anti-FHR-3 mAbs to specifically measure FHR-3 in human healthy donors of which we determined the gene copy number variation at the CFH/CFHR locus. Finally, we examined the acute-phase response characteristics of FHR-3 in a small sepsis cohort. We determined FHR-3 levels to have a mean of 19 nM and that under normal conditions the copy number of CFHR3 correlates to a very large extent with the FHR-3 serum levels. On average, FHR-3 was 132-fold lower compared to the FH concentration in the same serum samples and FHR-3 did not behave as a major acute phase response protein. PMID:27007437

  10. Target deletion of complement component 9 attenuates antibody-mediated hemolysis and lipopolysaccharide (LPS)-induced acute shock in mice

    PubMed Central

    Fu, Xiaoyan; Ju, Jiyu; Lin, Zhijuan; Xiao, Weiling; Li, Xiaofang; Zhuang, Baoxiang; Zhang, Tingting; Ma, Xiaojun; Li, Xiangyu; Ma, Chao; Su, Weiliang; Wang, Yuqi; Qin, Xuebin; Liang, Shujuan

    2016-01-01

    Terminal complement membrane attack complex (MAC) formation is induced initially by C5b, followed by the sequential condensation of the C6, C7, C8. Polymerization of C9 to the C5b-8 complex forms the C5b-9 (or MAC). The C5b-9 forms lytic or non lytic pores in the cell membrane destroys membrane integrity. The biological functionalities of MAC has been previously investigated by using either the mice deficient in C5 and C6, or MAC’s regulator CD59. However, there is no available C9 deficient mice (mC9−/−) for directly dissecting the role of C5b-9 in the pathogenesis of human diseases. Further, since C5b-7 and C5b-8 complexes form non lytic pore, it may also plays biological functionality. To better understand the role of terminal complement cascades, here we report a successful generation of mC9−/−. We demonstrated that lack of C9 attenuates anti-erythrocyte antibody-mediated hemolysis or LPS-induced acute shock. Further, the rescuing effect on the acute shock correlates with the less release of IL-1β in mC9−/−, which is associated with suppression of MAC-mediated inflammasome activation in mC9−/−. Taken together, these results not only confirm the critical role of C5b-9 in complement-mediated hemolysis and but also highlight the critical role of C5b-9 in inflammasome activation. PMID:27444648

  11. Recurrent infections in partial complement factor I deficiency: evaluation of three generations of a Brazilian family

    PubMed Central

    Grumach, A S; Leitão, M F; Arruk, V G; Kirschfink, M; Condino-Neto, A

    2006-01-01

    We report here on the evaluation of a factor I-deficient Brazilian family (three generations, 39 members) with strong consanguinity. The complete factor I-deficient patients (n = 3) presented recurrent respiratory infections, skin infections and meningitis; one of them died after sepsis. They presented an impaired total haemolytic activity (CH50), low C3, low factor H and undetectable C3dg/C3d. Partial factor I deficiency was detected in 16 family members (normal low cut-off value was 25 µg/ml). Respiratory infections were the most common clinical occurrence among partial factor I-deficient relatives. Two of them were submitted to nephrectomy following recurrent urinary tract infections. An additional two heterozygous relatives presented with arthritis and rheumatic fever. Apparently, patients with partial factor I deficiency are also at higher risk for recurrent infections. Vaccination against capsulated bacteria and the eventual use of prophylactic antibiotics should be considered individually in this patient group. PMID:16412054

  12. Engineering a ribozyme cleavage-induced split fluorescent aptamer complementation assay

    PubMed Central

    Ausländer, Simon; Fuchs, David; Hürlemann, Samuel; Ausländer, David; Fussenegger, Martin

    2016-01-01

    Hammerhead ribozymes are self-cleaving RNA molecules capable of regulating gene expression in living cells. Their cleavage performance is strongly influenced by intra-molecular loop–loop interactions, a feature not readily accessible through modern prediction algorithms. Ribozyme engineering and efficient implementation of ribozyme-based genetic switches requires detailed knowledge of individual self-cleavage performances. By rational design, we devised fluorescent aptamer-ribozyme RNA architectures that allow for the real-time measurement of ribozyme self-cleavage activity in vitro. The engineered nucleic acid molecules implement a split Spinach aptamer sequence that is made accessible for strand displacement upon ribozyme self-cleavage, thereby complementing the fluorescent Spinach aptamer. This fully RNA-based ribozyme performance assay correlates ribozyme cleavage activity with Spinach fluorescence to provide a rapid and straightforward technology for the validation of loop–loop interactions in hammerhead ribozymes. PMID:26939886

  13. Complement Test

    MedlinePlus

    ... helpful? Also known as: C1; C1q; C2; C3; C4; CH50; CH100 (among others) Formal name: Complement Activity; ... whether the system is functioning normally. C3 and C4 are the most frequently measured complement proteins. Total ...

  14. The Expression Profile of Complement Components in Podocytes.

    PubMed

    Li, Xuejuan; Ding, Fangrui; Zhang, Xiaoyan; Li, Baihong; Ding, Jie

    2016-01-01

    Podocytes are critical for maintaining the glomerular filtration barrier and are injured in many renal diseases, especially proteinuric kidney diseases. Recently, reports suggested that podocytes are among the renal cells that synthesize complement components that mediate glomerular diseases. Nevertheless, the profile and extent of complement component expression in podocytes remain unclear. This study examined the expression profile of complement in podocytes under physiological conditions and in abnormal podocytes induced by multiple stimuli. In total, 23/32 complement component components were detected in podocyte by conventional RT-PCR. Both primary cultured podocytes and immortalized podocytes expressed the complement factors C1q, C1r, C2, C3, C7, MASP, CFI, DAF, CD59, C4bp, CD46, Protein S, CR2, C1qR, C3aR, C5aR, and Crry (17/32), whereas C4, CFB, CFD, C5, C6, C8, C9, MBL1, and MBL2 (9/32) complement factors were not expressed. C3, Crry, and C1q-binding protein were detected by tandem mass spectrometry. Podocyte complement gene expression was affected by several factors (puromycin aminonucleoside (PAN), angiotensin II (Ang II), interleukin-6 (IL-6), and transforming growth factor-β (TGF-β)). Representative complement components were detected using fluorescence confocal microscopy. In conclusion, primary podocytes express various complement components at the mRNA and protein levels. The complement gene expressions were affected by several podocyte injury factors. PMID:27043537

  15. The Expression Profile of Complement Components in Podocytes

    PubMed Central

    Li, Xuejuan; Ding, Fangrui; Zhang, Xiaoyan; Li, Baihong; Ding, Jie

    2016-01-01

    Podocytes are critical for maintaining the glomerular filtration barrier and are injured in many renal diseases, especially proteinuric kidney diseases. Recently, reports suggested that podocytes are among the renal cells that synthesize complement components that mediate glomerular diseases. Nevertheless, the profile and extent of complement component expression in podocytes remain unclear. This study examined the expression profile of complement in podocytes under physiological conditions and in abnormal podocytes induced by multiple stimuli. In total, 23/32 complement component components were detected in podocyte by conventional RT-PCR. Both primary cultured podocytes and immortalized podocytes expressed the complement factors C1q, C1r, C2, C3, C7, MASP, CFI, DAF, CD59, C4bp, CD46, Protein S, CR2, C1qR, C3aR, C5aR, and Crry (17/32), whereas C4, CFB, CFD, C5, C6, C8, C9, MBL1, and MBL2 (9/32) complement factors were not expressed. C3, Crry, and C1q-binding protein were detected by tandem mass spectrometry. Podocyte complement gene expression was affected by several factors (puromycin aminonucleoside (PAN), angiotensin II (Ang II), interleukin-6 (IL-6), and transforming growth factor-β (TGF-β)). Representative complement components were detected using fluorescence confocal microscopy. In conclusion, primary podocytes express various complement components at the mRNA and protein levels. The complement gene expressions were affected by several podocyte injury factors. PMID:27043537

  16. The C-terminal tail of polycystin-1 regulates complement factor B expression by signal transducer and activator of transcription 1.

    PubMed

    Wu, Ming; Chen, Meihan; Jing, Ying; Gu, Junhui; Mei, Shuqin; Yao, Qing; Zhou, Jie; Yang, Ming; Sun, Lijun; Wang, Wutao; Hu, Huimin; Wüthrich, Rudolf P; Mei, Changlin

    2016-06-01

    Inhibition of the overactivated alternative complement pathway in autosomal dominant polycystic kidney disease (ADPKD) retards disease progression in animal models; however, it remains unknown how complement factor B (CFB) is upregulated in ADPKD. Here, we showed that the overexpression of CFB in cystic kidneys is associated with increased JAK2/STAT1 activity and enhanced expression of the polycystin-1 C-terminal tail (PC1-CTT). Overexpression or blockage of STAT1 increased or decreased CFB expression and CFB promoter activity. Moreover, overexpression of PC1-CTT induced JAK2/STAT1 activation and CFB upregulation in renal tubular epithelial cells. Furthermore, PC1-CTT overexpression increased human CFB promoter activity, whereas dominant negative STAT1 plasmids or mutation of putative STAT1 responsive elements decreased PC1-CTT-induced CFB promoter activity. The effect of CFB on macrophage differentiation was tested on a mouse macrophage cell line. Bioactive CFB dose dependently promoted macrophage M2 phenotype conversion. In addition, conditioned media from renal epithelial cells promoted macrophage M2 phenotype conversion which was blocked by STAT1 inhibition in a dose-dependent manner. Conditioned media from PC1-CTT-transfected renal epithelial cells further promoted macrophage M2 phenotype conversion, which was suppressed by fludarabine or a CFB antibody. In addition, we show that NF-κB acts downstream of PC1-CTT and may partly mediate PC1-CTT-induced CFB expression. In conclusion, our study reveals possible mechanisms of CFB upregulation in ADPKD and a novel role of PC1-CTT in ADPKD-associated inflammation. Furthermore, our study suggests that targeting STAT1 may be a new strategy to prevent inflammation in the kidney of patients with ADPKD. PMID:26984954

  17. Delta-short consensus repeat 4-decay accelerating factor (DAF: CD55) inhibits complement-mediated cytolysis but not NK cell-mediated cytolysis.

    PubMed

    Miyagawa, Shuji; Kubo, Tomoko; Matsunami, Katsuyoshi; Kusama, Tamiko; Beppu, Keiko; Nozaki, Hiroshi; Moritan, Toshiyuki; Ahn, Curie; Kim, Jae Young; Fukuta, Daisuke; Shirakura, Ryota

    2004-09-15

    NK cells play a critical role in the rejection of xenografts. In this study, we report on an investigation of the effect of complement regulatory protein, a decay accelerating factor (DAF: CD55), in particular, on NK cell-mediated cytolysis. Amelioration of human NK cell-mediated pig endothelial cell (PEC) and pig fibroblast cell lyses by various deletion mutants and point substitutions of DAF was tested, and compared with their complement regulatory function. Although wild-type DAF and the delta-short consensus repeat (SCR) 1-DAF showed clear inhibition of both complement-mediated and NK-mediated PEC lyses, delta-SCR2-DAF and delta-SCR3-DAF failed to suppress either process. However, delta-SCR4-DAF showed a clear complement regulatory effect, but had no effect on NK cells. Conversely, the point substitution of DAF (L147 x F148 to SS and KKK(125-127) to TTT) was half down-regulated in complement inhibitory function, but the inhibition of NK-mediated PEC lysis remained unchanged. Other complement regulatory proteins, such as the cell membrane-bound form factor H, fH-PI, and C1-inactivator, C1-INH-PI, and CD59 were also assessed, but no suppressive effect on NK cell-mediated PEC lysis was found. These data suggest, for DAF to function on NK cells, SCR2-4 is required but no relation to its complement regulatory function exists.

  18. Plasma-derived human C1-esterase inhibitor does not prevent mechanical ventilation-induced pulmonary complement activation in a rat model of Streptococcus pneumoniae pneumonia.

    PubMed

    de Beer, F M; Aslami, H; Hoeksma, J; van Mierlo, G; Wouters, D; Zeerleder, S; Roelofs, J J T H; Juffermans, N P; Schultz, M J; Lagrand, W K

    2014-11-01

    Mechanical ventilation has the potential to cause lung injury, and the role of complement activation herein is uncertain. We hypothesized that inhibition of the complement cascade by administration of plasma-derived human C1-esterase inhibitor (C1-INH) prevents ventilation-induced pulmonary complement activation, and as such attenuates lung inflammation and lung injury in a rat model of Streptococcus pneumoniae pneumonia. Forty hours after intratracheal challenge with S. pneumoniae causing pneumonia rats were subjected to ventilation with lower tidal volumes and positive end-expiratory pressure (PEEP) or high tidal volumes without PEEP, after an intravenous bolus of C1-INH (200 U/kg) or placebo (saline). After 4 h of ventilation blood, broncho-alveolar lavage fluid and lung tissue were collected. Non-ventilated rats with S. pneumoniae pneumonia served as controls. While ventilation with lower tidal volumes and PEEP slightly amplified pneumonia-induced complement activation in the lungs, ventilation with higher tidal volumes without PEEP augmented local complement activation more strongly. Systemic pre-treatment with C1-INH, however, failed to alter ventilation-induced complement activation with both ventilation strategies. In accordance, lung inflammation and lung injury were not affected by pre-treatment with C1-INH, neither in rats ventilated with lower tidal volumes and PEEP, nor rats ventilated with high tidal volumes without PEEP. Ventilation augments pulmonary complement activation in a rat model of S. pneumoniae pneumonia. Systemic administration of C1-INH, however, does not attenuate ventilation-induced complement activation, lung inflammation, and lung injury. PMID:24760631

  19. Structural basis of profactor D activation: from a highly flexible zymogen to a novel self-inhibited serine protease, complement factor D.

    PubMed Central

    Jing, H; Macon, K J; Moore, D; DeLucas, L J; Volanakis, J E; Narayana, S V

    1999-01-01

    The crystal structure of profactor D, determined at 2.1 A resolution with an Rfree and an R-factor of 25.1 and 20.4%, respectively, displays highly flexible or disordered conformation for five regions: N-22, 71-76, 143-152, 187-193 and 215-223. A comparison with the structure of its mature serine protease, complement factor D, revealed major conformational changes in the similar regions. Comparisons with the zymogen-active enzyme pairs of chymotrypsinogen, trypsinogen and prethrombin-2 showed a similar distribution of the flexible regions. However, profactor D is the most flexible of the four, and its mature enzyme displays inactive, self-inhibited active site conformation. Examination of the surface properties of the N-terminus-binding pocket indicates that Ile16 may play the initial positioning role for the N-terminus, and Leu17 probably also helps in inducing the required conformational changes. This process, perhaps shared by most chymotrypsinogen-like zymogens, is followed by a factor D-unique step, the re-orientation of an external Arg218 to an internal position for salt-bridging with Asp189, leading to the generation of the self-inhibited factor D. PMID:10022823

  20. Decay-accelerating factor (CD55), a glycosylphosphatidylinositol-anchored complement regulatory protein, is a receptor for several echoviruses.

    PubMed Central

    Bergelson, J M; Chan, M; Solomon, K R; St John, N F; Lin, H; Finberg, R W

    1994-01-01

    Echoviruses are human pathogens belonging to the picornavirus family. Decay-accelerating factor (DAF) is a glycosylphosphatidylinositol (GPI)-anchored surface protein that protects cells from lysis by autologous complement. Anti-DAF monoclonal antibodies prevented echovirus 7 attachment to susceptible cells and protected cells from infection. HeLa cells specifically lost the capacity to bind echovirus 7 when treated with phosphatidylinositol-specific phospholipase C, an enzyme that releases GPI-anchored proteins from the cell surface, indicating that the virus receptor, like DAF, is a GPI-anchored protein. Although Chinese hamster ovary cells do not bind echovirus 7, transfectants expressing human DAF bound virus efficiently, and binding was prevented by pretreatment with an anti-DAF monoclonal antibody. Anti-DAF antibodies prevented infection by at least six echovirus serotypes. These results indicate that DAF is the receptor mediating attachment and infection by several echoviruses. Images PMID:7517044

  1. Complement factor H polymorphism rs1061170 and the effect of cigarette smoking on the risk of lung cancer

    PubMed Central

    Ezzeldin, Nada; Darwish, Amira; El-Bastawissy, Ahmed; Shalaby, Alaa Eldin

    2015-01-01

    Aim of the study Complement factor H (CFH) has been known to inhibit the complement pathway and to contribute to tumour growth by suppressing the anti-tumour cell mediated response in cell lines from several malignancies. We examined the association of Try402His single nucleotide polymorphism in CFH gene with lung cancer and the interaction with cigarette smoking. Material and methods This case-control study included 80 primary lung cancer patients and 106 control subjects who were genotyped for Try402His (rs1061170) by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. Results Variant genotypes (Tyr/His and His/His) were overpresented among patients compared to controls (p = 0.03, OR = 2.510, 95% CI: 1.068–5.899), and the frequency of variant H allele was significantly overexpressed in cases compared to controls (p = 0.021). Tyr/His genotype was identified in 100% of small cell lung cancer (SCLC) patients vs. 34.5% of non-SCLC (NSCLC), while 20.7% of NSCLC patients were homozygous for the variant allele (His/His) (p = 0.001). Binary logistic regression analysis revealed a 2.5 times greater estimated risk for NSCLC than for SCLC among variant allele carriers, and a 7.3-fold increased risk of lung cancer among variant allele smoking carriers vs. 1.3-fold increased risk among wild allele smoking carriers. Moreover, the stage of cancer positively correlated with smoking and pack-years in allele H carriers, and the correlation was stronger among those who were homozygous for it (His/His) than those who were heterozygous (Tyr/His). Conclusions CFH 402H variant is a smoking-related risk factor for lung cancer, particularly the NSCLC. PMID:26843839

  2. Generation of embryos directly from embryonic stem cells by tetraploid embryo complementation reveals a role for GATA factors in organogenesis.

    PubMed

    Duncan, S A

    2005-12-01

    Gene targeting in ES (embryonic stem) cells has been used extensively to study the role of proteins during embryonic development. In the traditional procedure, this requires the generation of chimaeric mice by introducing ES cells into blastocysts and allowing them to develop to term. Once chimaeric mice are produced, they are bred into a recipient mouse strain to establish germline transmission of the allele of interest. Although this approach has been used very successfully, the breeding cycles involved are time consuming. In addition, genes that are essential for organogenesis often have roles in the formation of extra-embryonic tissues that are essential for early stages of post-implantation development. For example, mice lacking the GATA transcription factors, GATA4 or GATA6, arrest during gastrulation due to an essential role for these factors in differentiation of extra-embryonic endoderm. This lethality has frustrated the study of these factors during the development of organs such as the liver and heart. Extraembryonic defects can, however, be circumvented by generating clonal mouse embryos directly from ES cells by tetraploid complementation. Here, we describe the usefulness and efficacy of this approach using GATA factors as an example.

  3. Factor H binding as a complement evasion mechanism for an anaerobic pathogen, Fusobacterium necrophorum.

    PubMed

    Friberg, Nathalie; Carlson, Petteri; Kentala, Erna; Mattila, Petri S; Kuusela, Pentti; Meri, Seppo; Jarva, Hanna

    2008-12-15

    Fusobacterium necrophorum subspecies funduliforme is an obligate anaerobic Gram-negative rod causing invasive infections such as the life-threatening Lemierre's syndrome (sore throat, septicemia, jugular vein thrombosis, and disseminated infection). The aim of our study was to understand if and how F. necrophorum avoids C activation. We studied 12 F. necrophorum subsp. funduliforme strains isolated from patients with sepsis. All strains were resistant to serum killing after a 1-h incubation in 20% serum. The bacteria bound, at different levels, the C inhibitor factor H (fH). Binding was ionic and specific in nature and occurred via sites on both the N terminus and the C terminus of fH. Bound fH remained functionally active as a cofactor for factor I in the cleavage of C3b. Interestingly, patients with the most severe symptoms carried strains with the strongest ability to bind fH. An increased C3b deposition and membrane attack complex formation on the surface of a weakly fH-binding strain was observed and its survival in serum at 3.5 h was impaired. This strain had not caused a typical Lemierre's syndrome. These data, and the fact that fH-binding correlated with the severity of disease, suggest that the binding of fH contributes to virulence and survival of F. necrophorum subsp. funduliforme in the human host. Our data show, for the first time, that an anaerobic bacterium is able to bind the C inhibitor fH to evade C attack. PMID:19050282

  4. Role of Complement and Complement Regulatory Proteins in the Complications of Diabetes

    PubMed Central

    Ghosh, Pamela; Sahoo, Rupam; Vaidya, Anand; Chorev, Michael

    2015-01-01

    It is well established that the organ damage that complicates human diabetes is caused by prolonged hyperglycemia, but the cellular and molecular mechanisms by which high levels of glucose cause tissue damage in humans are still not fully understood. The prevalent hypothesis explaining the mechanisms that may underlie the pathogenesis of diabetes complications includes overproduction of reactive oxygen species, increased flux through the polyol pathway, overactivity of the hexosamine pathway causing intracellular formation of advanced glycation end products, and activation of protein kinase C isoforms. In addition, experimental and clinical evidence reported in past decades supports a strong link between the complement system, complement regulatory proteins, and the pathogenesis of diabetes complications. In this article, we summarize the body of evidence that supports a role for the complement system and complement regulatory proteins in the pathogenesis of diabetic vascular complications, with specific emphasis on the role of the membrane attack complex (MAC) and of CD59, an extracellular cell membrane-anchored inhibitor of MAC formation that is inactivated by nonenzymatic glycation. We discuss a pathogenic model of human diabetic complications in which a combination of CD59 inactivation by glycation and hyperglycemia-induced complement activation increases MAC deposition, activates pathways of intracellular signaling, and induces the release of proinflammatory, prothrombotic cytokines and growth factors. Combined, complement-dependent and complement-independent mechanisms induced by high glucose promote inflammation, proliferation, and thrombosis as characteristically seen in the target organs of diabetes complications. PMID:25859860

  5. Role of complement and complement regulatory proteins in the complications of diabetes.

    PubMed

    Ghosh, Pamela; Sahoo, Rupam; Vaidya, Anand; Chorev, Michael; Halperin, Jose A

    2015-06-01

    It is well established that the organ damage that complicates human diabetes is caused by prolonged hyperglycemia, but the cellular and molecular mechanisms by which high levels of glucose cause tissue damage in humans are still not fully understood. The prevalent hypothesis explaining the mechanisms that may underlie the pathogenesis of diabetes complications includes overproduction of reactive oxygen species, increased flux through the polyol pathway, overactivity of the hexosamine pathway causing intracellular formation of advanced glycation end products, and activation of protein kinase C isoforms. In addition, experimental and clinical evidence reported in past decades supports a strong link between the complement system, complement regulatory proteins, and the pathogenesis of diabetes complications. In this article, we summarize the body of evidence that supports a role for the complement system and complement regulatory proteins in the pathogenesis of diabetic vascular complications, with specific emphasis on the role of the membrane attack complex (MAC) and of CD59, an extracellular cell membrane-anchored inhibitor of MAC formation that is inactivated by nonenzymatic glycation. We discuss a pathogenic model of human diabetic complications in which a combination of CD59 inactivation by glycation and hyperglycemia-induced complement activation increases MAC deposition, activates pathways of intracellular signaling, and induces the release of proinflammatory, prothrombotic cytokines and growth factors. Combined, complement-dependent and complement-independent mechanisms induced by high glucose promote inflammation, proliferation, and thrombosis as characteristically seen in the target organs of diabetes complications.

  6. Meningococcal factor H-binding protein vaccines with decreased binding to human complement factor H have enhanced immunogenicity in human factor H transgenic mice.

    PubMed

    Rossi, Raffaella; Granoff, Dan M; Beernink, Peter T

    2013-11-01

    Factor H-binding protein (fHbp) is a component of a meningococcal vaccine recently licensed in Europe for prevention of serogroup B disease, and a second vaccine in clinical development. The protein specifically binds human factor H (fH), which down-regulates complement activation and enhances resistance to bactericidal activity. There are conflicting data from studies in human fH transgenic mice on whether binding of human fH to fHbp vaccines decreases immunogenicity, and whether mutant fHbp vaccines with decreased fH binding have enhanced immunogenicity. fHbp can be classified into two sub-families based on sequence divergence and immunologic cross-reactivity. Previous studies of mutant fHbp vaccines with low fH binding were from sub-family B, which account for approximately 60% of serogroup B case isolates. In the present study, we evaluated the immunogenicity of two mutant sub-family A fHbp vaccines containing single substitutions, T221A or D211A, which resulted in 15- or 30-fold lower affinity for human fH, respectively, than the corresponding control wild-type fHbp vaccine. In transgenic mice with high serum concentrations of human fH, both mutant vaccines elicited significantly higher IgG titers and higher serum bactericidal antibody responses than the control fHbp vaccine that bound human fH. Thus, mutations introduced into a sub-family A fHbp antigen to decrease fH binding can increase protective antibody responses in human fH transgenic mice. Collectively the data suggest that mutant fHbp antigens with decreased fH binding will result in superior vaccines in humans.

  7. Complement C4 Deficiency – A Plausible Risk Factor for Non-Tuberculous Mycobacteria (NTM) Infection in Apparently Immunocompetent Patients

    PubMed Central

    Kotilainen, Hannele; Lokki, Marja-Liisa; Paakkanen, Riitta; Seppänen, Mikko; Tukiainen, Pentti; Meri, Seppo; Poussa, Tuija; Eskola, Jussi; Valtonen, Ville; Järvinen, Asko

    2014-01-01

    Background Non-tuberculous mycobacteria (NTM) are ubiquitous in the environment and they infect mainly persons with underlying pulmonary diseases but also previously healthy elderly women. Defects in host resistance that lead to pulmonary infections by NTM are relatively unknown. A few genetic defects have been associated with both pulmonary and disseminated mycobacterial infections. Rare disseminated NTM infections have been associated with genetic defects in T-cell mediated immunity and in cytokine signaling in families. We investigated whether there was an association between NTM infections and deficiencies of complement components C4A or C4B that are encoded by major histocompatibility complex (MHC). Methods 50 adult patients with a positive NTM culture with symptoms and findings of a NTM disease were recruited. Patients' clinical history was collected and symptoms and clinical findings were categorized according to 2007 diagnostic criteria of The American Thoracic Society (ATS). To investigate the deficiencies of complement, C4A and C4B gene copy numbers and phenotype frequencies of the C4 allotypes were analyzed. Unselected, healthy, 149 Finnish adults were used as controls. Results NTM patients had more often C4 deficiencies (C4A or C4B) than controls (36/50 [72%] vs 83/149 [56%], OR = 2.05, 95%CI = 1.019–4.105, p = 0.042). C4 deficiencies for female NTM patients were more common than for controls (29/36 [81%] vs 55/100 [55%], OR = 3.39, 95% CI = 1.358–8.460, p = 0.007). C4 deficiences seemed not to be related to any specific underlying disease or C4 phenotype. Conclusions C4 deficiency may be a risk factor for NTM infection in especially elderly female patients. PMID:24638111

  8. A hybrid hydrologically complemented warning model for shallow landslides induced by extreme rainfall in Korean Mountain

    NASA Astrophysics Data System (ADS)

    Singh Pradhan, Ananta Man; Kang, Hyo-Sub; Kim, Yun-Tae

    2016-04-01

    This study uses a physically based approach to evaluate the factor of safety of the hillslope for different hydrological conditions, in Mt Umyeon, south of Seoul. The hydrological conditions were determined using intensity and duration of whole Korea of known landslide inventory data. Quantile regression statistical method was used to ascertain different probability warning levels on the basis of rainfall thresholds. Physically based models are easily interpreted and have high predictive capabilities but rely on spatially explicit and accurate parameterization, which is commonly not possible. Statistical probabilistic methods can include other causative factors which influence the slope stability such as forest, soil and geology, but rely on good landslide inventories of the site. In this study a hybrid approach has described that combines the physically-based landslide susceptibility for different hydrological conditions. A presence-only based maximum entropy model was used to hybrid and analyze relation of landslide with conditioning factors. About 80% of the landslides were listed among the unstable sites identified in the proposed model, thereby presenting its effectiveness and accuracy in determining unstable areas and areas that require evacuation. These cumulative rainfall thresholds provide a valuable reference to guide disaster prevention authorities in the issuance of warning levels with the ability to reduce losses and save lives.

  9. Attenuation of S. aureus-induced bacteremia by human mini-antibodies targeting the complement inhibitory protein Efb

    PubMed Central

    Georgoutsou-Spyridonos, Maria; Ricklin, Daniel; Pratsinis, Haris; Perivolioti, Eustathia; Pirmettis, Ioannis; Garcia, Brandon L.; Geisbrecht, Brian V.; Foukas, Periklis G.; Lambris, John D.; Mastellos, Dimitrios C.; Sfyroera, Georgia

    2015-01-01

    Staphylococcus aureus (S. aureus) can cause a broad range of potentially fatal inflammatory complications (e.g. sepsis, endocarditis). Its emerging antibiotic resistance and formidable immune evasion arsenal have emphasized the need for more effective antimicrobial approaches. Complement is an innate immune sensor that rapidly responds to bacterial infection eliciting C3-mediated opsonophagocytic and immunomodulatory responses. Extracellular Fibrinogen-binding Protein (Efb) is a key immune evasion protein of S. aureus that intercepts complement at the level of C3. To date, Efb has not been explored as a target for monoclonal antibody (mAb)-based antimicrobial therapeutics. Herein we have isolated donor-derived anti-Efb IgGs that attenuate S. aureus survival through enhanced neutrophil killing. A phage library screen yielded mAbs (miniAbs) that selectively inhibit the interaction of Efb with C3 partly by disrupting contacts essential for complex formation. Surface Plasmon Resonance-based kinetic analysis enabled the selection of miniAbs with favorable Efb-binding profiles as therapeutic leads. MiniAb-mediated blockade of Efb attenuated S aureus survival in a whole blood model of bacteremia. This neutralizing effect was associated with enhanced neutrophil-mediated killing of S. aureus, increased C5a release and modulation of IL-6 secretion. Finally, these miniAbs afforded protection from S. aureus-induced bacteremia in a murine renal abscess model, attenuating bacterial inflammation in kidneys. Overall, these findings are anticipated to pave the way towards novel antibody-based therapeutics for S. aureus-related diseases. PMID:26342032

  10. Effect of complement Factor H on anti-FHbp serum bactericidal antibody responses of infant rhesus macaques boosted with a licensed meningococcal serogroup B vaccine.

    PubMed

    Giuntini, Serena; Beernink, Peter T; Granoff, Dan M

    2015-12-16

    FHbp is a major serogroup B meningococcal vaccine antigen. Binding of complement Factor H (FH) to FHbp is specific for human and some non-human primate FH. In previous studies, FH binding to FHbp vaccines impaired protective anti-FHbp antibody responses. In this study we investigated anti-FHbp antibody responses to a third dose of a licensed serogroup B vaccine (MenB-4C) in infant macaques vaccinated in a previous study with MenB-4C. Six macaques with high binding of FH to FHbp (FH(high)), and six with FH(low) baseline phenotypes, were immunized three months after dose 2. After dose 2, macaques with the FH(low) baseline phenotype had serum anti-FHbp antibodies that enhanced FH binding to FHbp (functionally converting them to a FH(high) phenotype). In this group, activation of the classical complement pathway (C4b deposition) by serum anti-FHbp antibody, and anti-FHbp serum bactericidal titers were lower after dose 3 than after dose 2 (p<0.02). In macaques with the FH(high) baseline phenotype, the respective anti-FHbp C4b deposition and bactericidal titers were similar after doses 2 and 3. Two macaques developed serum anti-FH autoantibodies after dose 2, which were not detected after dose 3. In conclusion, in macaques with the FH(low) baseline phenotype whose post-dose 2 serum anti-FHbp antibodies had converted them to FH(high), the anti-FHbp antibody repertoire to dose 3 was skewed to less protective epitopes than after dose 2. Mutant FHbp vaccines that eliminate FH binding may avoid eliciting anti-FHbp antibodies that enhance FH binding, and confer greater protection with less risk of inducing anti-FH autoantibodies than FHbp vaccines that bind FH.

  11. CRP-mediated activation of complement in vivo: assessment by measuring circulating complement-C-reactive protein complexes.

    PubMed

    Wolbink, G J; Brouwer, M C; Buysmann, S; ten Berge, I J; Hack, C E

    1996-07-01

    The in vivo function of C-reactive protein (CRP) is unknown. Among the in vitro functions assigned to CRP is the ability to activate complement via the classical pathway. To date, there is no evidence supporting that CRP exerts this function in vivo. We here show a novel approach to assess CRP-mediated complement activation in vivo, which is based on the property that activated complement factors C3 and C4 fix to CRP during complement activation induced by this acute phase protein. We developed specific ELISAs for complexes between CRP and C4b, C4d, C3b, or C3d. We established that in vitro complement-CRP complexes were formed only during CRP-dependent activation, and not during activation by other activators, even in the presence of high CRP levels. Circulating levels of complement-CRP complexes were undetectable in normal donors, but significantly increased in nine patients following implantation of a renal allograft. Importantly, levels of complement-CRP complexes did not change in these patients upon a bolus infusion of mAb OKT3, which induces activation of the classical complement pathway, demonstrating in vivo that complement-CRP complexes are not formed during CRP-independent activation of complement, even when CRP is elevated. We conclude that measurement of complement-CRP complexes provides a suitable tool to study CRP-mediated activation of complement in vivo. Furthermore, increased levels of these complexes occur in clinical samples, indicating that CRP may induce activation of complement in vivo.

  12. Human embryonic and induced pluripotent stem cell research trends: complementation and diversification of the field.

    PubMed

    Kobold, Sabine; Guhr, Anke; Kurtz, Andreas; Löser, Peter

    2015-05-12

    Research in human induced pluripotent stem cells (hiPSCs) is rapidly developing and there are expectations that this research may obviate the need to use human embryonic stem cells (hESCs), the ethics of which has been a subject of controversy for more than 15 years. In this study, we investigated approximately 3,400 original research papers that reported an experimental use of these types of human pluripotent stem cells (hPSCs) and were published from 2008 to 2013. We found that research into both cell types was conducted independently and further expanded, accompanied by a growing intersection of both research fields. Moreover, an in-depth analysis of papers that reported the use of both cell types indicates that hESCs are still being used as a "gold standard," but in a declining proportion of publications. Instead, the expanding research field is diversifying and hESC and hiPSC lines are increasingly being used in more independent research and application areas.

  13. Human Embryonic and Induced Pluripotent Stem Cell Research Trends: Complementation and Diversification of the Field

    PubMed Central

    Kobold, Sabine; Guhr, Anke; Kurtz, Andreas; Löser, Peter

    2015-01-01

    Summary Research in human induced pluripotent stem cells (hiPSCs) is rapidly developing and there are expectations that this research may obviate the need to use human embryonic stem cells (hESCs), the ethics of which has been a subject of controversy for more than 15 years. In this study, we investigated approximately 3,400 original research papers that reported an experimental use of these types of human pluripotent stem cells (hPSCs) and were published from 2008 to 2013. We found that research into both cell types was conducted independently and further expanded, accompanied by a growing intersection of both research fields. Moreover, an in-depth analysis of papers that reported the use of both cell types indicates that hESCs are still being used as a “gold standard,” but in a declining proportion of publications. Instead, the expanding research field is diversifying and hESC and hiPSC lines are increasingly being used in more independent research and application areas. PMID:25866160

  14. Human embryonic and induced pluripotent stem cell research trends: complementation and diversification of the field.

    PubMed

    Kobold, Sabine; Guhr, Anke; Kurtz, Andreas; Löser, Peter

    2015-05-12

    Research in human induced pluripotent stem cells (hiPSCs) is rapidly developing and there are expectations that this research may obviate the need to use human embryonic stem cells (hESCs), the ethics of which has been a subject of controversy for more than 15 years. In this study, we investigated approximately 3,400 original research papers that reported an experimental use of these types of human pluripotent stem cells (hPSCs) and were published from 2008 to 2013. We found that research into both cell types was conducted independently and further expanded, accompanied by a growing intersection of both research fields. Moreover, an in-depth analysis of papers that reported the use of both cell types indicates that hESCs are still being used as a "gold standard," but in a declining proportion of publications. Instead, the expanding research field is diversifying and hESC and hiPSC lines are increasingly being used in more independent research and application areas. PMID:25866160

  15. Binding of complement factor H to PorB3 and NspA enhances resistance of Neisseria meningitidis to anti-factor H binding protein bactericidal activity.

    PubMed

    Giuntini, Serena; Pajon, Rolando; Ram, Sanjay; Granoff, Dan M

    2015-04-01

    Among 25 serogroup B Neisseria meningitidis clinical isolates, we identified four (16%) with high factor H binding protein (FHbp) expression that were resistant to complement-mediated bactericidal activity of sera from mice immunized with recombinant FHbp vaccines. Two of the four isolates had evidence of human FH-dependent complement downregulation independent of FHbp. Since alternative complement pathway recruitment is critical for anti-FHbp bactericidal activity, we hypothesized that in these two isolates binding of FH to ligands other than FHbp contributes to anti-FHbp bactericidal resistance. Knocking out NspA, a known meningococcal FH ligand, converted both resistant isolates to anti-FHbp susceptible isolates. The addition of a nonbactericidal anti-NspA monoclonal antibody to the bactericidal reaction also increased anti-FHbp bactericidal activity. To identify a role for FH ligands other than NspA or FHbp in resistance, we created double NspA/FHbp knockout mutants. Mutants from both resistant isolates bound 10-fold more recombinant human FH domains 6 and 7 fused to Fc than double knockout mutants prepared from two sensitive meningococcal isolates. In light of recent studies showing functional FH-PorB2 interactions, we hypothesized that PorB3 from the resistant isolates recruited FH. Allelic exchange of porB3 from a resistant isolate to a sensitive isolate increased resistance of the sensitive isolate to anti-FHbp bactericidal activity (and vice versa). Thus, some PorB3 variants functionally bind human FH, which in the presence of NspA enhances anti-FHbp resistance. Combining anti-NspA antibodies with anti-FHbp antibodies can overcome resistance. Meningococcal vaccines that target both NspA and FHbp are likely to confer greater protection than either antigen alone.

  16. MASP-3 is the exclusive pro-factor D activator in resting blood: the lectin and the alternative complement pathways are fundamentally linked

    PubMed Central

    Dobó, József; Szakács, Dávid; Oroszlán, Gábor; Kortvely, Elod; Kiss, Bence; Boros, Eszter; Szász, Róbert; Závodszky, Péter; Gál, Péter; Pál, Gábor

    2016-01-01

    MASP-3 was discovered 15 years ago as the third mannan-binding lectin (MBL)-associated serine protease of the complement lectin pathway. Lacking any verified substrate its role remained ambiguous. MASP-3 was shown to compete with a key lectin pathway enzyme MASP-2 for MBL binding, and was therefore considered to be a negative complement regulator. Later, knock-out mice experiments suggested that MASP-1 and/or MASP-3 play important roles in complement pro-factor D (pro-FD) maturation. However, studies on a MASP-1/MASP-3-deficient human patient produced contradicting results. In normal resting blood unperturbed by ongoing coagulation or complement activation, factor D is present predominantly in its active form, suggesting that resting blood contains at least one pro-FD activating proteinase that is not a direct initiator of coagulation or complement activation. We have recently showed that all three MASPs can activate pro-FD in vitro. In resting blood, however, using our previously evolved MASP-1 and MASP-2 inhibitors we proved that neither MASP-1 nor MASP-2 activates pro-FD. Other plasma proteinases, particularly MASP-3, remained candidates for that function. For this study we evolved a specific MASP-3 inhibitor and unambiguously proved that activated MASP-3 is the exclusive pro-FD activator in resting blood, which demonstrates a fundamental link between the lectin and alternative pathways. PMID:27535802

  17. MASP-3 is the exclusive pro-factor D activator in resting blood: the lectin and the alternative complement pathways are fundamentally linked.

    PubMed

    Dobó, József; Szakács, Dávid; Oroszlán, Gábor; Kortvely, Elod; Kiss, Bence; Boros, Eszter; Szász, Róbert; Závodszky, Péter; Gál, Péter; Pál, Gábor

    2016-01-01

    MASP-3 was discovered 15 years ago as the third mannan-binding lectin (MBL)-associated serine protease of the complement lectin pathway. Lacking any verified substrate its role remained ambiguous. MASP-3 was shown to compete with a key lectin pathway enzyme MASP-2 for MBL binding, and was therefore considered to be a negative complement regulator. Later, knock-out mice experiments suggested that MASP-1 and/or MASP-3 play important roles in complement pro-factor D (pro-FD) maturation. However, studies on a MASP-1/MASP-3-deficient human patient produced contradicting results. In normal resting blood unperturbed by ongoing coagulation or complement activation, factor D is present predominantly in its active form, suggesting that resting blood contains at least one pro-FD activating proteinase that is not a direct initiator of coagulation or complement activation. We have recently showed that all three MASPs can activate pro-FD in vitro. In resting blood, however, using our previously evolved MASP-1 and MASP-2 inhibitors we proved that neither MASP-1 nor MASP-2 activates pro-FD. Other plasma proteinases, particularly MASP-3, remained candidates for that function. For this study we evolved a specific MASP-3 inhibitor and unambiguously proved that activated MASP-3 is the exclusive pro-FD activator in resting blood, which demonstrates a fundamental link between the lectin and alternative pathways.

  18. MASP-3 is the exclusive pro-factor D activator in resting blood: the lectin and the alternative complement pathways are fundamentally linked.

    PubMed

    Dobó, József; Szakács, Dávid; Oroszlán, Gábor; Kortvely, Elod; Kiss, Bence; Boros, Eszter; Szász, Róbert; Závodszky, Péter; Gál, Péter; Pál, Gábor

    2016-01-01

    MASP-3 was discovered 15 years ago as the third mannan-binding lectin (MBL)-associated serine protease of the complement lectin pathway. Lacking any verified substrate its role remained ambiguous. MASP-3 was shown to compete with a key lectin pathway enzyme MASP-2 for MBL binding, and was therefore considered to be a negative complement regulator. Later, knock-out mice experiments suggested that MASP-1 and/or MASP-3 play important roles in complement pro-factor D (pro-FD) maturation. However, studies on a MASP-1/MASP-3-deficient human patient produced contradicting results. In normal resting blood unperturbed by ongoing coagulation or complement activation, factor D is present predominantly in its active form, suggesting that resting blood contains at least one pro-FD activating proteinase that is not a direct initiator of coagulation or complement activation. We have recently showed that all three MASPs can activate pro-FD in vitro. In resting blood, however, using our previously evolved MASP-1 and MASP-2 inhibitors we proved that neither MASP-1 nor MASP-2 activates pro-FD. Other plasma proteinases, particularly MASP-3, remained candidates for that function. For this study we evolved a specific MASP-3 inhibitor and unambiguously proved that activated MASP-3 is the exclusive pro-FD activator in resting blood, which demonstrates a fundamental link between the lectin and alternative pathways. PMID:27535802

  19. Segment spanning residues 727-768 of the complement C3 sequence contains a neoantigenic site and accommodates the binding of CR1, factor H, and factor B.

    PubMed

    Becherer, J D; Alsenz, J; Esparza, I; Hack, C E; Lambris, J D

    1992-02-18

    CR1, CR2, DAF, MCP, factor H, C4bp, factor B, and C3 are members of a family of structurally related molecules, the majority of which belong to the complement system. Several of these molecules also share functional features such as cofactor and decay/dissociation activity and compete with one another in binding to C3b. Since factor H appears to bind to multiple sites in C3, we investigated the relationship between the factor H- and CR1-binding sites in C3b. Factor H binding to C3b is inhibited by either the C3c or C3d fragments, and addition of both fragments together augments this inhibition. One monoclonal anti-C3c antibody, anti-C3-9, which recognizes a neoantigenic epitope expressed upon cleavage to C3 to C3b, inhibited both factor H and CR1 binding to EC3b cells. This monoclonal antibody (MoAb) also inhibited factor B binding to EC3b. Two observations further supported our hypothesis that these molecules bind to proximal sites in C3b. First, a synthetic peptide spanning this region of C3b (C3(727-768)) inhibited factor H binding. Second, antibodies raised against this peptide inhibited binding to CR1, factor H, and factor B to C3b. These data show that H binds to at least two sites in C3b: the site in the C3c fragment is within the identified CR1-binding domain while the site in the C3d fragment surrounds the CR2-binding site.(ABSTRACT TRUNCATED AT 250 WORDS)

  20. Isolation and characterization of a novel rat factor H-related protein that is up-regulated in glomeruli under complement attack.

    PubMed

    Ren, Guohui; Doshi, Mona; Hack, Bradley K; Alexander, Jessy J; Quigg, Richard J

    2002-12-13

    The factor H family in humans is composed of seven distinct proteins, including factor H-related proteins (FHR) 1-5. All members contain tandemly arranged short consensus repeats (SCR) typical of the regulators of complement activation gene family. FHR-5 is unusual for this group of proteins, as it was initially identified as a component of immune deposits in glomerular diseases. During our cloning of the cDNA for rat factor H from glomerular epithelial cells (GEC), we identified an alternative 2729-bp cDNA transcript. The translated sequence encoded a protein containing 11 SCRs, most similar to SCRs 7-15 and 19-20 in native rat factor H, which is the same basic structure of human FHR-5. As such, this rat protein was termed FHR. Recombinant rat FHR produced in a eukaryotic expression system had a molecular mass of 78 kDa. In functional studies, recombinant FHR bound C3b and inhibited the complement alternative pathway in a dose-dependent fashion. Given the prominent expression of FHR-5 in human membranous nephropathy, a disease in which complement activation occurs in the vicinity of GEC, the expression of FHR in a rat model of this disease was evaluated. In both in vitro and in vivo models of complement activation on the GEC, FHR mRNA was up-regulated by a factor of 3-6-fold compared with controls in which complement could not be activated. Thus, we have identified a novel factor H family member in rats. This FHR protein is analogous to human FHR-5, both in structure and in potential involvement in glomerular immune complex diseases.

  1. Human carcinomas variably express the complement inhibitory proteins CD46 (membrane cofactor protein), CD55 (decay-accelerating factor), and CD59 (protectin).

    PubMed

    Niehans, G A; Cherwitz, D L; Staley, N A; Knapp, D J; Dalmasso, A P

    1996-07-01

    Normal human tissues express membrane-associated complement inhibitory proteins that protect these tissues from damage by autologous complement. To determine whether neoplasms also express these proteins, we examined the distribution of the complement inhibitors decay-accelerating factor (DAF), CD59 (protectin), and membrane cofactor protein in frozen samples of human breast, colon, kidney, and lung carcinomas and in adjacent non-neoplastic tissues, using immunohistochemistry. All samples were also studied for deposition of C3 fragments and activated C5b-9. Differences between normal tissues and the corresponding neoplasms were often observed, with loss or gain of expression of one or more inhibitors. Ductal carcinomas of the breast showed the most variation in phenotype; some tumors expressed only one inhibitor while others expressed different combinations of two or three inhibitors. Colon carcinomas, by contrast, stained intensely for all inhibitors. Renal cell carcinomas had weak to moderate expression of one to three inhibitors, generally DAF and CD59, whereas non-small cell carcinomas of the lung usually expressed CD59 and membrane cofactor protein with variable DAF immunoreactivity. The two small cell carcinomas of the lung showed little or no staining for any inhibitor. Activated C5b-9 deposition was seen adjacent to tumor nests in a minority of carcinomas and showed no correlation with complement inhibitor expression. C3 fragment deposition was minimal. Our results demonstrate that most carcinomas, with the exception of small cell carcinomas of the lung, do express one or more complement inhibitors at a level likely to inhibit complement-mediated cellular damage. Unexpectedly, large quantities of DAF and CD59 were often observed in tumor stroma, with only limited deposition in normal connective tissue. This suggests that carcinomas may supplement the activity of membrane-associated complement inhibitors by release of soluble forms of DAF and CD59 into the

  2. Genetically engineered fusion of MAP-1 and factor H domains 1-5 generates a potent dual upstream inhibitor of both the lectin and alternative complement pathways.

    PubMed

    Nordmaj, Mie Anemone; Munthe-Fog, Lea; Hein, Estrid; Skjoedt, Mikkel-Ole; Garred, Peter

    2015-12-01

    Inhibition of the complement cascade has emerged as an option for treatment of a range of diseases. Mannose-binding lectin/ficolin/collectin-associated protein (MAP-1) is a pattern recognition molecule (PRM)-associated inhibitor of the lectin pathway. The central regulator of the alternative pathway (AP) is complement factor H (FH). Our aim was to design a dual upstream inhibitor of both human lectin and APs by fusing MAP-1 with a part of FH. There were 2 different recombinant chimeric proteins comprising full-length human MAP-1 and the first 5 N-terminal domains of human FH designed. The FH domains were orientated either in the N- or C-terminal part of MAP-1. The complement inhibition potential in human serum was assessed. Both chimeric constructs displayed the characteristics of the native molecules and bound to the PRMs with an EC50 of ∼ 2 nM. However, when added to serum diluted 1:4 in a solid-phase functional assay, only the first 5 N-terminal domains of complement FH fused to the C-terminal part of full-length MAP-1 chimeric construct were able to combine inhibition of lectin and AP activation with an half maximal inhibitory concentration of ∼ 100 and 20 nM, respectively. No effect was seen on the classical pathway. Fusion of MAP-1 with FH domains represents a novel therapeutic approach for selective targeting upstream and central complement activation at sites of inflammation.

  3. Vascular endothelial growth factor C complements the ability of positron emission tomography to predict nodal disease in lung cancer

    PubMed Central

    Farjah, Farhood; Madtes, David K.; Wood, Douglas E.; Flum, David R.; Zadworny, Megan E.; Waworuntu, Rachel; Hwang, Billanna; Mulligan, Michael S.

    2016-01-01

    Objective Vascular endothelial growth factors (VEGFs) C and D are biologically rational markers of nodal disease that could improve the accuracy of lung cancer staging. We hypothesized that these biomarkers would improve the ability of positron emission tomography (PET) to predict nodal disease among patients with suspected or confirmed non–small cell lung cancer (NSCLC). Methods A cross-sectional study (2010–2013) was performed of patients prospectively enrolled in a lung nodule biorepository, staged by computed tomography (CT) and PET, and who underwent pathologic nodal evaluation. Enzyme-linked immunosorbent assay was used to measure biomarker levels in plasma from blood drawn before anesthesia. Likelihood ratio testing was used to compare the following logistic regression prediction models: ModelPET, ModelPET/VEGF-C, ModelPET/VEGF-D, and ModelPET/VEGF-C/VEGF-D. To account for 5 planned pairwise comparisons, P values<.01 were considered significant. Results Among 62 patients (median age, 67 years; 48% men; 87% white; and 84% NSCLC), 58% had fluorodeoxyglucose uptake in hilar and/or mediastinal lymph nodes. The prevalence of pathologically confirmed lymph node metastases was 40%. Comparisons of prediction models revealed the following: ModelPET/VEGF-C versus ModelPET (P = .0069), ModelPET/VEGF-D versus ModelPET (P = .1886), ModelPET/VEGF-C/VEGF-D versus ModelPET (P = .0146), ModelPET/VEGF-C/VEGF-D versus ModelPET/VEGF-C (P = .2818), and ModelPET/VEGF-C/VEGF-D versus ModelPET/VEGF-D (P = .0095). In ModelPET/VEGF-C, higher VEGF-C levels were associated with an increased risk of nodal disease (odds ratio, 2.96; 95% confidence interval, 1.26–6.90). Conclusions Plasma levels of VEGF-C complemented the ability of PET to predict nodal disease among patients with suspected or confirmed NSCLC. VEGF-D did not improve prediction. PMID:26320776

  4. Role of tissue factor in a mouse model of thrombotic microangiopathy induced by antiphospholipid antibodies

    PubMed Central

    Seshan, Surya V.; Franzke, Claus-Werner; Redecha, Patricia; Monestier, Marc; Mackman, Nigel

    2009-01-01

    Using different mouse monoclonal and human antiphospholipid (aPL) antibodies, we developed a new animal model of renal injury that shares many features with thrombotic microangiopathy (TMA). We found that more than 1 mechanism/signaling pathway is involved in glomerular injury induced by aPL antibodies in this model. Both complement-dependent and complement-independent pathways were identified that lead to glomerular endothelial cell damage and renal function impairment. We also found that C5a-C5aR interaction is a crucial step for the activation of the coagulation cascade and glomerular injury induced by complement-activating antibodies. In addition, our studies demonstrated complement-independent mechanisms in which reactivity with β2 glycoprotein I (β2GPI) plays an important role in aPL-induced glomerular damage and renal failure. Independently of the mechanism responsible for aPL-induced TMA, mice that express low levels of tissue factor (TF) were protected from glomerular injury. That genetic reduction of TF prevents renal injury induced by different aPL antibodies indicates that TF is a common mediator of glomerular damage and a possible target for selective pharmacologic intervention. Treatment with pravastatin, which down-regulates glomerular TF synthesis, prevents aPL-induced TMA in this mouse model, thus emphasizing that targeting TF might be a good therapeutic intervention in patients with TMA. PMID:19535796

  5. Measles virus-induced down-regulation of CD46 is associated with enhanced sensitivity to complement-mediated lysis of infected cells.

    PubMed

    Schnorr, J J; Dunster, L M; Nanan, R; Schneider-Schaulies, J; Schneider-Schaulies, S; ter Meulen, V

    1995-04-01

    CD46, the major component of the measles virus (MV) receptor complex and a member of the regulators of complement activity (RCA) gene cluster, is down-regulated in MV-infected cells. We investigated whether the reduction of surface CD46 correlates with enhanced sensitivity of lymphoid and monocytic cells to lysis by activated complement. On human U937 cells, acutely or persistently infected with MV-Edmonston (ED) vaccine strain, infection-dependent down-regulation of CD46 confers sensitivity to activated complement, regardless of the pathway of activation and the specificity of the activating antibodies. Interestingly, down-regulation of CD46 alone is sufficient to confer susceptibility of cells to complement lysis despite the continued surface expression of other RCA proteins such as CD35 and CD55. In primary cultures, both peripheral blood lymphocytes and macrophages are efficiently lysed in the presence of complement activated via the alternative pathway after MV infection. In contrast to the MV-ED infection, infection of cells with the lymphotropic MV wild-type strain WTF does not down-regulate CD46. Cells infected with MV-WTF do not exhibit enhanced susceptibility to complement lysis. These data suggest that MV strains similar to WTF that do not down-regulate CD46 may have an enhanced potential for replication and dissemination within the human host, whereas complement-mediated elimination of cells infected with CD46-down-regulating strains of MV, such as ED, may limit the spread of MV infection, and could thus represent an attenuating factor for MV. PMID:7737301

  6. Identification of three physically and functionally distinct binding sites for C3b in human complement factor H by deletion mutagenesis.

    PubMed Central

    Sharma, A K; Pangburn, M K

    1996-01-01

    Human complement factor H controls spontaneous activation of complement in plasma and appears to play a role in distinguishing host cells from activators of the alternative pathway of complement. In both mice and humans, the protein is composed of 20 homologous short consensus repeat (SCR) domains. The size of the protein suggests that portions of the structure outside the known C3b binding site (SCR 1-4) possess a significant biological role. We have expressed the full-length cDNA of factor H in the baculovirus system and have shown the recombinant protein to be fully active. Mutants of this full-length protein have now been prepared, purified, and examined for cofactor activity and binding to C3b and heparin. The results demonstrate (i) that factor H has at least three sites that bind C3b, (ii) that one of these sites is located in SCR domains 1-4, as has been shown by others, (iii) that a second site exists in the domain 6-10 region, (iv) that a third site resides in the SCR 16-20 region, and (v) that two heparin binding sites exist in factor H, one near SCR 13 and another in the SCR 6-10 region. Functional assays demonstrated that only the first C3b site located in SCR 1-4 expresses factor I cofactor activity. Mutant proteins lacking any one of the three C3b binding sites exhibited 6- to 8-fold reductions in affinity for C3b on sheep erythrocytes, indicating that all three sites contribute to the control of complement activation on erythrocytes. The identification of multiple functionally distinct sites on factor H clarifies many of the heretofore unexplainable behaviors of this protein, including the heterogeneous binding of factor H to surface-bound C3b, the effects of trypsin cleavage, and the differential control of complement activation on activators and nonactivators of the alternative pathway of complement. Images Fig. 2 Fig. 3 PMID:8855297

  7. Regulation of the alternative pathway of complement modulates injury and immunity in a chronic model of dextran sulphate sodium-induced colitis

    PubMed Central

    Elvington, M; Schepp-Berglind, J; Tomlinson, S

    2015-01-01

    The role of complement in inflammatory bowel disease (IBD) has been studied primarily using acute models, and it is unclear how complement affects processes in more relevant chronic models of IBD in which modulation of adaptive immunity and development of fibrosis have pathogenic roles. Using mice deficient in C1q/mannose-binding lectin (MBL) or C3, we demonstrated an important role for these opsonins and/or the classical pathway C3 convertase in providing protection against mucosal injury and infection in a model of chronic dextran sulphate sodium (DSS)-induced colitis. In contrast, deficiency of the alternative pathway (fB–/– mice) had significantly less impact on injury profiles. Consequently, the effect of a targeted inhibitor of the alternative pathway was investigated in a therapeutic protocol. Following the establishment of colitis, mice were treated with CR2-fH during subsequent periods of DSS treatment and acute injury (modelling relapse). CR2-fH significantly reduced complement activation, inflammation and injury in the colon, and additionally reduced fibrosis. Alternative pathway inhibition also altered the immune response in the chronic state in terms of reducing numbers of B cells, macrophages and mature dendritic cells in the lamina propria. This study indicates an important role for the alternative pathway of complement in the pathogenesis and the shaping of an immune response in chronic DSS-induced colitis, and supports further investigation into the use of targeted alternative pathway inhibition for the treatment of IBD. PMID:25293413

  8. Complement Factor H Expressed by Retinal Pigment Epithelium Cells Can Suppress Neovascularization of Human Umbilical Vein Endothelial Cells: An in vitro Study

    PubMed Central

    Zhang, Yi; Huang, Qing; Tang, Min; Zhang, Junjun; Fan, Wei

    2015-01-01

    Complement factor H (CFH) is one of the most important soluble complement regulatory proteins and is closely associated with age-related macular degeneration (AMD), the leading cause of irreversible central vision loss in the elderly population in developed countries. Our study searches to investigate whether CFH expression is changed in oxidative damaged retinal pigment epithelium (RPE) cells and the role of CFH in the in vitro neovascularization. First, it was confirmed by immunofluorescence staining that CFH was expressed by ARPE-19 cells. CFH mRNA and protein in oxidative (H2O2) damaged ARPE-19 cells were both reduced, as determined by Real-time PCR and Western blotting analysis. Enzyme-linked immunosorbent assay (ELISA) also showed that ARPE-19 cells treated with H2O2 caused an increase in C3a content, which indicates complement activation. Then, wound assays were performed to show that CFH expression suppression promoted human umbilical vein endothelial cell (HUVECs) migration. Thereafter, ARPE-19 cells were transfected with CFH-specific siRNA and CFH knockdown was confirmed with the aid of Real-time PCR, immunofluorescence staining and Western blotting. The ELISA results showed that specific CFH knockdown in ARPE-19 cells activated the complement system. Finally, in vitro matrigel tube formation assay was performed to determine whether change of CFH expression in RPE would affect tube formation by HUVECs. More tubes were formed by HUVECs co-cultured with ARPE-19 cells transfected with CFH specific-siRNA when compared with controls. Our results suggested that RPE cells might be the local CFH source, and RPE cell injuries (such as oxidative stress) may cause CFH expression suppression, which in turn may lead to complement activation and promotion of tube formation by HUVECs. This finding is of importance in elucidating the role of complement in the pathogenesis of ocular neovascularization including choroidal neovascularization. PMID:26091360

  9. [In vitro and clinical study of complement activation during cardiopulmonary bypass with reference to types of oxygenator, primed autologous blood and patient's factor].

    PubMed

    Oda, K

    1991-09-01

    Complement activation during cardiopulmonary bypass (CPB) was studied in vitro and in vivo with regard to types of oxygenator, primed autologous blood and patient's factor. In vitro study was performed using human blood in a simple circuit involving an oxygenator, roller pump and connector tubing. In vivo study was carried out in 118 patients and divided into bubble (BO) and membrane oxygenator (MO) groups. The influence of primed homologous to circulating autologous blood volume (H/A) ratio was also examined. In vitro study, C3a and C4a increased steeply in the BO group. On the other hand, in the MO group, C3a and C4a increased up to minute of 60, and afterwards gradually decreased. In clinical study, complement was more activated in the BO group than in the MO group. These results supported that in the BO group, immunoglobulin denatured by blood-gas interface played an important role in complement activation. In membrane oxygenator, blood-material interface was a major cause of complement activation. In order to reduce these complement activation, we introduced fresh concentrated red cells, which was almost free of immunoglobulin, as a primed blood. Application of this method in clinical study, complement activation was reduced and postoperative lung function was improved significantly. These changes were more significant in the BO group. In the high H/A group, differences of anaphylatoxin level between BO and MO group had a tendency to increase. This method is useful to the CPB case of neonate and infant, which was subjected to be primed with a large amount of blood.

  10. The outer membrane protease PgtE of Salmonella enterica interferes with the alternative complement pathway by cleaving factors B and H

    PubMed Central

    Riva, Rauna; Korhonen, Timo K.; Meri, Seppo

    2015-01-01

    The virulence factor PgtE is an outer membrane protease (omptin) of the zoonotic pathogen Salmonella enterica that causes diseases ranging from gastroenteritis to severe enteric fever. It is surface exposed in bacteria that have a short-chain, i.e., rough LPS, as observed e.g., in bacteria residing inside macrophages or just emerging from them. We investigated whether PgtE cleaves the complement factors B (B) and H (H), key proteins controlling formation and inactivation of the complement protein C3b and thereby the activity of the complement system. S. enterica serovar Typhimurium or omptin-expressing recombinant E. coli bacteria were incubated with purified human complement proteins or recombinant H fragments. PgtE cleaved both B and H, whereas its close homolog Pla of Yersinia pestis cleaved only H. H was cleaved at both N- and C-termini, while the central region resisted proteolysis. Because of multiple effects of PgtE on complement components (cleavage of C3, C3b, B, and H) we assessed its effect on the opsonophagocytosis of Salmonella. In human serum, C3 cleavage was dependent on proteolytically active PgtE. Human neutrophils interacted less with serum-opsonized FITC-stained S. enterica 14028R than with the isogenic ΔpgtE strain, as analyzed by flow cytometry. In conclusion, cleavage of B and H by PgtE, together with C3 cleavage, affects the C3-mediated recognition of S. enterica by human neutrophils, thus thwarting the immune protection against Salmonella. PMID:25705210

  11. Distinct and separable roles of the complement system in factor H-deficient bone marrow chimeric mice with immune complex disease.

    PubMed

    Alexander, Jessy J; Aneziokoro, O G B; Chang, Anthony; Hack, Bradley K; Markaryan, Adam; Jacob, Alexander; Luo, Roger; Thirman, Michael; Haas, Mark; Quigg, Richard J

    2006-05-01

    Plasma complement factor H (Cfh) is a potent complement regulator, whereas Cfh on the surface of rodent platelets is responsible for immune complex processing. For dissection between the two, bone marrow chimeras between Cfh-deficient (Cfh(-/-)) and wild-type C57BL/6 mice were created. Platelet Cfh protein was tracked with the Cfh status of the bone marrow donor, indicating that platelet Cfh is of intrinsic origin. In an active model of immune complex disease, Cfh(-/-) mice that were reconstituted with wild-type bone marrow had levels of platelet-associated immune complexes comparable to those of wild-type mice and were protected against the excessive glomerular deposition of immune complexes seen in Cfh(-/-) mice, yet these mice still developed glomerular inflammation. In contrast, wild-type mice with Cfh(-/-) bone marrow had reduced platelet-associated immune complexes and extensive glomerular deposition of complement-activating immune complexes, but they did not develop glomerular pathology. The large quantities of glomerular C3 in wild-type mice with Cfh(-/-) bone marrow were in the form of iC3b and C3dg, whereas active C3b remained in Cfh(-/-) recipients of wild-type bone marrow. These data show that plasma Cfh limits complement activation in the circulation and other accessible sites such as the glomerulus, whereas platelet Cfh is responsible for immune complex processing.

  12. Environmental Factors Inducing Human Cancers

    PubMed Central

    Parsa, N

    2012-01-01

    Background An explosion of research has been done in discovering how human health is affected by environmental factors. I will discuss the impacts of environmental cancer causing factors and how they continue to cause multiple disruptions in cellular networking. Some risk factors may not cause cancer. Other factors initiate consecutive genetic mutations that would eventually alter the normal pathway of cellular proliferations and differentiation. Genetic mutations in four groups of genes; (Oncogenes, Tumor suppressor genes, Apoptosis genes and DNA repairing genes) play a vital role in altering the normal cell division. In recent years, molecular genetics have greatly increased our understanding of the basic mechanisms in cancer development and utilizing these molecular techniques for cancer screening, diagnosis, prognosis and therapies. Inhibition of carcinogenic exposures wherever possible should be the goal of cancer prevention programs to reduce exposures from all environmental carcinogens. PMID:23304670

  13. Inflammation-inducing Factors of Mycoplasma pneumoniae

    PubMed Central

    Shimizu, Takashi

    2016-01-01

    Mycoplasma pneumoniae, which causes mycoplasmal pneumonia in human, mainly causes pneumonia in children, although it occasionally causes disease in infants and geriatrics. Some pathogenic factors produced by M. pneumoniae, such as hydrogen peroxide and Community-Acquired Respiratory Distress Syndrome (CARDS) toxin have been well studied. However, these factors alone cannot explain this predilection. The low incidence rate of mycoplasmal pneumonia in infants and geriatrics implies that the strong inflammatory responses induced by M. pneumoniae coordinate with the pathogenic factors to induce pneumonia. However, M. pneumoniae lacks a cell wall and does not possess an inflammation-inducing endotoxin, such as lipopolysaccharide (LPS). In M. pneumoniae, lipoproteins were identified as an inflammation-inducing factor. Lipoproteins induce inflammatory responses through Toll-like receptors (TLR) 2. Because Mycoplasma species lack a cell wall and lipoproteins anchored in the membrane are exposed, lipoproteins and TLR2 have been thought to be important for the pathogenesis of M. pneumoniae. However, recent reports suggest that M. pneumoniae also induces inflammatory responses also in a TLR2-independent manner. TLR4 and autophagy are involved in this TLR2-independent inflammation. In addition, the CARDS toxin or M. pneumoniae cytadherence induces inflammatory responses through an intracellular receptor protein complex called the inflammasome. In this review, the inflammation-inducing factors of M. pneumoniae are summarized. PMID:27065977

  14. A novel antibody against human factor B that blocks formation of the C3bB proconvertase and inhibits complement activation in disease models.

    PubMed

    Subías, Marta; Tortajada, Agustín; Gastoldi, Sara; Galbusera, Miriam; López-Perrote, Andrés; Lopez, Lucia de Juana; González-Fernández, Fernando Ataúlfo; Villegas-Martínez, Ana; Dominguez, Mercedes; Llorca, Oscar; Noris, Marina; Morgan, B Paul; Rodríguez de Córdoba, Santiago

    2014-12-01

    The alternative pathway (AP) is critical for the efficient activation of complement regardless of the trigger. It is also a major player in pathogenesis, as illustrated by the long list of diseases in which AP activation contributes to pathology. Its relevance to human disease is further emphasized by the high prevalence of pathogenic inherited defects and acquired autoantibodies disrupting components and regulators of the AP C3-convertase. Because pharmacological downmodulation of the AP emerges as a broad-spectrum treatment alternative, there is a powerful interest in developing new molecules to block formation and/or activity of the AP C3-convertase. In this paper, we describe the generation of a novel mAb targeting human factor B (FB). mAb FB48.4.2, recognizing with high affinity an evolutionary-conserved epitope in the Ba fragment of FB, very efficiently inhibited formation of the AP C3-proconvertase by blocking the interaction between FB and C3b. In vitro assays using rabbit and sheep erythrocytes demonstrated that FB28.4.2 was a potent AP inhibitor that blocked complement-mediated hemolysis in several species. Using ex vivo models of disease we demonstrated that FB28.4.2 protected paroxysmal nocturnal hemoglobinuria erythrocytes from complement-mediated hemolysis and inhibited both C3 fragment and C5b-9 deposition on ADP-activated HMEC-1 cells, an experimental model for atypical hemolytic uremic syndrome. Moreover, i.v. injection of FB28.4.2 in rats blocked complement activation in rat serum and prevented the passive induction of experimental autoimmune Myasthenia gravis. As a whole, these data demonstrate the potential value of FB28.4.2 for the treatment of disorders associated with AP complement dysregulation in man and animal models.

  15. Expression of human complement factor H prevents age-related macular degeneration-like retina damage and kidney abnormalities in aged Cfh knockout mice.

    PubMed

    Ding, Jin-Dong; Kelly, Una; Landowski, Michael; Toomey, Christopher B; Groelle, Marybeth; Miller, Chelsey; Smith, Stephanie G; Klingeborn, Mikael; Singhapricha, Terry; Jiang, Haixiang; Frank, Michael M; Bowes Rickman, Catherine

    2015-01-01

    Complement factor H (CFH) is an important regulatory protein in the alternative pathway of the complement system, and CFH polymorphisms increase the genetic risk of age-related macular degeneration dramatically. These same human CFH variants have also been associated with dense deposit disease. To mechanistically study the function of CFH in the pathogenesis of these diseases, we created transgenic mouse lines using human CFH bacterial artificial chromosomes expressing full-length human CFH variants and crossed these to Cfh knockout (Cfh(-/-)) mice. Human CFH protein inhibited cleavage of mouse complement component 3 and factor B in plasma and in retinal pigment epithelium/choroid/sclera, establishing that human CFH regulates activation of the mouse alternative pathway. One of the mouse lines, which express relatively higher levels of CFH, demonstrated functional and structural protection of the retina owing to the Cfh deletion. Impaired visual function, detected as a deficit in the scotopic electroretinographic response, was improved in this transgenic mouse line compared with Cfh(-/-) mice, and transgenics had a thicker outer nuclear layer and less sub-retinal pigment epithelium deposit accumulation. In addition, expression of human CFH also completely protected the mice from developing kidney abnormalities associated with loss of CFH. These humanized CFH mice present a valuable model for study of the molecular mechanisms of age-related macular degeneration and dense deposit disease and for testing therapeutic targets.

  16. Expression of Human Complement Factor H Prevents Age-Related Macular Degeneration–Like Retina Damage and Kidney Abnormalities in Aged Cfh Knockout Mice

    PubMed Central

    Ding, Jin-Dong; Kelly, Una; Landowski, Michael; Toomey, Christopher B.; Groelle, Marybeth; Miller, Chelsey; Smith, Stephanie G.; Klingeborn, Mikael; Singhapricha, Terry; Jiang, Haixiang; Frank, Michael M.; Bowes Rickman, Catherine

    2016-01-01

    Complement factor H (CFH) is an important regulatory protein in the alternative pathway of the complement system, and CFH polymorphisms increase the genetic risk of age-related macular degeneration dramatically. These same human CFH variants have also been associated with dense deposit disease. To mechanistically study the function of CFH in the pathogenesis of these diseases, we created transgenic mouse lines using human CFH bacterial artificial chromosomes expressing full-length human CFH variants and crossed these to Cfh knockout (Cfh−/−) mice. Human CFH protein inhibited cleavage of mouse complement component 3 and factor B in plasma and in retinal pigment epithelium/choroid/sclera, establishing that human CFH regulates activation of the mouse alternative pathway. One of the mouse lines, which express relatively higher levels of CFH, demonstrated functional and structural protection of the retina owing to the Cfh deletion. Impaired visual function, detected as a deficit in the scotopic electroretinographic response, was improved in this transgenic mouse line compared with Cfh−/− mice, and transgenics had a thicker outer nuclear layer and less sub–retinal pigment epithelium deposit accumulation. In addition, expression of human CFH also completely protected the mice from developing kidney abnormalities associated with loss of CFH. These humanized CFH mice present a valuable model for study of the molecular mechanisms of age-related macular degeneration and dense deposit disease and for testing therapeutic targets. PMID:25447048

  17. Complement C5a induces PD-L1 expression and acts in synergy with LPS through Erk1/2 and JNK signaling pathways.

    PubMed

    An, Ling-Ling; Gorman, Jacob V; Stephens, Geoffrey; Swerdlow, Bonnie; Warrener, Paul; Bonnell, Jessica; Mustelin, Tomas; Fung, Michael; Kolbeck, Roland

    2016-01-01

    Severe bacterial infection results in both uncontrolled inflammation and immune suppression in septic patients. Although there is ample evidence that complement activation provokes overwhelming pro-inflammatory responses, whether or not it plays a role in immune suppression in this case is unclear. Here, we identify that complement C5a directly participates in negative regulation of immune responses to bacteria-induced inflammation in an ex vivo model of human whole blood. Challenge of whole blood with heat-killed Pseudomonas aeruginosa induces PD-L1 expression on monocytes and the production of IL-10 and TGF-β, which we show to be inhibited by C5a blockade. The induction of PD-L1 expression by C5a is via C5aR1but not C5aR2. Furthermore, C5a synergises with P. aeruginosa LPS in both PD-L1 expression and the production of IL-10 and TGF-β. Mechanistically, C5a contributes to the synergy in PD-L1 expression by specifically activating Erk1/2 and JNK signaling pathways. Our study reveals a new role for C5a in directly promoting immunosuppressive responses. Therefore, aberrant production of complement C5a during bacterial infection could have broader effect on compromising host defense including the induction of immune suppression. PMID:27624143

  18. Complement C5a induces PD-L1 expression and acts in synergy with LPS through Erk1/2 and JNK signaling pathways

    PubMed Central

    An, Ling-Ling; Gorman, Jacob V.; Stephens, Geoffrey; Swerdlow, Bonnie; Warrener, Paul; Bonnell, Jessica; Mustelin, Tomas; Fung, Michael; Kolbeck, Roland

    2016-01-01

    Severe bacterial infection results in both uncontrolled inflammation and immune suppression in septic patients. Although there is ample evidence that complement activation provokes overwhelming pro-inflammatory responses, whether or not it plays a role in immune suppression in this case is unclear. Here, we identify that complement C5a directly participates in negative regulation of immune responses to bacteria-induced inflammation in an ex vivo model of human whole blood. Challenge of whole blood with heat-killed Pseudomonas aeruginosa induces PD-L1 expression on monocytes and the production of IL-10 and TGF-β, which we show to be inhibited by C5a blockade. The induction of PD-L1 expression by C5a is via C5aR1but not C5aR2. Furthermore, C5a synergises with P. aeruginosa LPS in both PD-L1 expression and the production of IL-10 and TGF-β. Mechanistically, C5a contributes to the synergy in PD-L1 expression by specifically activating Erk1/2 and JNK signaling pathways. Our study reveals a new role for C5a in directly promoting immunosuppressive responses. Therefore, aberrant production of complement C5a during bacterial infection could have broader effect on compromising host defense including the induction of immune suppression. PMID:27624143

  19. The ancestral complement system in sea urchins.

    PubMed

    Smith, L C; Clow, L A; Terwilliger, D P

    2001-04-01

    The origin of adaptive immunity in the vertebrates can be traced to the appearance of the ancestral RAG genes in the ancestral jawed vertebrate; however, the innate immune system is more ancient. A central subsystem within innate immunity is the complement system, which has been identified throughout and seems to be restricted to the deuterostomes. The evolutionary history of complement can be traced from the sea urchins (members of the echinoderm phylum), which have a simplified system homologous to the alternative pathway, through the agnathans (hagfish and lamprey) and the elasmobranchs (sharks and rays) to the teleosts (bony fish) and tetrapods, with increases in the numbers of complement components and duplications in complement pathways. Increasing complexity in the complement system parallels increasing complexity in the deuterostome animals. This review focuses on the simplest of the complement systems that is present in the sea urchin. Two components have been identified that show significant homology to vertebrate C3 and factor B (Bf), called SpC3 and SpBf, respectively. Sequence analysis from both molecules reveals their ancestral characteristics. Immune challenge of sea urchins indicates that SpC3 is inducible and is present in coelomic fluid (the body fluids) in relatively high concentrations, while SpBf expression is constitutive and is present in much lower concentrations. Opsonization of foreign cells and particles followed by augmented uptake by phagocytic coelomocytes appears to be a central function for this simpler complement system and important for host defense in the sea urchin. These activities are similar to some of the functions of the homologous proteins in the vertebrate complement system. The selective advantage for the ancestral deuterostome may have been the amplification feedback loop that is still of central importance in the alternative pathway of complement in higher vertebrates. Feedback loop functions would quickly coat

  20. Microbe-specific C3b deposition in the horseshoe crab complement system in a C2/factor B-dependent or -independent manner.

    PubMed

    Tagawa, Keisuke; Yoshihara, Toyoki; Shibata, Toshio; Kitazaki, Kazuki; Endo, Yuichi; Fujita, Teizo; Koshiba, Takumi; Kawabata, Shun-ichiro

    2012-01-01

    Complement C3 plays an essential role in the opsonization of pathogens in the mammalian complement system, whereas the molecular mechanism underlying C3 activation in invertebrates remains unknown. To understand the molecular mechanism of C3b deposition on microbes, we characterized two types of C2/factor B homologs (designated TtC2/Bf-1 and TtC2/Bf-2) identified from the horseshoe crab Tachypleus tridentatus. Although the domain architectures of TtC2/Bf-1 and TtC2/Bf-2 were identical to those of mammalian homologs, they contained five-repeated and seven-repeated complement control protein domains at their N-terminal regions, respectively. TtC2/Bf-1 and TtC2/Bf-2 were synthesized and glycosylated in hemocytes and secreted to hemolymph plasma, which existed in a complex with C3 (TtC3), and their activation by microbes was absolutely Mg(2+)-dependent. Flow cytometric analysis revealed that TtC3b deposition was Mg(2+)-dependent on Gram-positive bacteria or fungi, but not on Gram-negative bacteria. Moreover, this analysis demonstrated that Ca(2+)-dependent lectins (C-reactive protein-1 and tachylectin-5A) were required for TtC3b deposition on Gram-positive bacteria, and that a Ca(2+)-independent lectin (Tachypleus plasma lectin-1) was definitely indispensable for TtC3b deposition on fungi. In contrast, a horseshoe crab lipopolysaccharide-sensitive protease factor C was necessary and sufficient to deposit TtC3b on Gram-negative bacteria. We conclude that plasma lectins and factor C play key roles in microbe-specific TtC3b deposition in a C2/factor B-dependent or -independent manner. PMID:22611464

  1. Microbe-specific C3b deposition in the horseshoe crab complement system in a C2/factor B-dependent or -independent manner.

    PubMed

    Tagawa, Keisuke; Yoshihara, Toyoki; Shibata, Toshio; Kitazaki, Kazuki; Endo, Yuichi; Fujita, Teizo; Koshiba, Takumi; Kawabata, Shun-ichiro

    2012-01-01

    Complement C3 plays an essential role in the opsonization of pathogens in the mammalian complement system, whereas the molecular mechanism underlying C3 activation in invertebrates remains unknown. To understand the molecular mechanism of C3b deposition on microbes, we characterized two types of C2/factor B homologs (designated TtC2/Bf-1 and TtC2/Bf-2) identified from the horseshoe crab Tachypleus tridentatus. Although the domain architectures of TtC2/Bf-1 and TtC2/Bf-2 were identical to those of mammalian homologs, they contained five-repeated and seven-repeated complement control protein domains at their N-terminal regions, respectively. TtC2/Bf-1 and TtC2/Bf-2 were synthesized and glycosylated in hemocytes and secreted to hemolymph plasma, which existed in a complex with C3 (TtC3), and their activation by microbes was absolutely Mg(2+)-dependent. Flow cytometric analysis revealed that TtC3b deposition was Mg(2+)-dependent on Gram-positive bacteria or fungi, but not on Gram-negative bacteria. Moreover, this analysis demonstrated that Ca(2+)-dependent lectins (C-reactive protein-1 and tachylectin-5A) were required for TtC3b deposition on Gram-positive bacteria, and that a Ca(2+)-independent lectin (Tachypleus plasma lectin-1) was definitely indispensable for TtC3b deposition on fungi. In contrast, a horseshoe crab lipopolysaccharide-sensitive protease factor C was necessary and sufficient to deposit TtC3b on Gram-negative bacteria. We conclude that plasma lectins and factor C play key roles in microbe-specific TtC3b deposition in a C2/factor B-dependent or -independent manner.

  2. Complement receptor 2-mediated targeting of complement inhibitors to sites of complement activation.

    PubMed

    Song, Hongbin; He, Chun; Knaak, Christian; Guthridge, Joel M; Holers, V Michael; Tomlinson, Stephen

    2003-06-01

    In a strategy to specifically target complement inhibitors to sites of complement activation and disease, recombinant fusion proteins consisting of a complement inhibitor linked to a C3 binding region of complement receptor (CR) 2 were prepared and characterized. Natural ligands for CR2 are C3 breakdown products deposited at sites of complement activation. Fusion proteins were prepared consisting of a human CR2 fragment linked to either the N terminus or C terminus of soluble forms of the membrane complement inhibitors decay accelerating factor (DAF) or CD59. The targeted complement inhibitors bound to C3-opsonized cells, and all were significantly more effective (up to 20-fold) than corresponding untargeted inhibitors at protecting target cells from complement. CR2 fusion proteins also inhibited CR3-dependent adhesion of U937 cells to C3 opsonized erythrocytes, indicating a second potential anti-inflammatory mechanism of CR2 fusion proteins, since CR3 is involved in endothelial adhesion and diapedesis of leukocytes at inflammatory sites. Finally, the in vivo validity of the targeting strategy was confirmed by the demonstration that CR2-DAF, but not soluble DAF, targets to the kidney in mouse models of lupus nephritis that are associated with renal complement deposition.

  3. Complement receptor 2-mediated targeting of complement inhibitors to sites of complement activation.

    PubMed

    Song, Hongbin; He, Chun; Knaak, Christian; Guthridge, Joel M; Holers, V Michael; Tomlinson, Stephen

    2003-06-01

    In a strategy to specifically target complement inhibitors to sites of complement activation and disease, recombinant fusion proteins consisting of a complement inhibitor linked to a C3 binding region of complement receptor (CR) 2 were prepared and characterized. Natural ligands for CR2 are C3 breakdown products deposited at sites of complement activation. Fusion proteins were prepared consisting of a human CR2 fragment linked to either the N terminus or C terminus of soluble forms of the membrane complement inhibitors decay accelerating factor (DAF) or CD59. The targeted complement inhibitors bound to C3-opsonized cells, and all were significantly more effective (up to 20-fold) than corresponding untargeted inhibitors at protecting target cells from complement. CR2 fusion proteins also inhibited CR3-dependent adhesion of U937 cells to C3 opsonized erythrocytes, indicating a second potential anti-inflammatory mechanism of CR2 fusion proteins, since CR3 is involved in endothelial adhesion and diapedesis of leukocytes at inflammatory sites. Finally, the in vivo validity of the targeting strategy was confirmed by the demonstration that CR2-DAF, but not soluble DAF, targets to the kidney in mouse models of lupus nephritis that are associated with renal complement deposition. PMID:12813023

  4. Complement Factor 3 Could Be an Independent Risk Factor for Mortality in Patients with HBV Related Acute-on-Chronic Liver Failure

    PubMed Central

    Zhang, Geng-lin; Zhang, Ting; Ye, Yi-nong; Liu, Jing; Zhang, Xiao-hong; Xie, Chan; Peng, Liang; Gao, Zhi-liang

    2016-01-01

    The complement is thought to be involved in the pathogenesis of multiple liver disorders. However, its role in patients with HBV related acute-on-chronic liver failure (HBV-ACLF) remains unclear. Serum levels of the third and fourth complement components (C3, C4) and complement function (CH50) were examined in this prospective, observational study. Associations between their expression and disease activity were analyzed. Survival was analyzed by Kaplan-Meier curves. Predictors of clinical outcome were determined by Cox regression analysis. C3, C4, and CH50 levels were significantly lower in HBV-ACLF patients compared to controls. C3, C4, and CH50 levels were negatively correlated with Tbil levels but positively associated with PTA levels. C3 levels were negatively associated with MELD-Na. C3 levels were significantly lower in HBV-ACLF patients who died compared to patients who survived. In a median hospital stay of 39 days, mortality occurred in 41 patients with a progressive increase based on C3 grade (P = 0.008). The actuarial probability of developing mortality was significantly higher in patients with low C3 grade compared to those with high C3 grade (P < 0.001). Multivariate Cox regression analysis showed that C3 levels were an independent predictor of mortality. Complement played a pathogenic role in HBV-ACLF patients and C3 was an independent predictor of mortality. PMID:27144164

  5. Protein engineering to target complement evasion in cancer.

    PubMed

    Carter, Darrick; Lieber, André

    2014-01-21

    The complement system is composed of soluble factors in plasma that enhance or "complement" immune-mediated killing through innate and adaptive mechanisms. Activation of complement causes recruitment of immune cells; opsonization of coated cells; and direct killing of affected cells through a membrane attack complex (MAC). Tumor cells up-regulate complement inhibitory factors - one of several strategies to evade the immune system. In many cases as the tumor progresses, dramatic increases in complement inhibitory factors are found on these cells. This review focuses on the classic complement pathway and the role of major complement inhibitory factors in cancer immune evasion as well as on how current protein engineering efforts are being employed to increase complement fixing or to reverse complement resistance leading to better therapeutic outcomes in oncology. Strategies discussed include engineering of antibodies to enhance complement fixation, antibodies that neutralize complement inhibitory proteins as well as engineered constructs that specifically target inhibition of the complement system.

  6. Tumor necrosis factor-inducing activities of Cryptococcus neoformans components.

    PubMed Central

    Delfino, D; Cianci, L; Migliardo, M; Mancuso, G; Cusumano, V; Corradini, C; Teti, G

    1996-01-01

    Cryptococcus neoformans-induced tumor necrosis factor alpha (TNF-alpha) production may lead to increased human immunodeficiency virus replication in patients with AIDS. In order to identify cryptococcal components that are predominantly responsible for stimulating TNF production, various concentrations of glucuronoxylomannan (GXM), galactoxylomannan (GalXM), mannoproteins (MP), and alpha(1-3) [corrected] glucan were added to whole-blood cultures. All of the cryptococcal components tested, as well as whole heat-killed cryptococci, were capable of inducing TNF-alpha release in a dose-dependent manner. MP were significantly more potent than any of the other cryptococcal components tested or heat-killed cryptococci in stimulating TNF-alpha production (P < 0.05). GXM, in contrast, was significantly less potent in this activity than either GalXM or MP (P < 0.05). As little as 0.5 microg of MP per ml was sufficient to produce moderate but significant elevations of TNF-alpha release. Maximal MP-induced TNF-alpha levels were similar to those induced by Salmonella enteritidis lipopolysaccharide, our positive control. Further experiments using isolated leukocytes suggested that monocytes were the cell population mainly responsible for TNF-alpha production, although the participation of other cell types could not be excluded. The presence of complement-sufficient plasma was a necessary requirement for TNF-alpha induction by GXM, GalXM, and low doses of MP. High MP concentrations (100 microg/ml) were also capable of stimulating TNF-alpha production in the absence of plasma. These data indicate that soluble products released by C. neoformans are capable of inducing TNF-alpha secretion in human leukocytes. This may be clinically relevant, since high concentrations of such products are frequently found in the body fluids of AIDS patients infected with C. neoformans. PMID:8945566

  7. Genetics of C-reactive protein and complement factor H have an epistatic effect on carotid artery compliance: The Cardiovascular Risk in Young Finns Study

    PubMed Central

    Jylhävä, J; Eklund, C; Pessi, T; Raitakari, O T; Juonala, M; Kähönen, M; Viikari, J S A; Lehtimäki, T; Hurme, M

    2009-01-01

    Atherosclerosis is characterized by a prominent inflammatory component and C-reactive protein (CRP) has been implicated to modulate the complement activity in atherosclerotic arteries via complement factor H (CFH) binding. In this study, we examined whether the gene-gene interactions between CRP haplotypes and CFH Tyr402His functional polymorphism exerted an effect on early atherosclerosis. Single nucleotide polymorphisms (SNPs) in CFH (Tyr402His) and CRP (−717A > G, −286C > T > A, +1059G > C, +1444C > T and +1846G > A) were genotyped in the participants of the Cardiovascular Risk in Young Finns Study (n = 1698, aged 24–39 years). The CRP SNPs were further constructed into haplotypes and their interactive effects with the CFH Tyr402His polymorphism on the early atherogenic vascular changes [i.e. carotid artery compliance (CAC) and intima-media thickness (IMT)] were examined. After risk factor adjustment, a significant gene-gene interaction (P = 0·007) on CAC was observed between CRP haplotype ATGTG and CFH Tyr402His polymorphism in males. Furthermore, logistic regression analysis verified the risk-modifying interactive effect on CAC between these loci (OR 3·70, 95% CI 1·37–10·02, P = 0·010). No effects on CAC were observed in females and no effects on IMT were detected in either sex. We conclude that the combined presence of CRP haplotype ATGTG and CFH 402His allele may be disadvantageous to carotid artery elasticity in males. PMID:19076828

  8. An ELISA assay with two monoclonal antibodies allows the estimation of free factor H and identifies patients with acquired deficiency of this complement regulator.

    PubMed

    Nozal, Pilar; Garrido, Sofía; Alba-Domínguez, María; Espinosa, Laura; Peña, Antonia; Córdoba, Santiago Rodríguez de; Sánchez-Corral, Pilar; López-Trascasa, Margarita

    2014-04-01

    Complement factor H (FH) serum levels can be affected by the presence of immune complexes of FH with autoantibodies like in autoimmune forms of atypical haemolytic uraemic syndrome (aHUS) or with C3b in homozygous factor I (FI) deficiency. These complexes reduce the amount of free functional circulating FH. In this study we aimed to determine whether FH levels measurement is disturbed in some pathological conditions and to establish a method for quantifying free and total FH in serum. For that purpose, FH levels were measured in serum samples from aHUS patients having anti-FH autoantibodies or mutations in FH gene, in patients with homozygous FI deficiency, and in healthy controls. Two anti-FH monoclonal antibodies, OX24 and A229, recognizing different functional regions in FH, were used as capture antibodies in an ELISA assay. In the control group and in the group of patients with FH mutations, the FH levels obtained with the two monoclonal antibodies were similar. In patients with anti-FH autoantibodies or with homozygous FI deficiency, however, FH levels measured with both antibodies were significantly different. As these patients had complexes of FH with autoantibodies or C3b, we interpreted that OX24 was detecting total FH and A229 was recognising free FH. Therefore, quantification of FH in plasma using these two monoclonal antibodies provides not only total FH level but also gives an estimation of how much FH circulates free and is thus available to properly control complement activation.

  9. N-Terminal Prodomain of Pfs230 Synthesized Using a Cell-Free System Is Sufficient To Induce Complement-Dependent Malaria Transmission-Blocking Activity▿

    PubMed Central

    Tachibana, Mayumi; Wu, Yimin; Iriko, Hideyuki; Muratova, Olga; MacDonald, Nicholas J.; Sattabongkot, Jetsumon; Takeo, Satoru; Otsuki, Hitoshi; Torii, Motomi; Tsuboi, Takafumi

    2011-01-01

    The aim of a malaria transmission-blocking vaccine is to block the development of malaria parasites in the mosquito and thus prevent subsequent infection of the human host. Previous studies have demonstrated that the gametocyte/gamete surface protein Pfs230 can induce transmission-blocking immunity and have evaluated Escherichia coli-produced Pfs230 as a transmission-blocking vaccine candidate. In this study, we used the wheat germ cell-free expression system to produce N-terminal fragments of Pfs230 and evaluated the transmission-blocking activity of antisera raised against the recombinant Pfs230 protein. The rabbit antisera reacted to the surface of cultured gametocytes and gametes of the Plasmodium falciparum NF54 line, recognized the 360-kDa form of parasite-produced Pfs230 by Western blot assay, and reduced the infectivity of NF54 parasites to Anopheles stefensi mosquitoes in the presence of complement in a standard membrane feeding assay. Thus, our data demonstrate that the N-terminal pro domain of Pfs230 is sufficient to induce complement-dependent transmission-blocking activity against P. falciparum. PMID:21715579

  10. Drug-mediated sensitization to TRAIL-induced apoptosis in caspase-8-complemented neuroblastoma cells proceeds via activation of intrinsic and extrinsic pathways and caspase-dependent cleavage of XIAP, Bcl-xL and RIP.

    PubMed

    Mühlethaler-Mottet, Annick; Bourloud, Katia Balmas; Auderset, Katya; Joseph, Jean-Marc; Gross, Nicole

    2004-07-15

    Neuroblastoma (NB) is a childhood neoplasm which heterogeneous behavior can be explained by differential regulation of apoptosis. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively induces rapid apoptosis in most tumor cells and thus represents a promising anticancer agent. We have reported silencing of caspase-8 expression in highly malignant NB cells as a possible mechanism of resistance to TRAIL-induced apoptosis. To explore the particular contribution of caspase-8 in such resistance, retroviral-mediated stable caspase-8 expression was induced in the IGR-N91 cells. As a result, sensitivity to TRAIL was fully restored in the caspase-8-complemented cells. TRAIL-induced cell death could be further enhanced by cotreatment of IGR-N91-C8 and SH-EP cells with cycloheximide or subtoxic concentrations of chemotherapeutic drugs in a caspase-dependent manner. Sensitization to TRAIL involved enhanced death receptor DR5 expression, activation of Bid and the complete caspases cascade. Interestingly, combined treatments also enhanced the cleavage-mediated inactivation of antiapoptotic molecules, XIAP, Bcl-x(L) and RIP. Our results show that restoration of active caspase-8 expression in a caspase-8-deficient NB cell line is necessary and sufficient to fully restore TRAIL sensitivity. Moreover, the synergistic effect of drugs and TRAIL results from activation of the caspase cascade via a mitochondrial pathway-mediated amplification loop and from the inactivation of apoptosis inhibitors. PMID:15094781

  11. Synthesis of complement factor B by the human monocyte U-937 cell line. Augmentation by immunostimulatory agents

    SciTech Connect

    Minta, J.O.

    1988-09-01

    The human pre-monocytic cell line U-937 was shown to synthesize and to secrete increasing amounts of factor B in short term cultures in serum-free medium containing BSA. The kinetics of factor B production were higher on day 2 than on days 1 and 3. The production of factor B was reversibly inhibited by cycloheximide, indicating de novo synthesis. Metabolic labeling with (/sup 35/S)-methionine and SDS-PAGE analysis revealed that both intracellular and secreted factor B were single-chain proteins with similar m.w. (90,000), which co-migrated with purified plasma factor B. Incubation of U-937 cells with the immunostimulants PMA, LPS, IFN-gamma, and IL-1 resulted in a dose-dependent augmentation of factor B production. A 24-h exposure to IL-1 was shown to be required for maximal stimulation. A combination of suboptimal doses of LPS and IFN-gamma was shown to exert a synergistic effect on factor B production. The U-937 cell line is thus a valuable model for the study of the regulation of the factor B gene expression.

  12. Decoration of Histophilus somni lipooligosaccharide with N-acetyl-5-neuraminic acid enhances bacterial binding of complement factor H and resistance to killing by serum and polymorphonuclear leukocytes.

    PubMed

    Inzana, Thomas J; Balyan, Rajiv; Howard, Michael D

    2012-12-28

    The incorporation of N-acetyl-5-neuraminic acid (Neu5Ac), or sialic acid, onto surface components of some bacterial species may enhance their virulence. We have previously shown that Neu5Ac can be incorporated onto the lipooligosaccharide (LOS) of the bovine pathogen Histophilus somni, resulting in diminished antibody binding and enhanced serum resistance (Inzana et al., 2002. Infect. Immun. 70, 4870). In the present study, we assessed the effect of sialylation of H. somni LOS on the interaction with bovine innate host defenses. Incubation of non-sialylated H. somni with pre-colostral calf serum (PCS) resulted in dose-dependent, complement-mediated killing of the bacteria by the alternative pathway. However, sialylated H. somni was significantly more resistant to killing at any of the concentrations of PCS used. Sialylated H. somni LOS activated and consumed less complement than non-sialylated LOS, as determined by reduction in hemolysis of opsonized red blood cells, and by Western blotting of C(3) activation products. Sialylated H. somni bound more factor H and iC(3)b and less C(3) than non-sialylated bacteria, as determined by enzyme-linked immunosorbent assay, supporting the deficiencies observed in complement activation and consumption by sialylated LOS. Sialylation of H. somni LOS inhibited both polymorphonuclear leukocyte phagocytosis of (3)H-thymidine-labeled bacteria and intracellular killing of the bacteria, compared to non-sialylated bacteria. Furthermore, sialylated H. somni bound less non-specific antibodies in normal bovine sera than non-sialylated bacteria. Therefore, sialylation of H. somni LOS had profound effects on resistance of the bacteria to innate bovine host defenses, which should be taken into consideration during in vitro studies of H. somni.

  13. Kinetics of acute inflammation induced by Escherichia coli in rabbits. II. The effect of hyperimmunization, complement depletion, and depletion of leukocytes.

    PubMed Central

    Kopaniak, M. M.; Movat, H. Z.

    1983-01-01

    The inflammatory response to Escherichia coli was quantitated in the skin of normal rabbits and the kinetics established as described previously. Hyperemia, measured with radiolabeled microspheres; vascular permeability, estimated with 125 I-albumin; and leukocyte infiltration, quantitated with 51Cr-labeled autologous leukocytes, reached maximal values 3 hours after the injection of bacteria and subsided almost completely by 6 hours. Hemorrhage, measured with homologous 59Fe-erythrocytes, continued to increase between 1 and 6 hours after injection and then reached plateau levels. The lesions were studied up to 8 hours, since in the previous study no changes were observed beyond that time. In the study described in this paper, the host mediation systems were manipulated in various groups of rabbits in order to elucidate the mechanisms underlying the development of the inflammatory reaction. One group of animals was hyperimmunized with the E coli organisms, another was partially depleted of hemolytic complement with cobra venom factor, and yet another was rendered leukopenic with nitrogen mustard. In hyperimmunized animals hyperemia in the dermal lesions induced by the microorganisms was significantly more intense than in normal rabbits. Vascular permeability increase occurred earlier in hyperimmunized rabbits and at 1 hour was significantly greater than in normals. Decomplemented rabbits had significantly less vascular permeability than normal animals, whereas in leukopenic rabbits no increase in vascular permeability could be elicited. Leukocyte accumulation was increased over the normal animals in the lesions of hyperimmunized rabbits. Hemorrhage was significantly decreased in leukopenic rabbits. Histologic examination of the lesions revealed that whereas in normal animals the infiltrating neutrophils ingested most of the bacteria and formed definite abscesses by 6-8 hours, these abscesses were absent in leukopenic animals, and free-lying bacteria were

  14. Inhibition of factor XII in septic baboons attenuates the activation of complement and fibrinolytic systems and reduces the release of interleukin-6 and neutrophil elastase.

    PubMed

    Jansen, P M; Pixley, R A; Brouwer, M; de Jong, I W; Chang, A C; Hack, C E; Taylor, F B; Colman, R W

    1996-03-15

    In previous studies, we have shown that administration of monoclonal antibody (MoAb) C6B7 against human factor XII to baboons challenged with a lethal dose of Escherichia coli abrogates activation of the contact system and modulates secondary hypotension. To evaluate the contribution of activated contact proteases to the appearance of other inflammatory mediators in this experimental model of sepsis, we studied the effect of administration of MoAb C6B7 on activation of complement and fibrinolytic cascades, stimulation of neutrophil degranulation, and release of the proinflammatory cytokines, tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6). Activation of the complement system, as reflected by circulating C3b/c and C4b/c levels, was significantly reduced in five animals that had received MoAb C6B7 before a lethal dose of E coli as compared with five control animals that had been given a lethal challenge only. Inhibition of contact activation also modulated the fibrinolytic response, since the release of tissue-type plasminogen activator (t-PA) and the appearance of plasmin-alpha2-antiplasmin (PAP) complexes into the circulation was significantly attenuated upon pretreatment with anti-factor XII MoAb. In contrast, plasma levels of plasminogen activator inhibitor (PAI) were modestly enhanced in the treatment group. Degranulation of neutrophils, as assessed by circulating elastase-alpha1-protease inhibitor complexes, and release of IL-6 but not of TNF-alpha was decreased in anti-factor XII-treated animals. Observed differences in the inflammatory response between treatment and control groups were not likely due to different challenges, since the number of E coli that had been infused, as well as circulating levels of endotoxin after the challenge, were similar for both groups. These data suggest that activation of the contact system modulates directly or indirectly various mediator systems involved in the inflammatory response during severe sepsis in

  15. Accelerated Tumor Growth Mediated by Sub-lytic Levels of Antibody-Induced Complement Activation is Associated with Activation of the PI3K/AKT Survival Pathway

    PubMed Central

    Wu, Xiaohong; Ragupathi, Govind; Panageas, Katherine; Hong, Feng; Livingston, Philip O.

    2013-01-01

    Purpose We addressed the possibility that low levels of tumor cell bound antibodies targeting gangliosides might accelerate tumor growth. Experimental Design To test this hypothesis, we treated mice with a range of mAb doses against GM2, GD2, GD3 and CD20 after challenge with tumors expressing these antigens and tested the activity of the same mAbs in-vitro. We also explored the mechanisms behind the complement-mediated tumor growth acceleration that we observed and an approach to overcome it. Results Serologically detectable levels of IgM-mAb against GM2 are able to delay or prevent tumor growth of high GM2-expressing cell lines both in-vitro and in a SCID mouse model, while very low levels of this mAb resulted in slight but consistent acceleration of tumor growth in both settings. Surprisingly, this is not restricted to IgM antibodies targeting GM2 but consistent against IgG-mAb targeting GD3 as well. These findings were mirrored by in-vitro studies with antibodies against these antigens as well as GD2 and CD20 (with Rituxan), and shown to be complement-dependent in all cases. Complement-mediated accelerated growth of cultured tumor cell lines initiated by low mAb levels was associated with activation of the PI3K/AKT survival pathway and significantly elevated levels of both p-AKT and p-PRAS40. This complement-mediated PI3K-activation and accelerated tumor growth in-vitro and in-vivo are eliminated by PI3K-inhibitors NVP-BEZ235 and Wortmannin. These PI3K-inhibitors also significantly increased efficacy of high doses of these 4 mAbs. Conclusion Our findings suggest that manipulation of the PI3K/AKT pathway and its signaling network can significantly increase the potency of passively administered mAbs and vaccine-induced-antibodies targeting a variety of tumor-cell-surface-antigens. PMID:23833306

  16. Alternative complement pathway activation is essential for inflammation and joint destruction in the passive transfer model of collagen-induced arthritis.

    PubMed

    Banda, Nirmal K; Thurman, Joshua M; Kraus, Damian; Wood, Allyson; Carroll, Michael C; Arend, William P; Holers, V Michael

    2006-08-01

    Activation of each complement initiation pathway (classical, alternative, and lectin) can lead to the generation of bioactive fragments with resulting inflammation in target organs. The objective of the current study was to determine the role of specific complement activation pathways in the pathogenesis of experimental anti-type II collagen mAb-passive transfer arthritis. C57BL/6 mice were used that were genetically deficient in either the alternative pathway protein factor B (Bf(-/-)) or in the classical pathway component C4 (C4(-/-)). Clinical disease activity was markedly decreased in Bf(-/-) compared with wild-type (WT) mice (0.5 +/- 0.22 (n = 6) in Bf(-/-) vs 8.83 +/- 0.41 (n = 6) in WT mice (p < 0.0001)). Disease activity scores were not different between C4(-/-) and WT mice. Analyses of joints showed that C3 deposition, inflammation, pannus, cartilage, and bone damage scores were all significantly less in Bf(-/-) as compared with WT mice. There were significant decreases in mRNA levels of C3, C4, CR2, CR3, C3aR, and C5aR in the knees of Bf(-/-) as compared with C4(-/-) and WT mice with arthritis; mRNA levels for complement regulatory proteins did not differ between the three strains. These results indicate that the alternative pathway is absolutely required for the induction of arthritis following injection of anti-collagen Abs. The mechanisms by which these target organ-specific mAbs bypass the requirements for engagement of the classical pathway remain to be defined but do not appear to involve a lack of alternative pathway regulatory proteins. PMID:16849503

  17. Surface antigen expression and complement susceptibility of differentiated neuroblastoma clones.

    PubMed

    Chen, S; Caragine, T; Cheung, N K; Tomlinson, S

    2000-03-01

    Human neuroblastoma cell lines typically consist of heterogenous subpopulations of cells that are morphologically and biochemically distinct. The cell types are characterized as neuroblastic (N-type), substrate-adherent Schwann-like (S-type), or intermediate (I). These cell types can undergo spontaneous or induced transdifferentiation in vitro. We investigated the complement sensitivity of different neuroblastoma cell lines and of matched sets of cloned N- and S-type neuroblastoma cell lines. Human neuroblastoma cell lines that consisted predominantly of a neuroblastic phenotype were shown to be significantly more susceptible to human complement-mediated lysis than cell lines of other cancer types. Complement sensitivity of neuroblastoma cell lines was correlated with low levels of CD59, decay-accelerating factor, and membrane cofactor protein expression. We found that cloned S-type neuroblastoma cells were much more resistant to complement-mediated lysis than cloned N-type cells. The increased complement resistance of S-type cells was shown to be due to increased expression of membrane-bound complement inhibitors. CD59 was the single most important protein in providing S-type cells with protection from complement lysis. S-type cells were also found to express lower levels of GD2, a target antigen for a complement activating monoclonal antibody currently in clinical trials for neuroblastoma immunotherapy. The ability of S-type cells to evade complement, and the ability of S-type cells to differentiate into the more tumorigenic N-type cells, may represent a mechanism of tumor survival and regrowth, a phenomenon often observed with this cancer.

  18. IL-4 and IL-13 induce protection from complement and melittin in endothelial cells despite initial loss of cytoplasmic proteins: membrane resealing impairs quantifying cytotoxicity with the lactate dehydrogenase permeability assay.

    PubMed

    Benson, Barbara A; Vercellotti, Gregory M; Dalmasso, Agustin P

    2015-01-01

    Endothelial cell activation and injury by the terminal pathway of complement is important in various pathobiological processes, including xenograft rejection. Protection against injury by human complement can be induced in porcine endothelial cells (ECs) with IL-4 and IL-13 through metabolic activation. However, despite this resistance, the complement-treated ECs were found to lose membrane permeability control assessed with the small molecule calcein. Therefore, to define the apparent discrepancy of permeability changes vis-à-vis the protection from killing, we now investigated whether IL-4 and IL-13 influence the release of the large cytoplasmic protein lactate dehydrogenase (LDH) in ECs incubated with complement or the pore-forming protein melittin. Primary cultures of ECs were pre-treated with IL-4 or IL-13 and then incubated with human serum as source of antibody and complement or melittin. Cell death was assessed using neutral red. Membrane permeability was quantitated measuring LDH release. We found that IL-4-/IL-13-induced protection of ECs from killing by complement or melittin despite loss of LDH in amounts similar to control ECs. However, the cytokine-treated ECs that were protected from killing rapidly regained effective control of membrane permeability. Moreover, the viability of the protected ECs was maintained for at least 2 days. We conclude that the protection induced by IL-4/IL-13 in ECs against lethal attack by complement or melittin is effective and durable despite severe initial impairment of membrane permeability. The metabolic changes responsible for protection allow the cells to repair the membrane injury caused by complement or melittin.

  19. Viral complement regulatory proteins.

    PubMed

    Rosengard, A M; Ahearn, J M

    1999-05-01

    The inactivation of complement provides cells and tissues critical protection from complement-mediated attack and decreases the associated recruitment of other inflammatory mediators. In an attempt to evade the host immune response, viruses have evolved two mechanisms to acquire complement regulatory proteins. They can directly seize the host cell complement regulators onto their outer envelope and/or they can produce their own proteins which are either secreted into the neighboring intercellular space or expressed as membrane-bound proteins on the infected host cell. The following review will concentrate on the viral homologues of the mammalian complement regulatory proteins, specifically those containing complement control protein (CCP) repeats. PMID:10408371

  20. Gene expression profiling of Gram-negative bacteria-induced inflammation in human whole blood: The role of complement and CD14-mediated innate immune response.

    PubMed

    Lau, Corinna; Olstad, Ole Kristoffer; Holden, Marit; Nygård, Ståle; Fure, Hilde; Lappegård, Knut Tore; Brekke, Ole-Lars; Espevik, Terje; Hovig, Eivind; Mollnes, Tom Eirik

    2015-09-01

    Non-sterile pathogen-induced sepsis and sterile inflammation like in trauma or ischemia-reperfusion injury may both coincide with the life threatening systemic inflammatory response syndrome and multi-organ failure. Consequently, there is an urgent need for specific biomarkers in order to distinguish sepsis from sterile conditions. The overall aim of this study was to uncover putative sepsis biomarkers and biomarker pathways, as well as to test the efficacy of combined inhibition of innate immunity key players complement and Toll-like receptor co-receptor CD14 as a possible therapeutic regimen for sepsis. We performed whole blood gene expression analyses using microarray in order to profile Gram-negative bacteria-induced inflammatory responses in an ex vivo human whole blood model. The experiments were performed in the presence or absence of inhibitors of complement proteins (C3 and CD88 (C5a receptor 1)) and CD14, alone or in combination. In addition, we used blood from a C5-deficient donor. Anti-coagulated whole blood was challenged with heat-inactivated Escherichia coli for 2 h, total RNA was isolated and microarray analyses were performed on the Affymetrix GeneChip Gene 1.0 ST Array platform. The initial experiments were performed in duplicates using blood from two healthy donors. C5-deficiency is very rare, and only one donor could be recruited. In order to increase statistical power, a technical replicate of the C5-deficient samples was run. Subsequently, log2-transformed intensities were processed by robust multichip analysis and filtered using a threshold of four. In total, 73 microarray chips were run and analyzed. The normalized and filtered raw data have been deposited in NCBI's Gene Expression Omnibus (GEO) and are accessible with GEO Series accession number GSE55537. Linear models for microarray data were applied to estimate fold changes between data sets and the respective multiple testing adjusted p-values (FDR q-values). The interpretation of the

  1. Role of complement in experiment silicosis

    SciTech Connect

    Callis, A.H.; Sohnle, P.G.; Mandel, G.S.; Mandel, N.S.

    1986-08-01

    The role of the complement system in the pathogenesis of crystal-induced pulmonary inflammation and fibrosis was evaluated using a mouse model of silicosis and congenitally complement-deficient mice. Mice lacking the fifth component of complement (B10.D2/o) were compared to C5-sufficient animals (B10.D2/n) for pulmonary changes following intratracheal instillation of silica crystals. Complement-deficient mice demonstrated a significant reduction compared to complement-sufficient mice in both cell number and protein content of lung lavage fluid throughout the 12 weeks following silica exposure. Lung hydroxyproline content (indicative of collagen deposition) was equivalent for both strains and significantly higher than controls at all times points following silica instillation. Moreover, studies in vitro have shown that silica crystals are capable of activating complement via the alternative pathway. These studies indicate that the complement system may be responsible for some of the pulmonary inflammation, but not fibrosis elicited by silica exposure.

  2. Complement system in zebrafish.

    PubMed

    Zhang, Shicui; Cui, Pengfei

    2014-09-01

    Zebrafish is recently emerging as a model species for the study of immunology and human diseases. Complement system is the humoral backbone of the innate immune defense, and our knowledge as such in zebrafish has dramatically increased in the recent years. This review summarizes the current research progress of zebrafish complement system. The global searching for complement components in genome database, together with published data, has unveiled the existence of all the orthologues of mammalian complement components identified thus far, including the complement regulatory proteins and complement receptors, in zebrafish. Interestingly, zebrafish complement components also display some distinctive features, such as prominent levels of extrahepatic expression and isotypic diversity of the complement components. Future studies should focus on the following issues that would be of special importance for understanding the physiological role of complement components in zebrafish: conclusive identification of complement genes, especially those with isotypic diversity; analysis and elucidation of function and mechanism of complement components; modulation of innate and adaptive immune response by complement system; and unconventional roles of complement-triggered pathways.

  3. Complement in Lupus Nephritis: New Perspectives

    PubMed Central

    Bao, Lihua; Cunningham, Patrick N.; Quigg, Richard J.

    2015-01-01

    Background Systemic lupus erythematosus (SLE) is an autoimmune disorder caused by loss of tolerance to self-antigens, the production of autoantibodies and deposition of complement-fixing immune complexes (ICs) in injured tissues. SLE is characterized by a wide range of clinical manifestations and targeted organs, with lupus nephritis being one of the most serious complications. The complement system consists of three pathways and is tightly controlled by a set of regulatory proteins to prevent injudicious complement activation on host tissue. The involvement of the complement system in the pathogenesis of SLE is well accepted; yet, its exact role is still not clear. Summary Complement plays dual roles in the pathogenesis of SLE. On the one hand, the complement system appears to have protective features in that hereditary homozygous deficiencies of classical pathway components, such as C1q and C4, are associated with an increased risk for SLE. On the other hand, IC-mediated activation of complement in affected tissues is clearly evident in both experimental and human SLE along with pathological features that are logical consequences of complement activation. Studies in genetically altered mice have shown that lack of complement inhibitors, such as complement factor H (CFH) or decay-accelerating factor (DAF) accelerates the development of experimental lupus nephritis, while treatment with recombinant protein inhibitors, such as Crry-Ig, CR2-Crry, CR2-DAF and CR2-CFH, ameliorates the disease development. Complement-targeted drugs, including soluble complement receptor 1 (TP10), C1 esterase inhibitor and a monoclonal anti-C5 antibody (eculizumab), have been shown to inhibit complement safely, and are now being investigated in a variety of clinical conditions. Key Messages SLE is an autoimmune disorder which targets multiple systems. Complement is centrally involved and plays dual roles in the pathogenesis of SLE. Studies from experimental lupus models and clinical

  4. Hypoxia-Inducible Factor in Thyroid Carcinoma

    PubMed Central

    Burrows, Natalie; Babur, Muhammad; Resch, Julia; Williams, Kaye J.; Brabant, Georg

    2011-01-01

    Intratumoural hypoxia (low oxygen tension) is associated with aggressive disease and poor prognosis. Hypoxia-inducible factor-1 is a transcription factor activated by hypoxia that regulates the expression of genes that promote tumour cell survival, progression, metastasis, and resistance to chemo/radiotherapy. In addition to hypoxia, HIF-1 can be activated by growth factor-signalling pathways such as the mitogen-activated protein kinases- (MAPK-) and phosphatidylinositol-3-OH kinases- (PI3K-) signalling cascades. Mutations in these pathways are common in thyroid carcinoma and lead to enhanced HIF-1 expression and activity. Here, we summarise current data that highlights the potential role of both hypoxia and MAPK/PI3K-induced HIF-1 signalling in thyroid carcinoma progression, metastatic characteristics, and the potential role of HIF-1 in thyroid carcinoma response to radiotherapy. Direct or indirect targeting of HIF-1 using an MAPK or PI3K inhibitor in combination with radiotherapy may be a new potential therapeutic target to improve the therapeutic response of thyroid carcinoma to radiotherapy and reduce metastatic burden. PMID:21765994

  5. The novel complement inhibitor human CUB and Sushi multiple domains 1 (CSMD1) protein promotes factor I-mediated degradation of C4b and C3b and inhibits the membrane attack complex assembly.

    PubMed

    Escudero-Esparza, Astrid; Kalchishkova, Nikolina; Kurbasic, Emila; Jiang, Wen G; Blom, Anna M

    2013-12-01

    CUB and Sushi multiple domains 1 (CSMD1) is a transmembrane protein containing 15 consecutive complement control protein (CCP) domains, which are characteristic for complement inhibitors. We expressed a membrane-bound fragment of human CSMD1 composed of the 15 C-terminal CCP domains and demonstrated that it inhibits deposition of C3b by the classical pathway on the surface of Chinese hamster ovary cells by 70% at 6% serum and of C9 (component of membrane attack complex) by 90% at 1.25% serum. Furthermore, this fragment of CSMD1 served as a cofactor to factor I-mediated degradation of C3b. In all functional assays performed, well-characterized complement inhibitors were used as positive controls, whereas Coxsackie adenovirus receptor, a protein with no effect on complement, was a negative control. Moreover, attenuation of expression in human T47 breast cancer cells that express endogenous CSMD1 significantly increased C3b deposition on these cells by 45% at 8% serum compared with that for the controls. Furthermore, by expressing a soluble 17-21 CCP fragment of CSMD1, we found that CSMD1 inhibits complement by promoting factor I-mediated C4b/C3b degradation and inhibition of MAC assembly at the level of C7. Our results revealed a novel complement inhibitor for the classical and lectin pathways.

  6. The novel complement inhibitor human CUB and Sushi multiple domains 1 (CSMD1) protein promotes factor I-mediated degradation of C4b and C3b and inhibits the membrane attack complex assembly.

    PubMed

    Escudero-Esparza, Astrid; Kalchishkova, Nikolina; Kurbasic, Emila; Jiang, Wen G; Blom, Anna M

    2013-12-01

    CUB and Sushi multiple domains 1 (CSMD1) is a transmembrane protein containing 15 consecutive complement control protein (CCP) domains, which are characteristic for complement inhibitors. We expressed a membrane-bound fragment of human CSMD1 composed of the 15 C-terminal CCP domains and demonstrated that it inhibits deposition of C3b by the classical pathway on the surface of Chinese hamster ovary cells by 70% at 6% serum and of C9 (component of membrane attack complex) by 90% at 1.25% serum. Furthermore, this fragment of CSMD1 served as a cofactor to factor I-mediated degradation of C3b. In all functional assays performed, well-characterized complement inhibitors were used as positive controls, whereas Coxsackie adenovirus receptor, a protein with no effect on complement, was a negative control. Moreover, attenuation of expression in human T47 breast cancer cells that express endogenous CSMD1 significantly increased C3b deposition on these cells by 45% at 8% serum compared with that for the controls. Furthermore, by expressing a soluble 17-21 CCP fragment of CSMD1, we found that CSMD1 inhibits complement by promoting factor I-mediated C4b/C3b degradation and inhibition of MAC assembly at the level of C7. Our results revealed a novel complement inhibitor for the classical and lectin pathways. PMID:23964079

  7. Complementation of growth factor receptor-dependent mitogenic signaling by a truncated type I phosphatidylinositol 4-phosphate 5-kinase.

    PubMed

    Davis, J N; Rock, C O; Cheng, M; Watson, J B; Ashmun, R A; Kirk, H; Kay, R J; Roussel, M F

    1997-12-01

    Substitution of phenylalanine for tyrosine at codon 809 (Y809F) of the human colony-stimulating factor 1 (CSF-1) receptor (CSF-1R) impairs ligand-stimulated tyrosine kinase activity, prevents induction of c-MYC and cyclin D1 genes, and blocks CSF-1-dependent progression through the G1 phase of the cell cycle. We devised an unbiased genetic screen to isolate genes that restore the ability of CSF-1 to stimulate growth in cells that express mutant CSF-1R (Y809F). This screen led us to identify a truncated form of the murine type Ibeta phosphatidylinositol 4-phosphate 5-kinase (mPIP5K-Ibeta). This truncated protein lacks residues 1 to 238 of mPIP5K-Ibeta and is catalytically inactive. When we transfected cells expressing CSF-1R (Y809F) with mPIP5K-Ibeta (delta1-238), CSF-1-dependent induction of c-MYC and cyclin D1 was restored and ligand-dependent cell proliferation was sustained. CSF-1 normally triggers the rapid disappearance of CSF-1R (Y809F) from the cell surface; however, transfection of cells with mPIP5K-Ibeta (delta1-238) stabilized CSF-1R (Y809F) expression on the cell surface, resulting in elevated levels of ligand-activated CSF-1R (Y809F). These results suggest a role for PIP5K-Ibeta in receptor endocytosis and that the truncated enzyme compensated for a mitogenically defective CSF-1R by interfering with this process.

  8. Molecular intercommunication between the complement and coagulation systems.

    PubMed

    Amara, Umme; Flierl, Michael A; Rittirsch, Daniel; Klos, Andreas; Chen, Hui; Acker, Barbara; Brückner, Uwe B; Nilsson, Bo; Gebhard, Florian; Lambris, John D; Huber-Lang, Markus

    2010-11-01

    The complement system as well as the coagulation system has fundamental clinical implications in the context of life-threatening tissue injury and inflammation. Associations between both cascades have been proposed, but the precise molecular mechanisms remain unknown. The current study reports multiple links for various factors of the coagulation and fibrinolysis cascades with the central complement components C3 and C5 in vitro and ex vivo. Thrombin, human coagulation factors (F) XIa, Xa, and IXa, and plasmin were all found to effectively cleave C3 and C5. Mass spectrometric analyses identified the cleavage products as C3a and C5a, displaying identical molecular weights as the native anaphylatoxins C3a and C5a. Cleavage products also exhibited robust chemoattraction of human mast cells and neutrophils, respectively. Enzymatic activity for C3 cleavage by the investigated clotting and fibrinolysis factors is defined in the following order: FXa > plasmin > thrombin > FIXa > FXIa > control. Furthermore, FXa-induced cleavage of C3 was significantly suppressed in the presence of the selective FXa inhibitors fondaparinux and enoxaparin in a concentration-dependent manner. Addition of FXa to human serum or plasma activated complement ex vivo, represented by the generation of C3a, C5a, and the terminal complement complex, and decreased complement hemolytic serum activity that defines exact serum concentration that results in complement-mediated lysis of 50% of sensitized sheep erythrocytes. Furthermore, in plasma from patients with multiple injuries (n = 12), a very early appearance and correlation of coagulation (thrombin-antithrombin complexes) and the complement activation product C5a was found. The present data suggest that coagulation/fibrinolysis proteases may act as natural C3 and C5 convertases, generating biologically active anaphylatoxins, linking both cascades via multiple direct interactions in terms of a complex serine protease system.

  9. Biologic effects of microwave exposure. II. Studies on the mechanisms controlling susceptibility to microwave-induced increases in complement receptor-positive spleen cells

    SciTech Connect

    Schlagel, C.J.; Sulek, K.; Ho, H.S.; Leach, W.M.; Ahmed, A.; Woody, J.N.

    1980-01-01

    In attempting to evaluate the mechanisms responsible for susceptibility to the inductive increase in splenic complement receptor-positive (CR+) cells following exposure to 2450-MHz microwaves, it was found that sensitivity to microwave-induced CR+ cell increases was under genetic control. In particular, evidence was accumulated suggesting that regulation was under the control of a gene or genes closely associated with but outside of the mouse major histocompatibility complex (H-2). All responsive strains of mice tested were of the H-2k haplotype, while mice of the H-2a, H-2b, H-2d and H-1i5 haplotypes were refractory to the microwave-induced increases in CR+ cells. By utilizing certain H-2k strains of mice that were genetically unable to respond to endotoxin, we were able to show that these strains of mice responded to microwaves, but not to endotoxin, by increasing CR+ cells. Microwave-induced increases in CR+ cells were not mimicked by the intraperitoneal injection of hydrocortisone. Athymic mice responded to microwave exposure, indicating that this event was not regulated by the T-cell population. Mice less than eight weeks old were found not to be susceptible to exposure to 2450-MHz microwaves. These studies indicate that microwaves do induce changes in the population of cells with specific cell-surface receptors, that susceptibility to these changes is under genetic control, and that it is unlikely that endotoxin, corticosteroids, or regulatory T cells play a significant role in the mechanisms regulating these increases.

  10. Identification of a Gene Product Induced by Hard-Surface Contact of Colletotrichum gloeosporioides Conidia as a Ubiquitin-Conjugating Enzyme by Yeast Complementation

    PubMed Central

    Liu, Zhi-Mei; Kolattukudy, Pappachan E.

    1998-01-01

    The germinating conidia of many phytopathogenic fungi on hosts must differentiate into an infection structure called the appressorium in order to penetrate their hosts. Chemical signals, such as the host’s surface wax or fruit ripening hormone, ethylene, trigger germination and appressorium formation of the avocado pathogen Colletotrichum gloeosporioides only after the conidia are in contact with a hard surface. What role this contact plays is unknown. Here, we describe isolation of genes expressed during the early stage of hard-surface treatment by a differential-display method and report characterization of one of these cloned genes, chip1 (Colletotrichum hard-surface induced protein 1 gene), which encodes a ubiquitin-conjugating enzyme. RNA blots clearly showed that it is induced by hard-surface contact and that ethylene treatment enhanced this induction. The predicted open reading frame (ubc1Cg) would encode a 16.2-kDa ubiquitin-conjugating enzyme, which shows 82% identity to the Saccharomyces cerevisiae UBC4-UBC5 E2 enzyme, comprising a major part of total ubiquitin-conjugating activity in stressed yeast cells. UBC1Cg can complement the proteolysis deficiency of the S. cerevisiae ubc4 ubc5 mutant, indicating that ubiquitin-dependent protein degradation is involved in conidial germination and appressorial differentiation. PMID:9658002

  11. Complement and autoimmunity.

    PubMed

    Ballanti, Eleonora; Perricone, Carlo; Greco, Elisabetta; Ballanti, Marta; Di Muzio, Gioia; Chimenti, Maria Sole; Perricone, Roberto

    2013-07-01

    The complement system is a component of the innate immune system. Its main function was initially believed to be limited to the recognition and elimination of pathogens through direct killing or stimulation of phagocytosis. However, in recent years, the immunoregulatory functions of the complement system were demonstrated and it was determined that the complement proteins play an important role in modulating adaptive immunity and in bridging innate and adaptive responses. When the delicate mechanisms that regulate this sophisticated enzymatic system are unbalanced, the complement system may cause damage, mediating tissue inflammation. Dysregulation of the complement system has been involved in the pathogenesis and clinical manifestations of several autoimmune diseases, such as systemic lupus erythematosus, vasculitides, Sjögren's syndrome, antiphospholipid syndrome, systemic sclerosis, dermatomyositis, and rheumatoid arthritis. Complement deficiencies have been associated with an increased risk to develop autoimmune disorders. Because of its functions, the complement system is an attractive therapeutic target for a wide range of diseases. Up to date, several compounds interfering with the complement cascade have been studied in experimental models for autoimmune diseases. The main therapeutic strategies are inhibition of complement activation components, inhibition of complement receptors, and inhibition of membrane attack complex. At present, none of the available agents was proven to be both safe and effective for treatment of autoimmune diseases in humans. Nonetheless, data from preclinical studies and initial clinical trials suggest that the modulation of the complement system could constitute a viable strategy for the treatment of autoimmune conditions in the decades to come.

  12. Antibody-Mediated Enhancement of Parvovirus B19 Uptake into Endothelial Cells Mediated by a Receptor for Complement Factor C1q

    PubMed Central

    von Kietzell, Kristina; Pozzuto, Tanja; Heilbronn, Regine; Grössl, Tobias; Fechner, Henry

    2014-01-01

    ABSTRACT Despite its strong host tropism for erythroid progenitor cells, human parvovirus B19 (B19V) can also infect a variety of additional cell types. Acute and chronic inflammatory cardiomyopathies have been associated with a high prevalence of B19V DNA in endothelial cells of the myocardium. To elucidate the mechanisms of B19V uptake into endothelium, we first analyzed the surface expression of the well-characterized primary B19V receptor P antigen and the putative coreceptors α5β1 integrins and Ku80 antigen on primary and permanent endothelial cells. The receptor expression pattern and also the primary attachment levels were similar to those in the UT7/Epo-S1 cell line regarded as functional for B19V entry, but internalization of the virus was strongly reduced. As an alternative B19V uptake mechanism in endothelial cells, we demonstrated antibody-dependent enhancement (ADE), with up to a 4,000-fold increase in B19V uptake in the presence of B19V-specific human antibodies. ADE was mediated almost exclusively at the level of virus internalization, with efficient B19V translocation to the nucleus. In contrast to monocytes, where ADE of B19V has been described previously, enhancement does not rely on interaction of the virus-antibody complexes with Fc receptors (FcRs), but rather, involves an alternative mechanism mediated by the heat-sensitive complement factor C1q and its receptor, CD93. Our results suggest that ADE represents the predominant mechanism of endothelial B19V infection, and it is tempting to speculate that it may play a role in the pathogenicity of cardiac B19V infection. IMPORTANCE Both efficient entry and productive infection of human parvovirus B19 (B19V) seem to be limited to erythroid progenitor cells. However, in vivo, the viral DNA can also be detected in additional cell types, such as endothelial cells of the myocardium, where its presence has been associated with acute and chronic inflammatory cardiomyopathies. In this study, we

  13. NETosing Neutrophils Activate Complement Both on Their Own NETs and Bacteria via Alternative and Non-alternative Pathways

    PubMed Central

    Yuen, Joshua; Pluthero, Fred G.; Douda, David N.; Riedl, Magdalena; Cherry, Ahmed; Ulanova, Marina; Kahr, Walter H. A.; Palaniyar, Nades; Licht, Christoph

    2016-01-01

    Neutrophils deposit antimicrobial proteins, such as myeloperoxidase and proteases on chromatin, which they release as neutrophil extracellular traps (NETs). Neutrophils also carry key components of the complement alternative pathway (AP) such as properdin or complement factor P (CFP), complement factor B (CFB), and C3. However, the contribution of these complement components and complement activation during NET formation in the presence and absence of bacteria is poorly understood. We studied complement activation on NETs and a Gram-negative opportunistic bacterial pathogen Pseudomonas aeruginosa (PA01, PAKwt, and PAKgfp). Here, we show that anaphylatoxin C5a, formyl-methionyl-leucyl-phenylalanine (fMLP) and phorbol myristate acetate (PMA), which activates NADPH oxidase, induce the release of CFP, CFB, and C3 from neutrophils. In response to PMA or P. aeruginosa, neutrophils secrete CFP, deposit it on NETs and bacteria, and induce the formation of terminal complement complexes (C5b–9). A blocking anti-CFP antibody inhibited AP-mediated but not non-AP-mediated complement activation on NETs and P. aeruginosa. Therefore, NET-mediated complement activation occurs via both AP- and non AP-based mechanisms, and AP-mediated complement activation during NETosis is dependent on CFP. These findings suggest that neutrophils could use their “AP tool kit” to readily activate complement on NETs and Gram-negative bacteria, such as P. aeruginosa, whereas additional components present in the serum help to fix non-AP-mediated complement both on NETs and bacteria. This unique mechanism may play important roles in host defense and help to explain specific roles of complement activation in NET-related diseases. PMID:27148258

  14. Complement regulates TLR4-mediated inflammatory responses during intestinal ischemia reperfusion.

    PubMed

    Pope, Michael R; Hoffman, Sara M; Tomlinson, Stephen; Fleming, Sherry D

    2010-01-01

    Innate immune responses including TLR4 and complement activation are required for mesenteric ischemia/reperfusion (IR)-induced tissue damage. We examined the regulation of TLR4 and complement activation in a mouse model of intestinal IR. Intestinal IR-induced C3 deposition in a TLR4 dependent manner. In addition, in wild-type but not TLR4 deficient mice, IR significantly increased C3 and Factor B (FB) mRNA expression within the intestine. To further examine the role of TLR4 and complement, we administered the complement inhibitor, CR2-Crry, to target local complement activation in wild-type C57Bl/10, and TLR4 deficient B10/ScN mice. TLR4 deficient mice sustained less damage and inflammation after IR than wild-type mice, but administration of CR2-Crry did not further reduce tissue damage. In contrast, CR2-Crry treatment of wild-type mice was accompanied by a reduction in complement activation and in C3 and FB transcription in response to IR. CR2-Crry also significantly decreased intestinal IL-6 and IL-12p40 production in both the wild-type and TLR4 deficient mice. These data indicate that TLR4 regulates extrahepatic complement production while complement regulates TLR4-mediated cytokine production during intestinal IR. PMID:20800895

  15. Insights into the Effects of Complement Factor H on the Assembly and Decay of the Alternative Pathway C3 Proconvertase and C3 Convertase.

    PubMed

    Bettoni, Serena; Bresin, Elena; Remuzzi, Giuseppe; Noris, Marina; Donadelli, Roberta

    2016-04-01

    The activated fragment of C3 (C3b) and factor B form the C3 proconvertase (C3bB), which is cleaved by factor D to C3 convertase (C3bBb). Older studies (Conrad, D. H., Carlo, J. R., and Ruddy, S. (1978)J. Exp. Med.147, 1792-1805; Pangburn, M. K., and Müller-Eberhard, H. J. (1978)Proc. Natl. Acad. Sci. U.S.A.75, 2416-2420; Kazatchkine, M. D., Fearon, D. T., and Austen, K. F. (1979)J. Immunol.122, 75-81) indicated that the complement alternative pathway regulator factor H (FH) competes with factor B for C3b binding; however, the capability of FH to prevent C3bB assembly has not been formally investigated. Moreover, in the few published studies FH did not favor C3bB dissociation. Whether FH may affect C3bBb formation from C3bB is unknown. We set up user-friendly assays based on combined microplate/Western blotting techniques that specifically detect either C3bB or C3bBb, with the aim of investigating the effect of FH on C3bB assembly and decay and C3bBb formation and decay. We document that FH does not affect C3bB assembly, indicating that FH does not efficiently compete with factor B for C3b binding. We also found that FH does not dissociate C3bB. FH showed a strong C3bBb decay-accelerating activity, as reported previously, and also exerted an apparent inhibitory effect on C3bBb formation. The latter effect was not fully attributable to a rapid FH-mediated dissociation of C3bBb complexes, because blocking decay with properdin and C3 nephritic factor did not restore C3bBb formation. FH almost completely prevented release of the smaller cleavage subunit of FB (Ba), without modifying the amount of C3bB complexes, suggesting that FH inhibits the conversion of C3bB to C3bBb. Thus, the inhibitory effect of FH on C3bBb formation is likely the sum of inhibition of C3bB conversion to C3bBb and of C3bBb decay acceleration. Further studies are required to confirm these findings in physiological cell-based settings.

  16. Complement factor H binding of monomeric C-reactive protein downregulates proinflammatory activity and is impaired with at risk polymorphic CFH variants

    PubMed Central

    Molins, Blanca; Fuentes-Prior, Pablo; Adán, Alfredo; Antón, Rosa; Arostegui, Juan I.; Yagüe, Jordi; Dick, Andrew D.

    2016-01-01

    Inflammation and immune-mediated processes are pivotal to the pathogenic progression of age-related macular degeneration (AMD). Although plasma levels of C-reactive protein (CRP) have been shown to be associated with an increased risk for AMD, the pathophysiological importance of the prototypical acute-phase reactant in the etiology of the disease is unknown, and data regarding the exact role of CRP in ocular inflammation are limited. In this study, we provide mechanistic insight into how CRP contributes to the development of AMD. In particular, we show that monomeric CRP (mCRP) but not the pentameric form (pCRP) upregulates IL-8 and CCL2 levels in retinal pigment epithelial cells. Further, we show that complement factor H (FH) binds mCRP to dampen its proinflammatory activity. FH from AMD patients carrying the “risk” His402 polymorphism displays impaired binding to mCRP, and therefore proinflammatory effects of mCRP remain unrestrained. PMID:26961257

  17. Binding of Complement Factor H (FH) Decreases Protective Anti-FH Binding Protein Antibody Responses of Infant Rhesus Macaques Immunized With a Meningococcal Serogroup B Vaccine

    PubMed Central

    Granoff, Dan M.; Costa, Isabella; Konar, Monica; Giuntini, Serena; Van Rompay, Koen K. A.; Beernink, Peter T.

    2015-01-01

    Background. The meningococcal vaccine antigen, factor H (FH)–binding protein (FHbp), binds human complement FH. In human FH transgenic mice, binding decreased protective antibody responses. Methods. To investigate the effect of primate FH binding, we immunized rhesus macaques with a 4-component serogroup B vaccine (4CMenB). Serum FH in 6 animals bound strongly to FHbp (FHbp-FHhigh) and, in 6 animals, bound weakly to FHbp (FHbp-FHlow). Results. There were no significant differences between the respective serum bactericidal responses of the 2 groups against meningococcal strains susceptible to antibody to the NadA or PorA vaccine antigens. In contrast, anti-FHbp bactericidal titers were 2-fold lower in FHbp-FHhigh macaques against a strain with an exact FHbp match to the vaccine (P = .08) and were ≥4-fold lower against 4 mutants with other FHbp sequence variants (P ≤ .005, compared with FHbp-FHlow macaques). Unexpectedly, postimmunization sera from all 12 macaques enhanced FH binding to meningococci. In contrast, serum anti-FHbp antibodies elicited by 4CMenB in mice whose mouse FH did not bind to the vaccine antigen inhibited FH binding. Conclusions. Binding of FH to FHbp decreases protective anti-FHbp antibody responses of macaques to 4CMenB. Even low levels of FH binding skew the antibody repertoire to FHbp epitopes outside of the FH-binding site, which enhance FH binding. PMID:25676468

  18. Complement activation product C5a is a selective suppressor of TLR4-induced, but not TLR3-induced, production of IL-27(p28) from macrophages

    PubMed Central

    Bosmann, Markus; Haggadone, Mikel D.; Hemmila, Mark R.; Zetoune, Firas S.; Sarma, J. Vidya; Ward, Peter A.

    2012-01-01

    There is accumulating evidence that the complement activation product, C5a, can orchestrate cellular immune functions. IL-27 (p28/EBI3) is an emerging key player essential for regulating inflammatory responses and T cells. Here, we report that C5a robustly suppressed IL-27(p28) gene expression and release in peritoneal macrophages. These cells from C57BL/6J mice abundantly produced IL-27(p28) after engagement of either the TLR3 (Poly I:C) or TLR4 (LPS) receptor. Genetic deficiency of either TLR4 or LPS-binding protein (LBP) completely incapacitated the ability of macrophages to secrete IL-27(p28) in response to LPS. IL-27(p28) producing macrophages also expressed the C5aR receptor, thus displaying an IL-27(p28)+F4/80+C5aR+ phenotype. C5a suppressed IL-27(p28) in LPS-stimulated macrophages via interactions with the C5aR receptor, rather than the C5L2 receptor. C5aR−/− mice after endotoxemia displayed higher plasma levels of IL-27(p28) when compared to C57BL/6J mice. C5a did not affect the release of IL-27(p28) or frequency of IL-27(p28)+F4/80+ macrophages after engagement of TLR3. Mechanistically, LPS activated both NFκB and the PI3K-Akt pathways, whereas C5a activated only the PI3K-Akt pathway. Engagement of PI3K-Akt was inhibitory for IL-27(p28) production, since PI3K-Akt pharmacologic blockade resulted in increased amounts of IL-27(p28) and reversed the suppressive effects of C5a. Blockade of PI3K-Akt in endotoxemic C57BL/6J mice resulted in higher generation of IL-27(p28). In contrast, for TLR3-mediated release of IL-27(p28), the PI3K-Akt pathway was not involved. These data provide new evidence on how complement activation may selectively interfere with production of T cell regulatory cytokines by antigen-presenting cells in the varying contexts of either bacterial (TLR4-pathway) or viral (TLR3-pathway) infection. PMID:22491257

  19. Complement-Mediated Death of Ciliate Tetrahymena pyriformis Caused by Human Blood Serum.

    PubMed

    Ivanov, P A; Faktor, M I; Karpova, N S; Cheremnykh, E G; Brusov, O S

    2016-04-01

    Toxicity of human blood serum for ciliate Tetrahymena pyriformis is determined by the complement system. When ciliate are dying after being exposed to blood serum, cell membrane permeability for low-molecular-weight compounds significantly increases, probably due to pore formation. Serine protease inhibitors or exposure to physical factors inducing complement inactivation (e.g., heating up to 56°C) completely prevented ciliate death under the effect of human serum. Activation of serum complement upon interaction with Tetrahymena cells occurred by the classical or lectin pathway, while the contribution of the alternative activation pathway was negligible.

  20. Complement and HIV-I infection/HIV-associated neurocognitive disorders.

    PubMed

    Liu, Fengming; Dai, Shen; Gordon, Jennifer; Qin, Xuebin

    2014-04-01

    The various neurological complications associated with HIV-1 infection, specifically HIV-associated neurocognitive disorders (HAND) persist as a major public health burden worldwide. Despite the widespread use of anti-retroviral therapy, the prevalence of HAND is significantly high. HAND results from the direct effects of an HIV-1 infection as well as secondary effects of HIV-1-induced immune reaction and inflammatory response. Complement, a critical mediator of innate and acquired immunity, plays important roles in defeating many viral infections by the formation of a lytic pore or indirectly by opsonization and recruitment of phagocytes. While the role of complement in the pathogenesis of HIV-1 infection and HAND has been previously recognized for over 15 years, it has been largely underestimated thus far. Complement can be activated through HIV-1 envelope proteins, mannose-binding lectins (MBL), and anti-HIV-1 antibodies. Complement not only fights against HIV-1 infection but also enhances HIV-1 infection. In addition, HIV-1 can hijack complement regulators such as CD59 and CD55 and can utilize these regulators and factor H to escape from complement attack. Normally, complement levels in brain are much lower than plasma levels and there is no or little complement deposition in brain cells. Interestingly, local production and deposition of complement are dramatically increased in HIV-1-infected brain, indicating that complement may contribute to the pathogenesis of HAND. Here, we review the current understanding of the role of complement in HIV-1 infection and HAND, as well as potential therapeutic approaches targeting the complement system for the treatment and eradications of HIV-1 infection.

  1. Versatility of the complement system in neuroinflammation, neurodegeneration and brain homeostasis

    PubMed Central

    Orsini, Franca; De Blasio, Daiana; Zangari, Rosalia; Zanier, Elisa R.; De Simoni, Maria-Grazia

    2014-01-01

    The immune response after brain injury is highly complex and involves both local and systemic events at the cellular and molecular level. It is associated to a dramatic over-activation of enzyme systems, the expression of proinflammatory genes and the activation/recruitment of immune cells. The complement system represents a powerful component of the innate immunity and is highly involved in the inflammatory response. Complement components are synthesized predominantly by the liver and circulate in the bloodstream primed for activation. Moreover, brain cells can produce complement proteins and receptors. After acute brain injury, the rapid and uncontrolled activation of the complement leads to massive release of inflammatory anaphylatoxins, recruitment of cells to the injury site, phagocytosis and induction of blood brain barrier (BBB) damage. Brain endothelial cells are particularly susceptible to complement-mediated effects, since they are exposed to both circulating and locally synthesized complement proteins. Conversely, during neurodegenerative disorders, complement factors play distinct roles depending on the stage and degree of neuropathology. In addition to the deleterious role of the complement, increasing evidence suggest that it may also play a role in normal nervous system development (wiring the brain) and adulthood (either maintaining brain homeostasis or supporting regeneration after brain injury). This article represents a compendium of the current knowledge on the complement role in the brain, prompting a novel view that complement activation can result in either protective or detrimental effects in brain conditions that depend exquisitely on the nature, the timing and the degree of the stimuli that induce its activation. A deeper understanding of the acute, subacute and chronic consequences of complement activation is needed and may lead to new therapeutic strategies, including the ability of targeting selective step in the complement cascade

  2. Comprehensive and comparative transcription analyses of the complement pathway in rainbow trout.

    PubMed

    Köbis, Judith M; Rebl, Alexander; Kühn, Carsten; Korytář, Tomáš; Köllner, Bernd; Goldammer, Tom

    2015-01-01

    The complement system is one of the most ancient and most essential innate immune cascades throughout the animal kingdom. Survival of aquatic animals, such as rainbow trout, depends on this early inducible, efficient immune cascade. Despite increasing research on genes coding for complement components in bony fish, some complement-related genes are still unknown in salmonid fish. In the present study, we characterize the genes encoding complement factor D (CFD), CD93 molecule (CD93), and C-type lectin domain family 4, member M (CLEC4M) from rainbow trout (Oncorhynchus mykiss). Subsequently, we performed comprehensive and comparative expression analyses of 36 complement genes including CFD, CD93, and CLEC4M and further putative complement-associated genes to obtain general information about the functional gene interaction within the complement pathway in fish. These quantification analyses were conducted in liver, spleen and gills of healthy fish of two rainbow trout strains, selected for survival (strain BORN) and growth (Import strain), respectively. The present expression study clearly confirms for rainbow trout that liver represents the primary site of complement expression. Spleen and gills also express most complement genes, although the mean transcript levels were generally lower than in liver. The transcription data suggest a contribution of spleen and gills to complement activity. The comparison of the two rainbow trout strains revealed a generally similar complement gene expression. However, a significantly lower expression of numerous genes especially in spleen seems characteristic for the BORN strain. This suggests a strain-specific complement pathway regulation under the selected rearing conditions.

  3. Apobec-1 Complementation Factor (A1CF) Inhibits Epithelial-Mesenchymal Transition and Migration of Normal Rat Kidney Proximal Tubular Epithelial Cells

    PubMed Central

    Huang, Liyuan; Wang, Honglian; Zhou, Yuru; Ni, Dongsheng; Hu, Yanxia; Long, Yaoshui; Liu, Jianing; Peng, Rui; Zhou, Li; Liu, Zhicheng; Lyu, Zhongshi; Mao, Zhaomin; Hao, Jin; Li, Yiman; Zhou, Qin

    2016-01-01

    Apobec-1 complementation factor (A1CF) is a member of the heterogeneous nuclear ribonucleoproteins (hnRNP) family, which participates in site-specific posttranscriptional RNA editing of apolipoprotein B (apoB) transcript. The posttranscriptional editing of apoB mRNA by A1CF in the small intestine is required for lipid absorption. Apart from the intestine, A1CF mRNA is also reported to be highly expressed in the kidneys. However, it is remained unknown about the functions of A1CF in the kidneys. The aim of this paper is to explore the potential functions of A1CF in the kidneys. Our results demonstrated that in C57BL/6 mice A1CF was weakly expressed in embryonic kidneys from E15.5dpc while strongly expressed in mature kidneys after birth, and it mainly existed in the tubules of inner cortex. More importantly, we identified A1CF negatively regulated the process of epithelial-mesenchymal transition (EMT) in kidney tubular epithelial cells. Our results found ectopic expression of A1CF up-regulated the epithelial markers E-cadherin, and down-regulated the mesenchymal markers vimentin and α-smooth muscle actin (α-SMA) in NRK52e cells. In addition, knockdown of A1CF enhanced EMT contrary to the overexpression effect. Notably, the two A1CF variants led to the similar trend in the EMT process. Taken together, these data suggest that A1CF may be an antagonistic factor to the EMT process of kidney tubular epithelial cells. PMID:26848653

  4. Dissection of CR1, factor H, membrane cofactor protein, and factor B binding and functional sites in the third complement component.

    PubMed

    Lambris, J D; Lao, Z; Oglesby, T J; Atkinson, J P; Hack, C E; Becherer, J D

    1996-06-15

    Previous studies have suggested that the residues 727-768 of human (Hu) C3 contain the binding sites for CR1, factor H, and factor B. Here, we have (1) characterized further some of the C3 structural requirements for its binding to CR1, H, and B, (2) investigated the functions associated with these C3-ligand interactions, and (3) studied the relationship of MCP-binding sites in C3 with those for CR1, H, and B. Hu C3 molecules in which residues 727-768 were deleted (designated C3delta727-768) or substituted with the corresponding segment of cobra venom factor, Xenopus, or trout C3 (chimeric C3s) were expressed in the baculovirus system and analyzed for their reactivity with C3-binding proteins. In contrast to wild-type iC3 which, in the presence of CR1, is cleaved by factor I to iC3b-a and C3c-a and C3dg, all chimeric C3s were cleaved only to iC3b-a. In addition, the cleavage of deleted (C3delta727-768) iC3 to iC3b-a by factor I in the presence of CR1 was significantly reduced, whereas it remained unaltered in the presence of MCP. Cleavage of iC3 to iC3b-a by factor I and H was similar in all expressed C3s except C3delta727-768, whose cleavage was significantly reduced. All of the expressed molecules except C3delta727-768 were capable of forming the fluid-phase alternative pathway C3 convertase, and all reacted with properdin. These results suggest that during cleavage of iC3 by factor I and CR1, or H, CR1 and H bind to at least two sites on C3 and that the MCP binding site(s) on C3b are different from those for CR1. They also indicate that some or all of the C3 residues that are directly involved in, or contribute to, the structure of one of the CR1 and H binding sites are located within residues 727-768. These studies also demonstrate that, although this segment of C3 may be involved in C3-factor B interaction, other residues in addition to 736EE (previously implicated in B binding) must also contribute significantly to this interaction.

  5. Dissecting NF-κB signaling induced by genotoxic agents via genetic complementation of NEMO-deficient 1.3E2 cells.

    PubMed

    Jackson, Shawn S; Miyamoto, Shigeki

    2015-01-01

    The transcription factor NF-κB regulates expression of a diverse set of genes to modulate multiple biological and pathological processes. Among these, NF-κB activation in response to genotoxic agents has received considerable attention due to its role in regulating cancer cell resistance to chemo- and radiation therapy. Furthermore, induction of this pathway by endogenous damage is further implicated in normal developmental processes, such as B cell development, and premature aging, among others. This pathway also serves as a signaling model in which nuclear initiated signals (DNA damage) are communicated to a cytoplasmic target (IκB kinase and NF-κB). Several of the critical molecular events of this nuclear to cytoplasmic NF-κB signaling cascade were discovered, in part, by genetic complementation analyses of the NEMO-deficient 1.3E2 mouse pre-B cell line. This chapter describes methods used to generate and analyze such reconstitution cell systems and certain caveats that are critical for proper interpretation of NEMO mutant defects.

  6. Structural integration in hypoxia-inducible factors

    SciTech Connect

    Wu, Dalei; Potluri, Nalini; Lu, Jingping; Kim, Youngchang; Rastinejad, Fraydoon

    2015-08-20

    The hypoxia-inducible factors (HIFs) coordinate cellular adaptations to low oxygen stress by regulating transcriptional programs in erythropoiesis, angiogenesis and metabolism. These programs promote the growth and progression of many tumours, making HIFs attractive anticancer targets. Transcriptionally active HIFs consist of HIF-alpha and ARNT (also called HIF-1 beta) subunits. Here we describe crystal structures for each of mouse HIF-2 alpha-ARNT and HIF-1 alpha-ARNT heterodimers in states that include bound small molecules and their hypoxia response element. A highly integrated quaternary architecture is shared by HIF-2 alpha-ARNT and HIF-1 alpha-ARNT, wherein ARNT spirals around the outside of each HIF-alpha subunit. Five distinct pockets are observed that permit small-molecule binding, including PAS domain encapsulated sites and an interfacial cavity formed through subunit heterodimerization. The DNA-reading head rotates, extends and cooperates with a distal PAS domain to bind hypoxia response elements. HIF-alpha mutations linked to human cancers map to sensitive sites that establish DNA binding and the stability of PAS domains and pockets.

  7. Complement in hemolytic anemia.

    PubMed

    Brodsky, Robert A

    2015-11-26

    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.

  8. Complement in hemolytic anemia.

    PubMed

    Brodsky, Robert A

    2015-01-01

    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.

  9. CspA from Borrelia burgdorferi Inhibits the Terminal Complement Pathway

    PubMed Central

    Hallström, Teresia; Siegel, Corinna; Mörgelin, Matthias; Kraiczy, Peter; Skerka, Christine; Zipfel, Peter F.

    2013-01-01

    ABSTRACT In order to survive and persist in an immunocompetent human host, Borrelia burgdorferi controls the human immune attack and blocks the damaging effects of the activated complement system. These Gram-negative spirochetes use CspA (CRASP-1) and four additional immune evasion proteins to bind combinations of human plasma regulators, including factor H, factor H-like protein 1 (FHL-1), complement factor H-related protein 1 (CFHR1), CFHR2, CFHR5, and plasminogen. As many microbial immune evasion proteins have multiple functions, we hypothesized that CspA has additional roles in complement or immune control. Here, we identify CspA as a terminal complement inhibitor. Borrelial CspA binds the human terminal complement components C7 and C9 and blocks assembly and membrane insertion of the terminal complement complex (TCC). CspA inhibits TCC assembly at the level of C7, as revealed by hemolytic assays, and inhibits polymerization of C9. CspA, when ectopically expressed on the surface of serum-sensitive Borrelia garinii, blocks TCC assembly on the level of C7 and induces serum resistance in the transformed bacteria. This CspA-mediated serum resistance and terminal complement pathway inhibition allow B. burgdorferi to survive in the hostile environment of human plasma. PMID:23943762

  10. Porcine complement regulatory protein CD46 and heparan sulfates are the major factors for classical swine fever virus attachment in vitro.

    PubMed

    Dräger, Carolin; Beer, Martin; Blome, Sandra

    2015-03-01

    Classical swine fever virus (CSFV) is the causative agent of a severe multi-systemic disease of pigs. While several aspects of virus-host-interaction are known, the early steps of infection remain unclear. For the closely related bovine viral diarrhea virus (BVDV), a cellular receptor is known: bovine complement regulatory protein CD46. Given that these two pestiviruses are closely related, porcine CD46 is also a candidate receptor for CSFV. In addition to CD46, cell-culture-adapted CSFV strains have been shown to use heparan sulfates as an additional cellular factor. In the present study, the interaction of field-type and cell-culture-adapted CSFV with a permanent porcine cell line or primary macrophages was assessed using anti-porcine CD46 monoclonal antibodies and a heparan-sulfate-blocking compound, DSTP-27. The influence of receptor blocking was assessed using virus titration and quantitative PCR. Treatment of cells with monoclonal antibodies against porcine CD46 led to a reduction of viral growth in both cell types. The effect was most pronounced with field-type CSFV. The blocking could be enhanced by addition of DSTP-27, especially for cell-culture-adapted CSFV. The combined use of both blocking agents led to a significant reduction of viral growth but was also not able to abolish infection completely. The results obtained in this study showed that both porcine CD46 and heparan sulfates play a major role in the initial steps of CSFV infection. Additional receptors might also play a role for attachment and entry; however, their impact is obviously limited in vitro in comparison to CD46 and heparan sulfates.

  11. Analysis of serum β-amyloid peptides, α2-macroglobulin, complement factor H, and clusterin levels in APP/PS1 transgenic mice during progression of Alzheimer's disease.

    PubMed

    Wang, Dejiang; Di, Xiangjun; Fu, Lu; Li, Yingnan; Han, Xiao; Wu, Hui; Cai, Linjun; Meng, Xiangyu; Jiang, Chunlai; Kong, Wei; Su, Weiheng

    2016-10-19

    As a progressive age-related neurodegenerative disorder, Alzheimer's disease (AD) is a global health concern. Despite the availability of psychological testing, neuroimaging, genetic testing, and biochemical assays of cerebrospinal fluid, convenient and accurate blood biomarkers for the prediction, diagnosis, and preclinical studies of AD are still lacking. The present study aims to longitudinally evaluate the feasibility of β-amyloid proteins, α2-macroglobulin (α-2M), complement factor H (CFH), and clusterin as blood biomarkers of AD. Using APP/PS1 transgenic and wild-type mice, cognitive impairment and amyloid plaque counts in the brain were evaluated over a range of ages using the Morris water maze test and immunohistochemistry methods, respectively. Serum Aβ40, Aβ42, α-2M, CFH, and clusterin levels were measured by enzyme-linked immunosorbent assay and correlated with progression of AD. APP/PS1 transgenic mice presented progressive AD characteristics at the ages of 3, 6, 9, and 12 months. Serum Aβ42 levels and Aβ42/Aβ40 ratios increased significantly in transgenic 3- and 6-month-old mice compared with controls. Serum CFH levels decreased significantly in 3- and 6-month-old transgenic mice compared with controls. Meanwhile, serum clusterin levels increased significantly in 12-month-old transgenic mice compared with controls. The α-2M level was not significantly different between transgenic and wild-type mice. The APP/PS1 transgenic mouse is a model of familial AD. The present study indicated that the serum Aβ42 level, Aβ42/Aβ40 ratio, and CFH level are potential biomarkers in preclinical and early stages of AD, whereas serum clusterin level is a potential biomarker in the late stage of AD. PMID:27541273

  12. Some Experiences with Nonoverlapping Schur Complement Parallel Preconditioning for CFD Calculations

    NASA Technical Reports Server (NTRS)

    Barth, Timothy J.; Chan, Tony F.; Tang, Wei-Pai; Kwak, Dochan (Technical Monitor)

    1996-01-01

    In this work we consider solving matrices which arise from the discretization of advection-diffusion field equations on arbitrary triangulated domains using stabilized numerical methods. The talk will discuss several candidate matrix preconditioning algorithms based on the 2 x 2 block factorization induced by an apriori partitioning of the triangulated domain. Application of the 2 x 2 block preconditioner requires the formation and inversion of the Schur complement submatrix. We consider several strategies for simplifying this task: incomplete Schur complement factorizations, drop tolerance element filling, Schur complement probing, and localized Schur complement inversion. Numerical results will be shown comparing performance and efficiency of these approximations. The matrix preconditioner has also been embedded into a Newton algorithm for solving the nonlinear Euler and Navier-Stokes equations governing compressible flow. The remainder of the talk will show numerous examples in CFD to demonstrate the efficiency and robustness of the techniques.

  13. Role of the complement system in rheumatoid arthritis and psoriatic arthritis: relationship with anti-TNF inhibitors.

    PubMed

    Ballanti, Eleonora; Perricone, Carlo; di Muzio, Gioia; Kroegler, Barbara; Chimenti, Maria Sole; Graceffa, Dario; Perricone, Roberto

    2011-08-01

    The complement system is an essential component of innate immunity and also plays an important role in modulating adaptive immunity. It comprises more than 30 plasma and membrane-bound proteins and can be activated through three pathways: the classical, the alternative and the lectin pathways. Its activation contributes to the pathogenesis of several autoimmune and inflammatory conditions. The evidence of complement activation in synovial fluid of Rheumatoid Arthritis (RA) patients is abundant, while few data exist in Psoriatic Arthritis (PsA) patients. Levels of complement proteins are generally depressed in the synovial fluid of patients with RA, reflecting consumption of complement. On the other hand, elevated levels of several complement cleavage products have been observed in synovial fluid. Involvement of complement in the pathogenesis of RA was also confirmed in animal models of arthritis: mice deficient for complement proteins are protected against the development of collagen-induced arthritis and administration of the anti-C5 monoclonal antibody prevents the onset of this arthritis. In the last decade anti-tumor necrosis factor agents have shown to be effective for the treatment of both RA and PsA and some studies suggest that the interaction between TNFα and complement system may contribute to the pathogenesis of these diseases. Reduction of the complement activation could be one of the mechanism by which TNFα-inhibitors exert their effectiveness in inflammatory arthritides. Because of these findings, complement could be an attractive therapeutic target both in RA and in PsA.

  14. Production of Tuber-Inducing Factor

    NASA Technical Reports Server (NTRS)

    Stutte, Gary W.; Yorio, Neil C.

    2006-01-01

    A process for making a substance that regulates the growth of potatoes and some other economically important plants has been developed. The process also yields an economically important by-product: potatoes. The particular growth-regulating substance, denoted tuber-inducing factor (TIF), is made naturally by, and acts naturally on, potato plants. The primary effects of TIF on potato plants are reducing the lengths of the main shoots, reducing the numbers of nodes on the main stems, reducing the total biomass, accelerating the initiation of potatoes, and increasing the edible fraction (potatoes) of the overall biomass. To some extent, these effects of TIF can override environmental effects that typically inhibit the formation of tubers. TIF can be used in the potato industry to reduce growth time and increase harvest efficiency. Other plants that have been observed to be affected by TIF include tomatoes, peppers, radishes, eggplants, marigolds, and morning glories. In the present process, potatoes are grown with their roots and stolons immersed in a nutrient solution in a recirculating hydroponic system. From time to time, a nutrient replenishment solution is added to the recirculating nutrient solution to maintain the required nutrient concentration, water is added to replace water lost from the recirculating solution through transpiration, and an acid or base is added, as needed, to maintain the recirculating solution at a desired pH level. The growing potato plants secrete TIF into the recirculating solution. The concentration of TIF in the solution gradually increases to a range in which the TIF regulates the growth of the plants.

  15. CSF coccidioides complement fixation

    MedlinePlus

    ... eds. Henry's Clinical Diagnosis and Management by Laboratory Methods . 22nd ed. Philadelphia, PA: Elsevier Saunders; 2011:chap 61. Read More Complement Update Date 5/1/2015 Updated by: Jatin M. Vyas, MD, ...

  16. Human seminal plasma inhibition of complement.

    PubMed

    Petersen, B H; Lammel, C J; Stites, D P; Brooks, G F

    1980-10-01

    Recent studies have shown that human seminal plasma contains chemically and biologically distinct factors which inhibit lymphocyte functions and the serum bactericidal and opsonic activities associated with the killing of gram-negative organisms. Because of the direct association between complement action and serum bactericidal and opsonic activities, inhibition of complement may be one of the possible mechanisms of action of seminal plasma immunoinhibitory factors. Complement hemolytic activity was measured for C3 and C4 in serum Neisseria gonorrhoeae and Escherichia coli bactericidal reaction mixtures with and without addition of seminal plasma. In the presence of seminal plasma, where there was no bactericidal action, C3 and titers were reduced to approximately 50% of the titers in the reactions with complement donor serum. The C3 titers were lower than in the reaction mixtures with immune serum and complement donor serum, where N. gonorrhoeae bactericidal activity occurred. Individual human seminal plasma specimens depressed CH50 activity of pooled normal human sera up to 50% of normal levels. There were no differences in inhibition by seminal plasma specimens from normal or vasectomized men. Treatment with seminal plasma depressed the functional activity of complement components C1 and C3 by more than 50%. Seminal plasma also inhibited alternate pathway activity. Cleavage of factor B was demonstrated. The seminal plasma factor which inhibited complement was of low molecular weight. DPF blocked the seminal plasma complement-inhibitory factor. However, amidolytic activity for serine protease substrates could not be demonstrated. It is likely that the seminal plasma complement inhibitor is a protease inhibitor acting singly or in combination.

  17. Complement activation in amyloid plaques in Alzheimer's dementia.

    PubMed

    Eikelenboom, P; Hack, C E; Rozemuller, J M; Stam, F C

    1989-01-01

    Amyloid plaques in Alzheimer's dementia contain complement factors C1q, C4 and C3. In the present study we demonstrate complement activation in amyloid plaques using immunoenzymatical techniques and specific antibodies against subunits of individual complement components and activated complement products. Amyloid plaques contain C1q and activated C3 fragments (C3c and C3d, g) but no C1s and C3a. These findings demonstrate that the complement components are not passively bound to the amyloid plaque structures but are the result of an activation process. The role of complement activation in the genesis of senile plaques is discussed.

  18. Complement fragments C3b and iC3b coupled to latex induce a respiratory burst in human neutrophils.

    PubMed

    Hoogerwerf, M; Weening, R S; Hack, C E; Roos, D

    1990-02-01

    The complement fragments C3b and iC3b were purified from human serum by affinity chromatography with Sepharose-coupled monoclonal antibody against the C3d region of C3. The resulting preparations were more than 95% pure and contained less than 0.1% native IgG. Purified C3b and iC3b were coupled to latex beads (0.8 micron diameter) by means of F(ab')2 fragments of monoclonal antibodies against the beta chain or the C3d region of C3, thus orienting the C3b and the iC3b on the latex with the C3b- and iC3b-specific regions outwards. These particles were found to activate the respiratory burst of freshly isolated human neutrophils to 20-30% of the maximal capacity. Latex particles randomly coated with C3b or iC3b were about 3 times less stimulatory. C3b, iC3b and IgG coupled to latex in an oriented fashion were about equally effective in stimulating the respiratory burst. Neutrophils from a patient with a total deficiency of CR3 responded normally to C3b-coated latex but did not respond to iC3b-coated latex. A monoclonal antibody against the alpha chain of CR3 inhibited the activation by iC3b-coated latex and a polyclonal antibody against CR1 partially inhibited the activation by C3b-coated latex. We found an additive effect between IgG-coated latex and C3b-coated latex, regardless of the presence of IgG and C3b on the same particle or on different particles. Thus, binding of ligands to either CR1 or CR3 per se is sufficient to induce an activating signal to the NADPH oxidase in human neutrophils.

  19. Complement System Part II: Role in Immunity

    PubMed Central

    Merle, Nicolas S.; Noe, Remi; Halbwachs-Mecarelli, Lise; Fremeaux-Bacchi, Veronique; Roumenina, Lubka T.

    2015-01-01

    The complement system has been considered for a long time as a simple lytic cascade, aimed to kill bacteria infecting the host organism. Nowadays, this vision has changed and it is well accepted that complement is a complex innate immune surveillance system, playing a key role in host homeostasis, inflammation, and in the defense against pathogens. This review discusses recent advances in the understanding of the role of complement in physiology and pathology. It starts with a description of complement contribution to the normal physiology (homeostasis) of a healthy organism, including the silent clearance of apoptotic cells and maintenance of cell survival. In pathology, complement can be a friend or a foe. It acts as a friend in the defense against pathogens, by inducing opsonization and a direct killing by C5b–9 membrane attack complex and by triggering inflammatory responses with the anaphylatoxins C3a and C5a. Opsonization plays also a major role in the mounting of an adaptive immune response, involving antigen presenting cells, T-, and B-lymphocytes. Nevertheless, it can be also an enemy, when pathogens hijack complement regulators to protect themselves from the immune system. Inadequate complement activation becomes a disease cause, as in atypical hemolytic uremic syndrome, C3 glomerulopathies, and systemic lupus erythematosus. Age-related macular degeneration and cancer will be described as examples showing that complement contributes to a large variety of conditions, far exceeding the classical examples of diseases associated with complement deficiencies. Finally, we discuss complement as a therapeutic target. PMID:26074922

  20. Scatter factor induces blood vessel formation in vivo.

    PubMed Central

    Grant, D S; Kleinman, H K; Goldberg, I D; Bhargava, M M; Nickoloff, B J; Kinsella, J L; Polverini, P; Rosen, E M

    1993-01-01

    Scatter factor (also known as hepatocyte growth factor) is a glycoprotein secreted by stromal cells that stimulates cell motility and proliferation. In vitro, scatter factor stimulates vascular endothelial cell migration, proliferation, and organization into capillary-like tubes. Using two different in vivo assays, we showed that physiologic quantities of purified native mouse scatter factor and recombinant human hepatocyte growth factor induce angiogenesis (the formation of new blood vessels). The angiogenic activity was blocked by specific anti-scatter factor antibodies. Scatter factor induced cultured microvascular endothelial cells to accumulate and secrete significantly increased quantities of urokinase, an enzyme associated with development of an invasive endothelial phenotype during angiogenesis. We further showed that immunoreactive scatter factor is present surrounding sites of blood vessel formation in psoriatic skin. These findings suggest that scatter factor may act as a paracrine mediator in pathologic angiogenesis associated with human inflammatory disease. Images Fig. 1 Fig. 2 Fig. 3 Fig. 5 PMID:7680481

  1. Complement facilitates macrophage phagocytosis of inhaled iron particles but has little effect in mediating silica-induced lung inflammatory and clearance responses

    SciTech Connect

    Warheit, D.B.; Carakostas, M.C.; Bamberger, J.R.; Hartsky, M.A. )

    1991-12-01

    The present studies were undertaken to investigate the role of complement in mediating pulmonary inflammation and/or phagocytosis as a function of particle clearance in rats exposed to silica or carbonyl iron (CI) particles. Both particle types were shown to be weak activators of serum complement in vitro. In these studies, normal and complement-depressed (CVFD-treated) rats were exposed to aerosols of Ci or silica particles for 6 hr at 100 mg/m{sup 3}. Following exposure, alveolar fluids and cells from sham and dust-exposed animals were recovered by bronchoalveolar lavage (BAL) at several time periods postexposure and measured for a variety of biochemical and cellular indices. In addition, pulmonary macrophages were cultured and studied for morphology and phagocytosis. The authors results showed that CI exposure did not produce cellular or biochemical indices of pulmonary inflammation, either in normal or complement-depleted rats. However, fewer phagocytic macrophages were recovered from the lungs of CVF-treated, CI-exposed rats than from normal exposed animals. In contrast, silica inhalation produced a sustained PMN inflammatory response in the lungs of exposed rats, measured up through 1 month postexposure, along with significant increases in BAL fluid levels of LDH, protein, and alkaline phosphatase and deficits in pulmonary macrophage phagocytic functions.

  2. Complement expression in the retina is not influenced by short-term pressure elevation

    PubMed Central

    Dong, Cecilia Q.; Panagis, Lampros; Kamthan, Gautam; Ren, Lizhen; Rozenboym, Anna; Perera, Tarique D.; Coplan, Jeremy D.; Danias, John

    2014-01-01

    Purpose To determine whether short-term pressure elevation affects complement gene expression in the retina in vitro and in vivo. Methods Muller cell (TR-MUL5) cultures and organotypic retinal cultures from adult mice and monkeys were subjected to either 24-h or 72-h of pressure at 0, 15, 30, and 45 mmHg above ambient. C57BL/6 mice were subjected to microbead-induced intraocular pressure (IOP) elevation for 7 days. RNA and protein were extracted and used for analysis of expression levels of complement component genes and complement component 1, q subcomponent (C1q) and complement factor H (CFH) immunoblotting. Results mRNA levels of complement genes and C1q protein levels in Muller cell cultures remained the same for all pressure levels after exposure for either 24 or 72 h. In primate and murine organotypic cultures, pressure elevation did not produce changes in complement gene expression or C1q and CFH protein levels at either the 24-h or 72-h time points. Pressure-related glial fibrillary acidic protein (GFAP) mRNA expression changes were detected in primate retinal organotypic cultures (analysis of variance [ANOVA]; p<0.05). mRNA expression of several other genes changed as a result of time in culture. Eyes subjected to microbead-induced IOP elevation had no differences in mRNA expression of complement genes and C1q protein levels (ANOVA; p>0.05 for both) with contralateral control and naïve control eyes. Conclusions Short-term elevation of pressure in vitro as well as short-term (1 week) IOP elevation in vivo does not seem to dramatically alter complement system gene expression in the retina. Prolonged expression to elevated pressure may be necessary to affect the complement system expression. PMID:24505213

  3. Laboratory tests for disorders of complement and complement regulatory proteins.

    PubMed

    Shih, Angela R; Murali, Mandakolathur R

    2015-12-01

    The complement pathway is a cascade of proteases that is involved in immune surveillance and innate immunity, as well as adaptive immunity. Dysfunction of the complement cascade may be mediated by aberrations in the pathways of activation, complement regulatory proteins, or complement deficiencies, and has been linked to a number of hematologic disorders, including paroxysmal noctural hemoglobinuria (PNH), hereditary angioedema (HAE), and atypical hemolytic-uremic syndrome (aHUS). Here, current laboratory tests for disorders of the complement pathway are reviewed, and their utility and limitations in hematologic disorders and systemic diseases are discussed. Current therapeutic advances targeting the complement pathway in treatment of complement-mediated hematologic disorders are also reviewed.

  4. C-reactive protein activates complement in infarcted human myocardium.

    PubMed

    Nijmeijer, Remco; Lagrand, Wim K; Lubbers, Yvonne T P; Visser, Cees A; Meijer, Chris J L M; Niessen, Hans W M; Hack, C Erik

    2003-07-01

    Circulating levels of C-reactive protein (CRP) constitute a cardiovascular risk marker. Immunohistochemical studies have revealed co-localization of CRP and activated complement in human infarcted myocardium suggesting CRP to enhance inflammation in ischemic myocardium by inducing local complement activation. The aim was to establish whether CRP activates complement in infarcted human myocardium and to assess the relationship between this activation and the duration of infarction. Myocardial tissue samples from 56 patients that had died from acute myocardial infarction were evaluated. Specimens were taken from infarcted as well as noninfarcted sites of the heart. CRP-mediated complement activation was assessed by immunohistochemistry and by measuring levels of complement, CRP, and CRP-complement complexes, specific markers for CRP-mediated activation, in homogenates of the heart. Infarctions of 12 hours to 5 days had significantly more extensive depositions of complement and CRP and contained significantly more CRP, activated complement, and CRP-complement complexes than infarctions that were less than 12 hours old. Levels of CRP complexes correlated significantly with CRP and complement concentrations in the infarctions, as well as with the extent of complement and CRP depositions as measured via immunohistochemistry. Specific activation products of CRP-mediated activation of complement are increased in infarcts of more than 12 hours in duration and correlate with the extent of complement depositions. Hence, CRP seems to enhance local inflammatory reactions ensuing in human myocardial infarcts of more than 12 hours duration.

  5. Complement and thrombosis in the antiphospholipid syndrome.

    PubMed

    Oku, Kenji; Nakamura, Hiroyuki; Kono, Michihiro; Ohmura, Kazumasa; Kato, Masaru; Bohgaki, Toshiyuki; Horita, Tetsuya; Yasuda, Shinsuke; Amengual, Olga; Atsumi, Tatsuya

    2016-10-01

    The involvement of complement activation in the pathophysiology of antiphospholipid syndrome (APS) was first reported in murine models of antiphospholipid antibody (aPL)-related pregnancy morbidities. We previously reported that complement activation is prevalent and may function as a source of procoagulant cell activation in the sera of APS patients. Recently, autoantibodies against C1q, a component of complement 1, were reported to be correlated with complement activation in systemic lupus erythematosus. These antibodies target neoepitopes of deformed C1q bound to various molecules (i.e., anionic phospholipids) and induce accelerated complement activation. We found that anti-C1q antibodies are more frequently detected in primary APS patients than in control patients and in refractory APS patients with repeated thrombotic events. The titer of anti-C1q antibodies was significantly higher in refractory APS patients than in APS patients without flare. The binding of C1q to anionic phospholipids may be associated with the surge in complement activation in patients with anti-C1q antibodies when triggered by 'second-hit' biological stressors such as infection. Such stressors will induce overexpression of anionic phospholipids, with subsequent increases in deformed C1q that is targeted by anti-C1q antibodies.

  6. Complement component 3 (C3)

    MedlinePlus

    C3 and C4 are the most commonly measured complement components. A complement test may be used to monitor people with an ... normal levels of the complement proteins C3 and C4 . Complement activity varies throughout the body. For example, ...

  7. Regulation of humoral immunity by complement.

    PubMed

    Carroll, Michael C; Isenman, David E

    2012-08-24

    The complement system of innate immunity is important in regulating humoral immunity largely through the complement receptor CR2, which forms a coreceptor on B cells during antigen-induced activation. However, CR2 also retains antigens on follicular dendritic cells (FDCs). Display of antigen on FDCs is critical for clonal selection and affinity maturation of activated B cells. This review will discuss the role of complement in adaptive immunity in general with a focus on the interplay between CR2-associated antigen on B cells with CR2 expressed on FDCs. This latter interaction provides an opportunity for memory B cells to sample antigen over prolonged periods. The cocrystal structure of CR2 with its ligand C3d provides insight into how the complement system regulates access of antigen by B cells with implications for therapeutic manipulations to modulate aberrant B cell responses in the case of autoimmunity.

  8. [Biological roles of complement and recent topics in clinical medicine].

    PubMed

    Wakamiya, Nobutaka

    2012-08-01

    The complement has been identified as a complementation factor to compensate for the function of an antibody. The complement consists of C1-C9, a complement-related molecule, and its regulating molecules. Three major biological roles of the complement have been classified: First: opsonization following phagocytosis and the elimination of microbes; second: direct destruction of bacteria due to membrane attack complex (MAC); third: complement activation following the induction of anaphylactoid factors and local recruitment and activation of neutrophilic leukocytes. In this review, the basic findings and recent treatments of paroxysmal nocturnal hemoglobinuria (PNH) and hereditary angioedema (HAE) are summarized. Finally, there is a short review of a rare autosomal recessive disorder of 3MC syndrome and new biological functions of complement factors except for that of innate immunity are proposed.

  9. Verbal Complementizers in Arabic

    ERIC Educational Resources Information Center

    Ahmed, Hossam Eldin Ibrahim

    2015-01-01

    A class of Modern Standard Arabic complementizers known as "'?inna' and its sisters" demonstrate unique case and word order restrictions. While CPs in Arabic allow both Subject-Verb (SV) and Verb-Subject (VS) word order and their subjects show nominative morphology, CPs introduced by "?inna" ban a verb from directly following…

  10. Regulation of Complement and Contact System Activation via C1 Inhibitor Potentiation and Factor XIIa Activity Modulation by Sulfated Glycans – Structure-Activity Relationships

    PubMed Central

    Schoenfeld, Ann-Kathrin; Lahrsen, Eric; Alban, Susanne

    2016-01-01

    The serpin C1 inhibitor (C1-INH) is the only regulator of classical complement activation as well as the major regulator of the contact system. Its importance is demonstrated by hereditary angioedema (HAE), a severe disease with potentially life-threatening attacks due to deficiency or dysfunction of C1-INH. C1-INH replacement is the therapy of choice in HAE. In addition, C1-INH showed to have beneficial effects in other diseases characterized by inappropriate complement and contact system activation. Due to some limitations of its clinical application, there is a need for improving the efficacy of therapeutically applied C1-INH or to enhance the activity of endogenous C1-INH. Given the known potentiating effect of heparin on C1-INH, sulfated glycans (SG) may be such candidates. The aim of this study was to characterize suitable SG by evaluating structure-activity relationships. For this, more than 40 structurally distinct SG were examined for their effects on C1-INH, C1s and FXIIa. The SG turned out to potentiate the C1s inhibition by C1-INH without any direct influence on C1s. Their potentiating activity proved to depend on their degree of sulfation, molecular mass as well as glycan structure. In contrast, the SG had no effect on the FXIIa inhibition by C1-INH, but structure-dependently modulated the activity of FXIIa. Among the tested SG, β-1,3-glucan sulfates with a Mr ≤ 10 000 were identified as most promising lead candidates for the development of a glycan-based C1-INH amplifier. In conclusion, the obtained information on structural characteristics of SG favoring C1-INH potentiation represent an useful elementary basis for the development of compounds improving the potency of C1-INH in diseases and clinical situations characterized by inappropriate activation of complement and contact system. PMID:27783665

  11. The XX sex chromosome complement is required in male and female mice for enhancement of immunity induced by exposure to 3,4-dichloropropionanilide

    PubMed Central

    Holásková, Ida; Franko, Jennifer; Goodman, Robert L.; Arnold, Arthur P.; Schafer, Rosana

    2015-01-01

    Problem The chemical propanil enhances antibody responses to a heat killed-Streptococcus pneumoniae (HKSP) vaccine. The enhanced response is dependent on gonads in females, but independent of gonads in males. The sex differences in the immune response may be due to sexual differentiation of the immune system or sex chromosome complement. Method of Study To test the hypothesis that the immune system is sexually differentiated, newborn C57BL/6 pups were treated with testosterone propionate (TP) or placebo. The role of sex chromosome complement was investigated using the 4-core genotypes (FCG) model of XXF and XYF gonadal females (ovaries), and XXM and XYM gonadal males (testes). For some experiments mice were gonadectomized or sham gonadectomized. All mice were vaccinated with HKSP, treated with propanil, and the antibody response determined at day seven. Results. Neonatal TP did not alter the response to HKSP. In FCG mice propanil significantly enhanced the immune response in XXF females and XXM males, but not in XYF females or XYM males. Conclusion The immune system of females was not masculinized by neonatal TP treatment. Sex chromosome complement significantly contributes to the sexually dimorphic immune response after propanil exposure. PMID:25765220

  12. Complement regulation: physiology and disease relevance

    PubMed Central

    2015-01-01

    The complement system is part of the innate immune response and as such defends against invading pathogens, removes immune complexes and damaged self-cells, aids organ regeneration, confers neuroprotection, and engages with the adaptive immune response via T and B cells. Complement activation can either benefit or harm the host organism; thus, the complement system must maintain a balance between activation on foreign or modified self surfaces and inhibition on intact host cells. Complement regulators are essential for maintaining this balance and are classified as soluble regulators, such as factor H, and membrane-bound regulators. Defective complement regulators can damage the host cell and result in the accumulation of immunological debris. Moreover, defective regulators are associated with several autoimmune diseases such as atypical hemolytic uremic syndrome, dense deposit disease, age-related macular degeneration, and systemic lupus erythematosus. Therefore, understanding the molecular mechanisms by which the complement system is regulated is important for the development of novel therapies for complement-associated diseases. PMID:26300937

  13. [Complement system regulation and C3 glomerulopathy].

    PubMed

    Xiao, Hui-jie; He, Rui-juan

    2013-04-18

    Complement system is a key system for immune surveillance and homeostasis. Excessive activation of complement system,especially the activation of alternative pathway may play a very important role in the pathogenesis of primary and secondary glomerulonephritis. C3 glomerulopathy is a newly named disease characterized by evident C3 deposition in the glomeruli with little or no immunoglobulin under immunofluorescence (IF). Its clinical and pathological manifestations vary a lot. The decreased plasma C3 and Factor H(FH)suggest that abnormal regulation of complement system plays an importment role in its pathogenesis. C3 glomerulopathy varies a lot as to its clinical manifestation, treatment and prognosis. The inhibition of excessive complement activation might be the key to treating C3 glomerulopathy.

  14. [Risk factors and subjective symptoms of drug-induced leucopenia].

    PubMed

    Hayashi, Kyoko; Ohtsu, Fumiko; Yano, Reiko; Sakakibara, Jinsaku; Goto, Nobuyuki

    2011-01-01

    The present study investigated risk factors and subjective symptoms associated with drug-induced leucopenia. We selected 248 patients with drug-induced leucopenia from the Case Reports of Adverse Drug Reactions and Poisoning Information System (CARPIS) database of over 47000 case reports of adverse drug reactions and assigned them to a case group. We also randomly selected 743 cases of adverse drug reactions not associated with leucopenia as a control group. A comparison of patient characteristic data between the two groups using logistic-regression analysis revealed that female sex, autoimmune disease and renal damage were background risk factors for drug-induced leucopenia. In addition, thiamazole, ritodrine, propylthiouracil, ticlopidine, allopurinol, minocycline and captopril administration significantly increased the risk of drug-induced leucopenia. A significant association was also found for fever, chills and pharyngeal abnormalities. Based on these findings, we developed two estimated regression equations to help prevent drug-induced leucopenia in the community pharmacy setting. PMID:21212623

  15. Transient cefuroxime/metronidazole treatment induced factor V antibodies.

    PubMed

    Van den Berg, Sjoerd Adrianus Antonius; Verwer, Patricia E; Idema, René N; Van Guldener, Coen

    2014-01-01

    A 29-year-old patient presented with an appendicular infiltrate, initially treated with intravenous antibiotics, but later requiring percutaneous drainage. Both prothrombin time (PT) and activated partial thromboplastin time (aPTT) were prolonged on 3 days of antibiotic treatment and unresponsive to vitamin K or prothrombin complex concentrate. Laboratory investigation ultimately showed reduced factor V activity and factor V antibodies. In contrast to previously described cases of factor V antibodies, PT and aPTT were only mildly prolonged and residual factor V activity was still >20%. Draining of the abscess did not induce significant bleeding. Afterwards, no haemostatic medication was required. The patient was discharged from the hospital without complications. One week after cessation of the antibiotic treatment, PT and aPTT were within normal range again, with a factor V activity level of 36%. In conclusion, we present a patient with transient factor V antibodies, induced by antibiotics, without clinical bleeding tendency.

  16. Genetic factors and manganese-induced neurotoxicity

    PubMed Central

    Chen, Pan; Parmalee, Nancy; Aschner, Michael

    2014-01-01

    Manganese (Mn), is a trace metal required for normal physiological processes in humans. Mn levels are tightly regulated, as high levels of Mn result in accumulation in the brain and cause a neurological disease known as manganism. Manganism shares many similarities with Parkinson’s disease (PD), both at the physiological level and the cellular level. Exposure to high Mn-containing environments increases the risk of developing manganism. Mn is absorbed primarily through the intestine and then released in the blood. Excessive Mn is secreted in the bile and excreted in feces. Mn enters and exits cells through a number of non-specific importers localized on the cell membrane. Mutations in one of the Mn exporters, SLC30A10 (solute carrier family 30, member 10), result in Mn induced toxicity with liver impairments and neurological dysfunction. Four PD genes have been identified in connection to regulation of Mn toxicity, shedding new light on potential links between manganism and PD. PMID:25136353

  17. Risk factors for ganciclovir-induced thrombocytopenia and leukopenia.

    PubMed

    Matsumoto, Kazuaki; Shigemi, Akari; Ikawa, Kazuro; Kanazawa, Naoko; Fujisaki, Yuko; Morikawa, Norifumi; Takeda, Yasuo

    2015-01-01

    Ganciclovir is a nucleoside guanosine analogue that exhibits therapeutic activity against human cytomegalovirus infection, and is primarily excreted via glomerular filtration and active tubular secretion. The adverse effects induced by ganciclovir therapy are generally of a hematological nature and include thrombocytopenia and leukopenia. Low marrow cellularity and elevated serum creatinine have been identified as risk factors for ganciclovir-induced neutropenia. However, the risk factors for thrombocytopenia have yet to be determined. Therefore, this study investigated patients administered ganciclovir to determine the risk factors for thrombocytopenia and leukopenia. Thrombocytopenia occurred in 41 of these patients (30.6%). Multivariate logistic regression analysis identified three independent risk factors for thrombocytopenia: cancer chemotherapy (odds ratio (OR)=3.1), creatinine clearance (<20 mL/min) (OR=12.8), and the ganciclovir dose (≥12 mg/kg/d) (OR=15.1). Leukopenia occurred in 36 patients (28.6%), and white blood cell count (<6000 cells/mm(3)) (OR=3.7) and the ganciclovir dose (≥12 mg/kg/d) (OR=7.8) were identified as risk factors. These results demonstrated that several factors influenced the occurrence of ganciclovir-induced thrombocytopenia and leukopenia, and suggest that special attention should be paid to patients receiving cancer chemotherapy with a low creatinine clearance (<20 mL/min) and high dose (≥12 mg/kg/d) in order to avoid ganciclovir-induced thrombocytopenia. PMID:25747982

  18. Hypoxia-Inducible Factor as an Angiogenic Master Switch

    PubMed Central

    Hashimoto, Takuya; Shibasaki, Futoshi

    2015-01-01

    Hypoxia-inducible factors (HIFs) regulate the transcription of genes that mediate the response to hypoxia. HIFs are constantly expressed and degraded under normoxia, but stabilized under hypoxia. HIFs have been widely studied in physiological and pathological conditions and have been shown to contribute to the pathogenesis of various vascular diseases. In clinical settings, the HIF pathway has been studied for its role in inhibiting carcinogenesis. HIFs might also play a protective role in the pathology of ischemic diseases. Clinical trials of therapeutic angiogenesis after the administration of a single growth factor have yielded unsatisfactory or controversial results, possibly because the coordinated activity of different HIF-induced factors is necessary to induce mature vessel formation. Thus, manipulation of HIF activity to simultaneously induce a spectrum of angiogenic factors offers a superior strategy for therapeutic angiogenesis. Because HIF-2α plays an essential role in vascular remodeling, manipulation of HIF-2α is a promising approach to the treatment of ischemic diseases caused by arterial obstruction, where insufficient development of collateral vessels impedes effective therapy. Eukaryotic initiation factor 3 subunit e (eIF3e)/INT6 interacts specifically with HIF-2α and induces the proteasome inhibitor-sensitive degradation of HIF-2α, independent of hypoxia and von Hippel-Lindau protein. Treatment with eIF3e/INT6 siRNA stabilizes HIF-2α activity even under normoxic conditions and induces the expression of several angiogenic factors, at levels sufficient to produce functional arteries and veins in vivo. We have demonstrated that administration of eIF3e/INT6 siRNA to ischemic limbs or cold-injured brains reduces ischemic damage in animal models. This review summarizes the current understanding of the relationship between HIFs and vascular diseases. We also discuss novel oxygen-independent regulatory proteins that bind HIF-α and the implications

  19. Complement activation plays a key role in the side-effects of rituximab treatment.

    PubMed

    van der Kolk, L E; Grillo-López, A J; Baars, J W; Hack, C E; van Oers, M H

    2001-12-01

    Treatment with rituximab, a chimaeric anti-CD20 monoclonal antibody, can be associated with moderate to severe first-dose side-effects, notably in patients with high numbers of circulating tumour cells. The aim of this study was to elucidate the mechanism of these side-effects. At multiple early time points during the first infusion of rituximab, complement activation products (C3b/c and C4b/c) and cytokines [tumour necrosis factor alpha (TNF-alpha), interleukin 6 (IL-6) and IL-8] were measured in five relapsed low-grade non-Hodgkin's lymphoma (NHL) patients. Infusion of rituximab induced rapid complement activation, preceding the release of TNF-alpha, IL-6 and IL-8. Although the study group was small, the level of complement activation appeared to be correlated both with the number of circulating B cells prior to the infusion (r = 0.85; P = 0.07) and with the severity of the side-effects. We conclude that complement plays a pivotal role in the pathogenesis of side-effects of rituximab treatment. As complement activation can not be prevented by corticosteroids, it might be relevant to study the possible role of complement inhibitors during the first administration of rituximab.

  20. Hypoxia inducible factor-1 alpha and multiple myeloma

    PubMed Central

    Tiwary, Bhupendra Nath

    2016-01-01

    Rapid tumor growth creates a state of hypoxia in the tumor microenvironment and results in release of hypoxia inducible factor-1 alpha (HiF-1α) in the local milieu. Hypoxia inducible factor activity is deregulated in many human cancers, especially those that are highly hypoxic. In multiple myeloma (MM) in initial stages of disease establishment, the hypoxic bone marrow microenvironment supports the initial survival and growth of the myeloma cells. Hypoxic tumour cells are usually resistant to radiotherapy and most conventional chemotherapeutic agents, rendering them highly aggressive and metastatic. Therefore, HIF is an attractive, although challenging, therapeutic target in MM directly or indirectly in recent years. PMID:26900575

  1. HUS and the case for complement.

    PubMed

    Conway, Edward M

    2015-10-29

    Hemolytic-uremic syndrome (HUS) is a thrombotic microangiopathy that is characterized by microangiopathic hemolytic anemia, thrombocytopenia, and renal failure. Excess complement activation underlies atypical HUS and is evident in Shiga toxin-induced HUS (STEC-HUS). This Spotlight focuses on new knowledge of the role of Escherichia coli-derived toxins and polyphosphate in modulating complement and coagulation, and how they affect disease progression and response to treatment. Such new insights may impact on current and future choices of therapies for STEC-HUS.

  2. Serum-induced potentiation of tumor necrosis factor alpha production by human monocytes in response to staphylococcal peptidoglycan: involvement of different serum factors.

    PubMed Central

    Mattsson, E; Rollof, J; Verhoef, J; Van Dijk, H; Fleer, A

    1994-01-01

    Peptidoglycan from a Staphylococcus epidermidis strain, isolated from a patient with septicemia, was preincubated with human serum. This mixture was then investigated for its potency to induce tumor necrosis factor (TNF) secretion by human blood monocytes. TNF was measured in the supernatants by using a bioassay and/or an enzyme-linked immunosorbent assay specific for TNF alpha (TNF-alpha). Although earlier studies indicated that staphylococcal peptidoglycan alone is a relatively poor stimulator of TNF-alpha production, the present study shows that human serum highly potentiates peptidoglycan-induced TNF-alpha release by human monocytes. In the presence of serum and in the low-dose range, peptidoglycan was almost as potent as endotoxin. At high peptidoglycan concentrations, monocytes showed an extremely high TNF-alpha response, but again only in the presence of serum. At low peptidoglycan doses, the stimulatory effect of serum was abrogated by heat treatment or depleting serum of complement components C1 and C3/C4, which suggests a role for the classical complement pathway. At high doses of peptidoglycan, the serum stimulatory effect depended mainly on immunoglobulin G. PMID:8063400

  3. Tumor Necrosis Factor Related Apoptosis Inducing Ligand (Trail) in endothelial response to biomechanical and biochemical stresses in arteries.

    PubMed

    D'Auria, F; Centurione, L; Centurione, M A; Angelini, A; Di Pietro, R

    2015-11-01

    Shear stress is determined by three physical components described in a famous triad: blood flow, blood viscosity and vessel geometry. Through the direct action on endothelium, shear stress is able to radically interfere with endothelial properties and the physiology of the vascular wall. Endothelial cells (ECs) have also to sustain biochemical stresses represented by chemokines, growth factors, cytokines, complement, hormones, nitric oxide (NO), oxygen and reactive oxygen species (ROS). Many growth factors, cytokines, chemokines, hormones, and chemical substances, like NO, act and regulate endothelium functions and homeostasis. Among these cytokines Tumor Necrosis Factor Related Apoptosis Inducing Ligand (TRAIL) has been assigned a regulatory role in ECs physiology and physiopathology. Thus, the aim of this review is to provide a general overview of the endothelial response pathways after different types of biomechanical and biochemical stress in in vitro models and to analyze the crucial role of TRAIL under pathological conditions of the cardiocirculatory system like atherosclerosis, coronary artery disease, and diabetes.

  4. Complement Opsonization of HIV-1 Results in Decreased Antiviral and Inflammatory Responses in Immature Dendritic Cells via CR3

    PubMed Central

    Ellegård, Rada; Crisci, Elisa; Burgener, Adam; Sjöwall, Christopher; Birse, Kenzie; Westmacott, Garrett; Hinkula, Jorma; Lifson, Jeffrey D.

    2014-01-01

    Immature dendritic cells (iDCs) in genital and rectal mucosa may be one of the first cells to come into contact with HIV-1 during sexual transmission of virus. HIV-1 activates the host complement system, which results in opsonization of virus by inactivated complement fragments, for example, iC3b. We investigated antiviral and inflammatory responses induced in human iDCs after exposure to free HIV-1 (F-HIV), complement-opsonized HIV-1 (C-HIV), and complement and Ab–opsonized HIV-1 (CI-HIV). F-HIV gave rise to a significantly higher expression of antiviral factors such as IFN-β, myxovirus resistance protein A, and IFN-stimulated genes, compared with C-HIV and CI-HIV. Additionally, F-HIV induced inflammatory factors such as IL-1β, IL-6, and TNF-α, whereas these responses were weakened or absent after C-HIV or CI-HIV exposure. The responses induced by F-HIV were TLR8-dependent with subsequent activation of IFN regulatory factor 1, p38, ERK, PI3K, and NF-κB pathways, whereas these responses were not induced by C-HIV, which instead induced activation of IFN regulatory factor 3 and Lyn. This modulation of TLR8 signaling was mediated by complement receptor 3 and led to enhanced infection. The impact that viral hijacking of the complement system has on iDC function could be an important immune evasion mechanism used by HIV-1 to establish infection in the host. PMID:25252956

  5. Complement analysis 2016: Clinical indications, laboratory diagnostics and quality control.

    PubMed

    Prohászka, Zoltán; Nilsson, Bo; Frazer-Abel, Ashley; Kirschfink, Michael

    2016-11-01

    In recent years, complement analysis of body fluids and biopsies, going far beyond C3 and C4, has significantly enhanced our understanding of the disease process. Such expanded complement analysis allows for a more precise differential diagnosis and for critical monitoring of complement-targeted therapy. These changes are a result of the growing understanding of the involvement of complement in a diverse set of disorders. To appreciate the importance of proper complement analysis, it is important to understand the role it plays in disease. Historically, it was the absence of complement as manifested in severe infection that was noted. Since then complement has been connected to a variety of inflammatory disorders, such as autoimmune diseases and hereditary angioedema. While the role of complement in the rejection of renal grafts has been known longer, the significant impact of complement. In certain nephropathies has now led to the reclassification of some rare kidney diseases and an increased role for complement analysis in diagnosis. Even more unexpected is that complement has also been implicated in neural, ophtalmological and dermatological disorders. With this level of involvement in some varied and impactful health issues proper complement testing is clearly important; however, analysis of the complement system varies widely among laboratories. Except for a few proteins, such as C3 and C4, there are neither well-characterized standard preparations nor calibrated assays available. This is especially true for the inter-laboratory variation of tests which assess classical, alternative, or lectin pathway function. In addition, there is a need for the standardization of the measurement of complement activation products that are so critical in determining whether clinically relevant complement activation has occurred in vivo. Finally, autoantibodies to complement proteins (e.g. anti-C1q), C3 and C4 convertases (C3 and C4 nephritic factor) or to regulatory proteins

  6. Complement analysis 2016: Clinical indications, laboratory diagnostics and quality control.

    PubMed

    Prohászka, Zoltán; Nilsson, Bo; Frazer-Abel, Ashley; Kirschfink, Michael

    2016-11-01

    In recent years, complement analysis of body fluids and biopsies, going far beyond C3 and C4, has significantly enhanced our understanding of the disease process. Such expanded complement analysis allows for a more precise differential diagnosis and for critical monitoring of complement-targeted therapy. These changes are a result of the growing understanding of the involvement of complement in a diverse set of disorders. To appreciate the importance of proper complement analysis, it is important to understand the role it plays in disease. Historically, it was the absence of complement as manifested in severe infection that was noted. Since then complement has been connected to a variety of inflammatory disorders, such as autoimmune diseases and hereditary angioedema. While the role of complement in the rejection of renal grafts has been known longer, the significant impact of complement. In certain nephropathies has now led to the reclassification of some rare kidney diseases and an increased role for complement analysis in diagnosis. Even more unexpected is that complement has also been implicated in neural, ophtalmological and dermatological disorders. With this level of involvement in some varied and impactful health issues proper complement testing is clearly important; however, analysis of the complement system varies widely among laboratories. Except for a few proteins, such as C3 and C4, there are neither well-characterized standard preparations nor calibrated assays available. This is especially true for the inter-laboratory variation of tests which assess classical, alternative, or lectin pathway function. In addition, there is a need for the standardization of the measurement of complement activation products that are so critical in determining whether clinically relevant complement activation has occurred in vivo. Finally, autoantibodies to complement proteins (e.g. anti-C1q), C3 and C4 convertases (C3 and C4 nephritic factor) or to regulatory proteins

  7. [BPO-Specific, complement-dependant cell-lysis of differently sensitized sheep red cells: evaluation of haptenic groups and their influence on IgM and IgG-induced lysis (author's transl)].

    PubMed

    Wiedermann, G; Stemberger, H; Förster, O; Müller, M

    1976-04-01

    Sheep erythrocytes were coated with bencylpenicilloyl-(BPO)groups. Different incubation periods resulted in erythrocyte preparations with different hapten density. Complement dependent lysis induced by IgM or IgG antibodies was studied with the cell preparations. The calculation of hapten density on the erythrocyte surface was not possible by direct measurement of coupled radioactive BPO since more than 90% of radioactive material was found in the soluble supernatant after osmotic cell lysis and less than 10% was fixed to the cellular membrane. Measurement of membrane bound immunologically relevant BPO-groups was achieved, therefore, by comparison of the inhibitory capacity of the test cells with that of a standard cell preparation. The latter consisted of tannic acid treated erythrocytes coated with protein complexed radioactive BPO. Surface hapten density of the different target cell preparations varied between 1.9 x 10(5) and 4.8 10(5) BPO-groups per cell depending on the time of incubation. Complement dependent antibody mediated cell lysis was significantly reduced by reduction of haptenic sites per target cell, IgG induced lysis being much more affected than hemolysis induced by IgM antibodies. Statistical calculations led to the conclusion that 18,000 protein islets per cell bearing 4 or more BPO-groups are not sufficient for hemolysis induced by IgG antibodies. 48,000 protein islets with this hapten density are necessary for "optimal" sensitization. IgG antibodies must be apparently bound to the cell surface in bivalent form.

  8. Complement activation in chronic liver disease.

    PubMed Central

    Munoz, L E; De Villiers, D; Markham, D; Whaley, K; Thomas, H C

    1982-01-01

    Patients with HBsAg positive chronic active liver disease (CALD) and primary biliary cirrhosis (PBC) exhibit increased C3d concentrations and changes in the serum concentrations of the complement components consistent with activation of the classical and alternative pathways. In these patients the concentrations of the regulatory proteins, C3b inactivator (C3bINA) and beta IH globulin, are normal. Patients with HBsAg negative CALD and alcohol induced liver disease (ALD) exhibit no evidence of an increased level of complement system activation. In these patients diminished serum concentrations of complement components appear to be related to diminished hepatic synthetic function. C4 synthesis may be specifically reduced in autoimmune chronic active liver disease. PMID:7083631

  9. Supramolecular Control over Split-Luciferase Complementation.

    PubMed

    Bosmans, Ralph P G; Briels, Jeroen M; Milroy, Lech-Gustav; de Greef, Tom F A; Merkx, Maarten; Brunsveld, Luc

    2016-07-25

    Supramolecular split-enzyme complementation restores enzymatic activity and allows for on-off switching. Split-luciferase fragment pairs were provided with an N-terminal FGG sequence and screened for complementation through host-guest binding to cucurbit[8]uril (Q8). Split-luciferase heterocomplex formation was induced in a Q8 concentration dependent manner, resulting in a 20-fold upregulation of luciferase activity. Supramolecular split-luciferase complementation was fully reversible, as revealed by using two types of Q8 inhibitors. Competition studies with the weak-binding FGG peptide revealed a 300-fold enhanced stability for the formation of the ternary heterocomplex compared to binding of two of the same fragments to Q8. Stochiometric binding by the potent inhibitor memantine could be used for repeated cycling of luciferase activation and deactivation in conjunction with Q8, providing a versatile module for in vitro supramolecular signaling networks.

  10. Growth factor involvement in tension-induced skeletal muscle growth

    NASA Technical Reports Server (NTRS)

    Vandenburgh, Herman H.

    1993-01-01

    Long-term manned space travel will require a better understanding of skeletal muscle atrophy which results from microgravity. Astronaut strength and dexterity must be maintained for normal mission operations and for emergency situations. Although exercise in space slows the rate of muscle loss, it does not prevent it. A biochemical understanding of how gravity/tension/exercise help to maintain muscle size by altering protein synthesis and/or degradation rate should ultimately allow pharmacological intervention to prevent muscle atrophy in microgravity. The overall objective is to examine some of the basic biochemical processes involved in tension-induced muscle growth. With an experimental in vitro system, the role of exogenous and endogenous muscle growth factors in mechanically stimulated muscle growth are examined. Differentiated avian skeletal myofibers can be 'exercised' in tissue culture using a newly developed dynamic mechanical cell stimulator device which simulates different muscle activity patterns. Patterns of mechanical activity which significantly affect muscle growth and metabolic characteristics were found. Both exogenous and endogenous growth factors are essential for tension-induced muscle growth. Exogenous growth factors found in serum, such as insulin, insulin-like growth factors, and steroids, are important regulators of muscle protein turnover rates and mechanically-induced muscle growth. Endogenous growth factors are synthesized and released into the culture medium when muscle cells are mechanically stimulated. At least one family of mechanically induced endogenous factors, the prostaglandins, help to regulate the rates of protein turnover in muscle cells. Endogenously synthesized IGF-1 is another. The interaction of muscle mechanical activity and these growth factors in the regulation of muscle protein turnover rates with our in vitro model system is studied.

  11. CLINICAL FACTORS FOR DEVELOPING SHOCK IN RADIOCONTRAST MEDIA INDUCED ANAPHYLAXIS.

    PubMed

    Kim, Sang Min; Ko, Byuk Sung; Kim, Ji Yeon; Ha, Sang Ook; Ahn, Shin; Sohn, Chang Hwan; Seo, Dong Woo; Kim, Tae-Bum; Kim, Won Young

    2016-03-01

    The aim of this study was to investigate the time interval between radiocontrast media (RCM) administration and the development of anaphylactic shock, and risk factors associated with RCM-induced anaphylactic shock. We reviewed the medical records of 154 patients with RCM-induced anaphylaxis presenting to the emergency department of a tertiary care hospital between January 2005 and December 2014. Clinical features of RCM-induced anaphylaxis were analyzed, and patients were categorized into shock and non-shock groups to identify associated factors in affected patients. Of the 154 cases of RCM-induced anaphylaxis, 101 (65.9%) patients experienced shock. The median time between RCM exposure and the onset of shock was 11 min (interquartile range, 7.0-18.8). In patients with RCM-induced anaphylaxis accompanying shock, the median time from RCM to the first symptom onset was 6 min (interquartile range, 5.0-10.0). In the multivariate analysis, age, neurological manifestations, and allergy history except RCM were associated with the development of shock. RCM-induced anaphylaxis was commonly accompanied with shock, and the time interval between RCM exposure and the onset of shock was short. Physicians should pay attention to the development of potential cardiovascular collapse in anaphylaxis patients of old age and with neurologic manifestations. PMID:26506069

  12. Hypoxia Inducible Factors and Hypertension: Lessons from Sleep Apnea Syndrome

    PubMed Central

    Nanduri, Jayasri; Peng, Ying-Jie; Yuan, Guoxiang; Kumar, Ganesh K.; Prabhakar, Nanduri R.

    2015-01-01

    Systemic hypertension is one of the most prevalent cardiovascular diseases. Sleep disordered breathing (SDB) with recurrent apnea is a major risk factor for developing essential hypertension. Chronic intermittent hypoxia (CIH) is a hallmark manifestation of recurrent apnea. Rodent models patterned after the O2 profiles seen with SDB patients showed that CIH is the major stimulus for causing systemic hypertension. This article reviews the physiological and molecular basis of CIH-induced hypertension. Physiological studies have identified that augmented carotid body chemosensory reflex and the resulting increase in sympathetic nerve activity is a major contributor to CIH-induced hypertension. Analysis of molecular mechanisms revealed that CIH activates hypoxia-inducible factor (HIF)-1 and suppresses HIF-2- mediated transcription. Dysregulation of HIF-1- and HIF-2- mediated transcription leads to imbalance of pro-oxidant and anti-oxidant enzyme gene expression resulting in increased reactive species (ROS) generation in the chemosensory reflex which is central for developing hypertension. PMID:25772710

  13. Inhibition of Complement Retards Ankylosing Spondylitis Progression

    PubMed Central

    Yang, Chaoqun; Ding, Peipei; Wang, Qingkai; Zhang, Long; Zhang, Xin; Zhao, Jianquan; Xu, Enjie; Wang, Na; Chen, Jianfeng; Yang, Guang; Hu, Weiguo; Zhou, Xuhui

    2016-01-01

    Ankylosing spondylitis (AS) is a chronic axial spondyloarthritis (SpA) resulting in back pain and progressive spinal ankyloses. Currently, there are no effective therapeutics targeting AS largely due to elusive pathogenesis mechanisms, even as potential candidates such as HLA-B27 autoantigen have been identified. Herein, we employed a proteoglycan (PG)-induced AS mouse model together with clinical specimens, and found that the complement system was substantially activated in the spinal bone marrow, accompanied by a remarkable proportion alteration of neutrophils and macrophage in bone marrow and spleen, and by the significant increase of TGF-β1 in serum. The combined treatment with a bacteria-derived complement inhibitor Efb-C (C-terminal of extracellular fibrinogen-binding protein of Staphylococcus aureus) remarkably retarded the progression of mouse AS by reducing osteoblast differentiation. Furthermore, we demonstrated that two important modulators involved in AS disease, TGF-β1 and RANKL, were elevated upon in vitro complement attack in osteoblast and/or osteoclast cells. These findings further unravel that complement activation is closely related with the pathogenesis of AS, and suggest that complement inhibition may hold great potential for AS therapy. PMID:27698377

  14. Complement in monoclonal antibody therapy of cancer.

    PubMed

    Rogers, Laura M; Veeramani, Suresh; Weiner, George J

    2014-08-01

    Monoclonal antibodies (mAb) have been used as targeted treatments against cancer for more than a decade, with mixed results. Research is needed to understand mAb mechanisms of action with the goal of improving the efficacy of currently used mAbs and guiding the design of novel mAbs. While some mAb-induced tumor cell killing is a result of direct effects on tumor cell signaling, mAb opsonization of tumor cells also triggers activation of immune responses due to complement activation and engagement of antibody receptors on immune effector cells. In fact, complement has been shown to play an important role in modulating the anti-tumor activity of many mAb through complement-dependent cytotoxicity, antibody-dependent cytotoxicity, and through indirect effects by modulating the tumor microenvironment. Complement activity can have both agonistic and antagonistic effects on these processes. How the balance of such effects impacts on the clinical efficacy of mAb therapy remains unclear. In this review, we discuss the mAbs currently approved for cancer treatment and examine how complement can impact their efficacy with a focus on how this information might be used to improve the clinical efficacy of mAb treatment.

  15. Sequence and Role in Virulence of the Three Plasmid Complement of the Model Tumor-Inducing Bacterium Pseudomonas savastanoi pv. savastanoi NCPPB 3335

    PubMed Central

    Bardaji, Leire; Pérez-Martínez, Isabel; Rodríguez-Moreno, Luis; Rodríguez-Palenzuela, Pablo; Sundin, George W.; Ramos, Cayo; Murillo, Jesús

    2011-01-01

    Pseudomonas savastanoi pv. savastanoi NCPPB 3335 is a model for the study of the molecular basis of disease production and tumor formation in woody hosts, and its draft genome sequence has been recently obtained. Here we closed the sequence of the plasmid complement of this strain, composed of three circular molecules of 78,357 nt (pPsv48A), 45,220 nt (pPsv48B), and 42,103 nt (pPsv48C), all belonging to the pPT23A-like family of plasmids widely distributed in the P. syringae complex. A total of 152 coding sequences were predicted in the plasmid complement, of which 38 are hypothetical proteins and seven correspond to putative virulence genes. Plasmid pPsv48A contains an incomplete Type IVB secretion system, the type III secretion system (T3SS) effector gene hopAF1, gene ptz, involved in cytokinin biosynthesis, and three copies of a gene highly conserved in plant-associated proteobacteria, which is preceded by a hrp box motif. A complete Type IVA secretion system, a well conserved origin of transfer (oriT), and a homolog of the T3SS effector gene hopAO1 are present in pPsv48B, while pPsv48C contains a gene with significant homology to isopentenyl-diphosphate delta-isomerase, type 1. Several potential mobile elements were found on the three plasmids, including three types of MITE, a derivative of IS801, and a new transposon effector, ISPsy30. Although the replication regions of these three plasmids are phylogenetically closely related, their structure is diverse, suggesting that the plasmid architecture results from an active exchange of sequences. Artificial inoculations of olive plants with mutants cured of plasmids pPsv48A and pPsv48B showed that pPsv48A is necessary for full virulence and for the development of mature xylem vessels within the knots; we were unable to obtain mutants cured of pPsv48C, which contains five putative toxin-antitoxin genes. PMID:22022435

  16. Overexpression of gankyrin in mouse hepatocytes induces hemangioma by suppressing factor inhibiting hypoxia-inducible factor-1 (FIH-1) and activating hypoxia-inducible factor-1.

    PubMed

    Liu, Yu; Higashitsuji, Hiroaki; Higashitsuji, Hisako; Itoh, Katsuhiko; Sakurai, Toshiharu; Koike, Kazuhiko; Hirota, Kiichi; Fukumoto, Manabu; Fujita, Jun

    2013-03-01

    Gankyrin (also called p28 or PSMD10) is an oncoprotein commonly overexpressed in hepatocellular carcinomas. It consists of 7 ankyrin repeats and interacts with multiple proteins including Rb, Cdk4, MDM2 and NF-κB. To assess the oncogenic activity in vivo, we produced transgenic mice that overexpress gankyrin specifically in the hepatocytes. Unexpectedly, 5 of 7 F2 transgenic mice overexpressing hepatitis B virus X protein (HBX) promoter-driven gankyrin, and one of 3 founder mice overexpressing serum amyloid P component (SAP) promoter-driven gankyrin developed hepatic vascular neoplasms (hemangioma/hemangiosarcomas) whereas none of the wild-type mice did. Endothelial overgrowth was more frequent in the livers of diethylnitrosamine-treated transgenic mice than wild-type mice. Mouse hepatoma Hepa1-6 cells overexpressing gankyrin formed tumors with more vascularity than parental Hepa1-6 cells in the transplanted mouse skin. We found that gankyrin binds to and sequester factor inhibiting hypoxia-inducible factor-1 (FIH-1), which results in decreased interaction between FIH-1 and hypoxia-inducible factor-1α (HIF-1α) and increased activity of HIF-1 to promote VEGF production. The effects of gankyrin were more prominent under 3% O2 than 1% or 20% O2 conditions. Thus, the present study clarified, at least partly, mechanisms of vascular tumorigenesis, and suggests that gankyrin might play a physiological role in hypoxic responses besides its roles as an oncoprotein. PMID:23376718

  17. Engineering of human complement component C3 for catalytic inhibition of complement.

    PubMed

    Kölln, Johanna; Bredehorst, Reinhard; Spillner, Edzard

    2005-04-15

    As a novel therapeutic approach in complement-mediated pathologies, we recently developed a human C3 derivative capable of obliterating functional complement by a catalytic, non-inhibitory mechanism. In this derivative, the C-terminal region of hC3 was substituted by a 275 amino acid sequence derived from the corresponding sequence of cobra venom factor (CVF), a complement-activating C3b homologue from snake venom. In this study, we replaced shorter C-terminal sequences of hC3 by corresponding CVF sequences to further reduce potential immunogenicity and to identify domains essential for the formation of functionally stable C3 convertases. In one of these derivatives that is still capable of obliterating functional complement in vitro, the non-human portion could be reduced to a small domain located in the C-terminus of different complement proteins. This conserved NTR/C345C motif is known to be involved in assembly of different convertases of the complement system. These results suggest a major role of the C345C domain in the regulation of the half-life of the C3 convertase. Moreover, its overall identity of 96% to human C3 renders this derivative a promising candidate for therapeutic intervention in complement-mediated pathologies. PMID:15790508

  18. Complementing Gender Analysis Methods.

    PubMed

    Kumar, Anant

    2016-01-01

    The existing gender analysis frameworks start with a premise that men and women are equal and should be treated equally. These frameworks give emphasis on equal distribution of resources between men and women and believe that this will bring equality which is not always true. Despite equal distribution of resources, women tend to suffer and experience discrimination in many areas of their lives such as the power to control resources within social relationships, and the need for emotional security and reproductive rights within interpersonal relationships. These frameworks believe that patriarchy as an institution plays an important role in women's oppression, exploitation, and it is a barrier in their empowerment and rights. Thus, some think that by ensuring equal distribution of resources and empowering women economically, institutions like patriarchy can be challenged. These frameworks are based on proposed equality principle which puts men and women in competing roles. Thus, the real equality will never be achieved. Contrary to the existing gender analysis frameworks, the Complementing Gender Analysis framework proposed by the author provides a new approach toward gender analysis which not only recognizes the role of economic empowerment and equal distribution of resources but suggests to incorporate the concept and role of social capital, equity, and doing gender in gender analysis which is based on perceived equity principle, putting men and women in complementing roles that may lead to equality. In this article the author reviews the mainstream gender theories in development from the viewpoint of the complementary roles of gender. This alternative view is argued based on existing literature and an anecdote of observations made by the author. While criticizing the equality theory, the author offers equity theory in resolving the gender conflict by using the concept of social and psychological capital. PMID:25941756

  19. Complementing Gender Analysis Methods.

    PubMed

    Kumar, Anant

    2016-01-01

    The existing gender analysis frameworks start with a premise that men and women are equal and should be treated equally. These frameworks give emphasis on equal distribution of resources between men and women and believe that this will bring equality which is not always true. Despite equal distribution of resources, women tend to suffer and experience discrimination in many areas of their lives such as the power to control resources within social relationships, and the need for emotional security and reproductive rights within interpersonal relationships. These frameworks believe that patriarchy as an institution plays an important role in women's oppression, exploitation, and it is a barrier in their empowerment and rights. Thus, some think that by ensuring equal distribution of resources and empowering women economically, institutions like patriarchy can be challenged. These frameworks are based on proposed equality principle which puts men and women in competing roles. Thus, the real equality will never be achieved. Contrary to the existing gender analysis frameworks, the Complementing Gender Analysis framework proposed by the author provides a new approach toward gender analysis which not only recognizes the role of economic empowerment and equal distribution of resources but suggests to incorporate the concept and role of social capital, equity, and doing gender in gender analysis which is based on perceived equity principle, putting men and women in complementing roles that may lead to equality. In this article the author reviews the mainstream gender theories in development from the viewpoint of the complementary roles of gender. This alternative view is argued based on existing literature and an anecdote of observations made by the author. While criticizing the equality theory, the author offers equity theory in resolving the gender conflict by using the concept of social and psychological capital.

  20. Production of Multiple Transgenic Yucatan Miniature Pigs Expressing Human Complement Regulatory Factors, Human CD55, CD59, and H-Transferase Genes

    PubMed Central

    Jang, Gun-Hyuk; Jeong, Yeun-Ik; Hwang, In-Sung; Jeong, Yeon-woo; Kim, Yu-Kyung; Shin, Taeyoung; Kim, Nam-Hyung; Hyun, Sang-Hwan; Jeung, Eui-Bae; Hwang, Woo-Suk

    2013-01-01

    The present study was conducted to generate transgenic pigs coexpressing human CD55, CD59, and H-transferase (HT) using an IRES-mediated polycistronic vector. The study focused on hyperacute rejection (HAR) when considering clinical xenotransplantation as an alternative source for human organ transplants. In total, 35 transgenic cloned piglets were produced by somatic cell nuclear transfer (SCNT) and were confirmed for genomic integration of the transgenes from umbilical cord samples by PCR analysis. Eighteen swine umbilical vein endothelial cells (SUVEC) were isolated from umbilical cord veins freshly obtained from the piglets. We observed a higher expression of transgenes in the transgenic SUVEC (Tg SUVEC) compared with the human umbilical vein endothelial cells (HUVEC). Among these genes, HT and hCD59 were expressed at a higher level in the tested Tg organs compared with non-Tg control organs, but there was no difference in hCD55 expression between them. The transgenes in various organs of the Tg clones revealed organ-specific and spatial expression patterns. Using from 0 to 50% human serum solutions, we performed human complement-mediated cytolysis assays. The results showed that, overall, the Tg SUVEC tested had greater survival rates than did the non-Tg SUVEC, and the Tg SUVEC with higher HT expression levels tended to have more down-regulated α-Gal epitope expression, resulting in greater protection against cytotoxicity. By contrast, several Tg SUVEC with low CD55 expression exhibited a decreased resistance response to cytolysis. These results indicated that the levels of HT expression were inversely correlated with the levels of α-Gal epitope expression and that the combined expression of hCD55, hCD59, and HT proteins in SUVECs markedly enhances a protective response to human serum-mediated cytolysis. Taken together, these results suggest that combining a polycistronic vector system with SCNT methods provides a fast and efficient alternative for the

  1. Factors that modify risks of radiation-induced cancer

    SciTech Connect

    Fabrikant, J.I.

    1988-11-01

    The collective influence of biologic and physical factors that modify risks of radiation-induced cancer introduces uncertainties sufficient to deny precision of estimates of human cancer risk that can be calculated for low-dose radiation in exposed populations. The important biologic characteristics include the tissue sites and cell types, baseline cancer incidence, minimum latent period, time-to-tumor recognition, and the influence of individual host (age and sex) and competing etiologic influences. Physical factors include radiation dose, dose rate, and radiation quality. Statistical factors include time-response projection models, risk coefficients, and dose-response relationships. Other modifying factors include other carcinogens, and other biological sources (hormonal status, immune status, hereditary factors).

  2. Characterization of the third component of complement (C3) after activation by cigarette smoke

    SciTech Connect

    Kew, R.R.; Ghebrehiwet, B.; Janoff, A.

    1987-08-01

    Activation of lung complement by tobacco smoke may be an important pathogenetic factor in the development of pulmonary emphysema in smokers. We previously showed that cigarette smoke can modify C3 and activate the alternative pathway of complement in vitro. However, the mechanism of C3 activation was not fully delineated in these earlier studies. In the present report, we show that smoke-treated C3 induces cleavage of the alternative pathway protein, Factor B, when added to serum containing Mg-EGTA. This effect of cigarette smoke is specific for C3 since smoke-treated C4, when added to Mg-EGTA-treated serum, fails to activate the alternative pathway and fails to induce Factor B cleavage. Smoke-modified C3 no longer binds significant amounts of (/sup 14/C)methylamine (as does native C3), and relatively little (/sup 14/C)methylamine is incorporated into its alpha-chain. Thus, prior internal thiolester bond cleavage appears to have occurred in C3 activated by cigarette smoke. Cigarette smoke components also induce formation of noncovalently associated, soluble C3 multimers, with a Mr ranging from 1 to 10 million. However, prior cleavage of the thiolester bond in C3 with methylamine prevents the subsequent formation of these smoke-induced aggregates. These data indicate that cigarette smoke activates the alternative pathway of complement by specifically modifying C3 and that these modifications include cleavage of the thiolester bond in C3 and formation of noncovalently linked C3 multimers.

  3. Characterization of the third component of complement (C3) after activation by cigarette smoke.

    PubMed

    Kew, R R; Ghebrehiwet, B; Janoff, A

    1987-08-01

    Activation of lung complement by tobacco smoke may be an important pathogenetic factor in the development of pulmonary emphysema in smokers. We previously showed that cigarette smoke can modify C3 and activate the alternative pathway of complement in vitro. However, the mechanism of C3 activation was not fully delineated in these earlier studies. In the present report, we show that smoke-treated C3 induces cleavage of the alternative pathway protein, Factor B, when added to serum containing Mg-EGTA. This effect of cigarette smoke is specific for C3 since smoke-treated C4, when added to Mg-EGTA-treated serum, fails to activate the alternative pathway and fails to induce Factor B cleavage. Smoke-modified C3 no longer binds significant amounts of [14C]methylamine (as does native C3), and relatively little [14C]methylamine is incorporated into its alpha-chain. Thus, prior internal thiolester bond cleavage appears to have occurred in C3 activated by cigarette smoke. Cigarette smoke components also induce formation of noncovalently associated, soluble C3 multimers, with a Mr ranging from 1 to 10 million. However, prior cleavage of the thiolester bond in C3 with methylamine prevents the subsequent formation of these smoke-induced aggregates. These data indicate that cigarette smoke activates the alternative pathway of complement by specifically modifying C3 and that these modifications include cleavage of the thiolester bond in C3 and formation of noncovalently linked C3 multimers.

  4. Initiation and Regulation of Complement during Hemolytic Transfusion Reactions

    PubMed Central

    Stowell, Sean R.; Winkler, Anne M.; Maier, Cheryl L.; Arthur, C. Maridith; Smith, Nicole H.; Girard-Pierce, Kathryn R.; Cummings, Richard D.; Zimring, James C.; Hendrickson, Jeanne E.

    2012-01-01

    Hemolytic transfusion reactions represent one of the most common causes of transfusion-related mortality. Although many factors influence hemolytic transfusion reactions, complement activation represents one of the most common features associated with fatality. In this paper we will focus on the role of complement in initiating and regulating hemolytic transfusion reactions and will discuss potential strategies aimed at mitigating or favorably modulating complement during incompatible red blood cell transfusions. PMID:23118779

  5. Complement-mediated antiinflammatory effect of bisbenzylisoquinoline alkaloid fangchinoline.

    PubMed

    Hristova, M; Istatkova, R

    1999-11-01

    Complement-mediated mode of action of bisbenzylisoquinoline alkaloid fangchinoline was investigated in vivo and in vitro. The application of fangchinoline intraperitoneally (i.p.) to complement normal mice, strain ICR, inhibited the complement activity in serum and peritoneal exudate. The substance activated serum complement of C5-deficient DBA/2 mice. Fangchinoline was able to provoke local inflammatory reaction in both strains after subcutaneous (s.c.) injection. The alkaloid suppressed paw swelling induced by live Candida albicans in ICR and DBA/2 mice. Its effect depended on the dose and time of injection prior to inflammatory reaction. The in vitro experiments proved the interference of fangchinoline action with post-C5 reactions. The substance augmented C5-convertase formation and functional activity. These results are in correspondence with our previous investigations, proving the complement-mediated action of fangchinoline. The antiinflammatory effect could be a consequence of the caused complement exhaustion. PMID:11962544

  6. Utilizing complement evasion strategies to design complement-based antibacterial immunotherapeutics: Lessons from the pathogenic Neisseriae.

    PubMed

    Ram, Sanjay; Shaughnessy, Jutamas; DeOliveira, Rosane B; Lewis, Lisa A; Gulati, Sunita; Rice, Peter A

    2016-10-01

    Novel therapies are urgently needed to combat the global threat of multidrug-resistant pathogens. Complement forms an important arm of innate defenses against infections. In physiological conditions, complement activation is tightly controlled by soluble and membrane-associated complement inhibitors, but must be selectively activated on invading pathogens to facilitate microbial clearance. Many pathogens, including Neisseria gonorrhoeae and N. meningitidis, express glycans, including N-acetylneuraminic acid (Neu5Ac), that mimic host structures to evade host immunity. Neu5Ac is a negatively charged 9-cabon sugar that inhibits complement, in part by enhancing binding of the complement inhibitor factor H (FH) through C-terminal domains (19 and 20) on FH. Other microbes also bind FH, in most instances through FH domains 6 and 7 or 18-20. Here we describe two strategies to target complement activation on Neisseriae. First, microbial binding domains of FH were fused to IgG Fc to create FH18-20/Fc (binds gonococci) and FH6,7/Fc (binds meningococci). A point mutation in FH domain 19 eliminated hemolysis caused by unmodified FH18-20, but retained binding to gonococci. FH18-20/Fc and FH6,7/Fc mediated complement-dependent killing in vitro and showed efficacy in animal models of gonorrhea and meningococcal bacteremia, respectively. The second strategy utilized CMP-nonulosonate (CMP-NulO) analogs of sialic acid that were incorporated into LOS and prevented complement inhibition by physiologic CMP-Neu5Ac and resulted in attenuated gonococcal infection in mice. While studies to establish the safety of these agents are needed, enhancing complement activation on microbes may represent a promising strategy to treat antimicrobial resistant organisms. PMID:27297292

  7. Utilizing complement evasion strategies to design complement-based antibacterial immunotherapeutics: Lessons from the pathogenic Neisseriae.

    PubMed

    Ram, Sanjay; Shaughnessy, Jutamas; DeOliveira, Rosane B; Lewis, Lisa A; Gulati, Sunita; Rice, Peter A

    2016-10-01

    Novel therapies are urgently needed to combat the global threat of multidrug-resistant pathogens. Complement forms an important arm of innate defenses against infections. In physiological conditions, complement activation is tightly controlled by soluble and membrane-associated complement inhibitors, but must be selectively activated on invading pathogens to facilitate microbial clearance. Many pathogens, including Neisseria gonorrhoeae and N. meningitidis, express glycans, including N-acetylneuraminic acid (Neu5Ac), that mimic host structures to evade host immunity. Neu5Ac is a negatively charged 9-cabon sugar that inhibits complement, in part by enhancing binding of the complement inhibitor factor H (FH) through C-terminal domains (19 and 20) on FH. Other microbes also bind FH, in most instances through FH domains 6 and 7 or 18-20. Here we describe two strategies to target complement activation on Neisseriae. First, microbial binding domains of FH were fused to IgG Fc to create FH18-20/Fc (binds gonococci) and FH6,7/Fc (binds meningococci). A point mutation in FH domain 19 eliminated hemolysis caused by unmodified FH18-20, but retained binding to gonococci. FH18-20/Fc and FH6,7/Fc mediated complement-dependent killing in vitro and showed efficacy in animal models of gonorrhea and meningococcal bacteremia, respectively. The second strategy utilized CMP-nonulosonate (CMP-NulO) analogs of sialic acid that were incorporated into LOS and prevented complement inhibition by physiologic CMP-Neu5Ac and resulted in attenuated gonococcal infection in mice. While studies to establish the safety of these agents are needed, enhancing complement activation on microbes may represent a promising strategy to treat antimicrobial resistant organisms.

  8. Hepatocyte growth factor triggers distinct mechanisms of Asef and Tiam1 activation to induce endothelial barrier enhancement.

    PubMed

    Higginbotham, Katherine; Tian, Yufeng; Gawlak, Grzegorz; Moldobaeva, Nurgul; Shah, Alok; Birukova, Anna A

    2014-11-01

    Previous reports described an important role of hepatocyte growth factor (HGF) in mitigation of pulmonary endothelial barrier dysfunction and cell injury induced by pathologic agonists and mechanical forces. HGF protective effects have been associated with Rac-GTPase signaling pathway activated by Rac-specific guanine nucleotide exchange factor Tiam1 and leading to enhancement of intercellular adherens junctions. This study tested involvement of a novel Rac-specific activator, Asef, in endothelial barrier enhancement by HGF and investigated a mechanism of HGF-induced Asef activation. Si-RNA-based knockdown of Tiam1 and Asef had an additive effect on attenuation of HGF-induced Rac activation and endothelial cell (EC) barrier enhancement. Tiam1 and Asef activation was abolished by pharmacologic inhibitors of HGF receptor and PI3-kinase. In contrast to Tiam1, Asef interacted with APC and associated with microtubule fraction upon HGF stimulation. EC treatment by low dose nocodazole to inhibit peripheral microtubule dynamics partially attenuated HGF-induced Asef peripheral translocation, but had negligible effect on Tiam1 translocation. These effects were associated with attenuation of HGF-induced barrier enhancement in EC pretreated with low ND dose and activation of Rac and its cytoskeletal effectors PAK1 and cortactin. These data demonstrate, that in addition to microtubule-independent Tiam1 activation, HGF engages additional microtubule- and APC-dependent pathway of Asef activation. These mechanisms may complement each other to provide the fine tuning of Rac signaling and endothelial barrier enhancement in response to various agonists.

  9. Placental Growth Factor Administration Abolishes Placental Ischemia-Induced Hypertension.

    PubMed

    Spradley, Frank T; Tan, Adelene Y; Joo, Woo S; Daniels, Garrett; Kussie, Paul; Karumanchi, S Ananth; Granger, Joey P

    2016-04-01

    Preeclampsia is a pregnancy-specific disorder of new-onset hypertension. Unfortunately, the most effective treatment is early delivery of the fetus and placenta. Placental ischemia appears central to the pathogenesis of preeclampsia because placental ischemia/hypoxia induced in animals by reduced uterine perfusion pressure (RUPP) or in humans stimulates release of hypertensive placental factors into the maternal circulation. The anti-angiogenic factor soluble fms-like tyrosine kinase-1 (sFlt-1), which antagonizes and reduces bioavailable vascular endothelial growth factor and placental growth factor (PlGF), is elevated in RUPP rats and preeclampsia. Although PlGF and vascular endothelial growth factor are both natural ligands for sFlt-1, vascular endothelial growth factor also has high affinity to VEGFR2 (Flk-1) causing side effects like edema. PlGF is specific for sFlt-1. We tested the hypothesis that PlGF treatment reduces placental ischemia-induced hypertension by antagonizing sFlt-1 without adverse consequences to the mother or fetus. On gestational day 14, rats were randomized to 4 groups: normal pregnant or RUPP±infusion of recombinant human PlGF (180 μg/kg per day; AG31, a purified, recombinant human form of PlGF) for 5 days via intraperitoneal osmotic minipumps. On day 19, mean arterial blood pressure and plasma sFlt-1 were higher and glomerular filtration rate lower in RUPP than normal pregnant rats. Infusion of recombinant human PlGF abolished these changes seen with RUPP along with reducing oxidative stress. These data indicate that the increased sFlt-1 and reduced PlGF resulting from placental ischemia contribute to maternal hypertension. Our novel finding that recombinant human PlGF abolishes placental ischemia-induced hypertension, without major adverse consequences, suggests a strong therapeutic potential for this growth factor in preeclampsia. PMID:26831193

  10. The Complement System in Lupus Nephritis.

    PubMed

    Birmingham, Daniel J; Hebert, Lee A

    2015-09-01

    The complement system is composed of a family of soluble and membrane-bound proteins that historically has been viewed as a key component of the innate immune system, with a primary role of providing a first-line defense against microorganisms. Although this role indeed is important, complement has many other physiological roles, including the following: (1) influencing appropriate immune responses, (2) disposing of waste in the circulation (immune complexes, cellular debris), and (3) contributing to damage of self-tissue through inflammatory pathways. These three roles are believed to be significant factors in the pathogenesis of systemic lupus erythematosus, particularly its renal manifestation (lupus nephritis), contributing both protective and damaging effects. In this review, we provide an overview of the human complement system and its functions, and discuss its intricate and seemingly contradictory roles in the pathogenesis of lupus nephritis.

  11. Interallelic complementation at the mouse Mitf locus.

    PubMed Central

    Steingrímsson, Eiríkur; Arnheiter, Heinz; Hallsson, Jón Hallsteinn; Lamoreux, M Lynn; Copeland, Neal G; Jenkins, Nancy A

    2003-01-01

    Mutations at the mouse microphthalmia locus (Mitf) affect the development of different cell types, including melanocytes, retinal pigment epithelial cells of the eye, and osteoclasts. The MITF protein is a member of the MYC supergene family of basic-helix-loop-helix-leucine-zipper (bHLHZip) transcription factors and is known to regulate the expression of cell-specific target genes by binding DNA as homodimer or as heterodimer with related proteins. The many mutations isolated at the locus have different effects on the phenotype and can be arranged in an allelic series in which the phenotypes range from near normal to white microphthalmic animals with osteopetrosis. Previous investigations have shown that certain combinations of Mitf alleles complement each other, resulting in a phenotype more normal than that of each homozygote alone. Here we analyze this interallelic complementation in detail and show that it is limited to one particular allele, Mitf(Mi-white) (Mitf(Mi-wh)), a mutation affecting the DNA-binding domain. Both loss- and gain-of-function mutations are complemented, as are other Mitf mutations affecting the DNA-binding domain. Furthermore, this behavior is not restricted to particular cell types: Both eye development and coat color phenotypes are complemented. Our analysis suggests that Mitf(Mi-wh)-associated interallelic complementation is due to the unique biochemical nature of this mutation. PMID:12586714

  12. T-Cell Immune Response Assessment as a Complement to Serology and Intranasal Protection Assays in Determining the Protective Immunity Induced by Acellular Pertussis Vaccines in Mice

    PubMed Central

    Ausiello, C. M.; Lande, R.; Stefanelli, P.; Fazio, C.; Fedele, G.; Palazzo, R.; Urbani, F.; Mastrantonio, P.

    2003-01-01

    The relative value of antibodies and/or T-cell immune responses to Bordetella pertussis antigens in the immunity induced by acellular pertussis (aP) vaccines is still an open issue, probably due to the incomplete knowledge on the mechanisms of protective immunity to pertussis. The relevance of T-cell immune responses in protection from pertussis has been demonstrated in murine and human models of infection; thus, in this study, the ability of different vaccine preparations of three component (pertussis toxin, filamentous hemagglutinin, and pertactin) aP vaccines to induce T-cell responses was investigated in mice. All vaccine preparations examined passed the immunogenicity control test, based on antibody titer assessment, according to European Pharmacopoeia standards, and protected mice from B. pertussis intranasal challenge, but not all preparations were able to prime T cells to pertussis toxin, the specific B. pertussis antigen. In particular, one vaccine preparation was unable to induce proliferation and gamma interferon (IFN-γ) production while the other two gave borderline results. The evaluation of T-cell responses to pertussis toxin antigen may provide information on the protective immunity induced by aP vaccines in animal models. Considering the critical role of the axis interleukin-12-IFN-γ for protection from pertussis, our results suggest that testing the induction of a key protective cytokine such as IFN-γ could be an additional tool for the evaluation of the immune response induced by aP vaccines. PMID:12853397

  13. Hypoxia inducible factors and the response to hypoxic stress

    PubMed Central

    Majmundar, Amar J.; Wong, Waihay J.; Simon, M. Celeste

    2011-01-01

    Oxygen (O2) is an essential nutrient that serves as a key substrate in cellular metabolism and bioenergetics. In a variety of physiological and pathological states, organisms encounter insufficient O2 availability, or hypoxia. In order to cope with this stress, evolutionarily conserved responses are engaged. In mammals, the primary transcriptional response to hypoxic stress is mediated by the Hypoxia-inducible factors (HIFs). While canonically regulated by prolyl hydroxylase domain-containing enzymes (PHDs), the HIFα subunits are intricately responsive to numerous other factors including Factor Inhibiting HIF-1α (FIH1), sirtuins, and metabolites. These transcription factors function in normal tissue homeostasis and impinge on critical aspects of disease progression and recovery. Insights from basic HIF biology are being translated into pharmaceuticals targeting the HIF pathway. PMID:20965423

  14. Trichinella spiralis Paramyosin Binds Human Complement C1q and Inhibits Classical Complement Activation

    PubMed Central

    Sun, Ran; Zhao, Xi; Wang, Zixia; Yang, Jing; Zhao, Limei; Zhan, Bin; Zhu, Xinping

    2015-01-01

    Background Trichinella spiralis expresses paramyosin (Ts-Pmy) as a defense mechanism. Ts-Pmy is a functional protein with binding activity to human complement C8 and C9 and thus plays a role in evading the attack of the host’s immune system. In the present study, the binding activity of Ts-Pmy to human complement C1q and its ability to inhibit classical complement activation were investigated. Methods and Findings The binding of recombinant and natural Ts-Pmy to human C1q were determined by ELISA, Far Western blotting and immunoprecipitation, respectively. Binding of recombinant Ts-Pmy (rTs-Pmy) to C1q inhibited C1q binding to IgM and consequently inhibited C3 deposition. The lysis of antibody-sensitized erythrocytes (EAs) elicited by the classical complement pathway was also inhibited in the presence of rTs-Pmy. In addition to inhibiting classical complement activation, rTs-Pmy also suppressed C1q binding to THP-1-derived macrophages, thereby reducing C1q-induced macrophages migration. Conclusion Our results suggest that T. spiralis paramyosin plays an important role in immune evasion by interfering with complement activation through binding to C1q in addition to C8 and C9. PMID:26720603

  15. Complement: the Iceman of immunology?

    PubMed

    Würzner, R

    1997-12-01

    Complement provides an important host defence system involved in a multitude of immune reactions, including opsonisation of micro-organisms, enhancement of inflammatory response, immunomodulation, clearance of immune complexes and cell lysis. The 6th European Meeting on Complement in Human Disease, 12-15 March 1997 in Innsbruck, Austria, the preservation site of the neolithic Iceman, addressed the functional role of complement and its regulators in human disease. The scientific presentations clearly demonstrated that complement is not a redundant fossil, evolving since the dawn of vertebrate existence on the earth, but remains continuously important for mankind.

  16. Complement system in lung disease.

    PubMed

    Pandya, Pankita H; Wilkes, David S

    2014-10-01

    In addition to its established contribution to innate immunity, recent studies have suggested novel roles for the complement system in the development of various lung diseases. Several studies have demonstrated that complement may serve as a key link between innate and adaptive immunity in a variety of pulmonary conditions. However, the specific contributions of complement to lung diseases based on innate and adaptive immunity are just beginning to emerge. Elucidating the role of complement-mediated immune regulation in these diseases will help to identify new targets for therapeutic interventions.

  17. Hypoxia inducible factor pathway inhibitors as anticancer therapeutics

    PubMed Central

    Burroughs, Sarah K; Kaluz, Stefan; Wang, Danzhu; Wang, Ke

    2013-01-01

    Hypoxia is a significant feature of solid tumor cancers. Hypoxia leads to a more malignant phenotype that is resistant to chemotherapy and radiation, is more invasive and has greater metastatic potential. Hypoxia activates the hypoxia inducible factor (HIF) pathway, which mediates the biological effects of hypoxia in tissues. The HIF complex acts as a transcription factor for many genes that increase tumor survival and proliferation. To date, many HIF pathway inhibitors indirectly affect HIF but there have been no clinically approved direct HIF inhibitors. This can be attributed to the complexity of the HIF pathway, as well as to the challenges of inhibiting protein–protein interactions. PMID:23573973

  18. Hypoxia and Hypoxia Inducible Factors: Diverse Roles in Liver Diseases

    PubMed Central

    Nath, Bharath; Szabo, Gyongyi

    2011-01-01

    Hypoxia has been shown to have a role in the pathogenesis of several forms of liver disease. The Hypoxia Inducible Factors (HIFs) are a family of evolutionarily conserved transcriptional regulators that affect a homeostatic response to low oxygen tension and have been identified as key mediators of angiogenesis, inflammation, and metabolism. In this review, we summarize the evidence for a role of HIFs across a range of hepatic pathophysiology. We describe regulation of the hypoxia inducible factors and review investigations that demonstrate a role for HIFs in the development of liver fibrosis, activation of innate immune pathways, hepatocellular carcinoma, as well as other liver diseases in both human disease as well as murine models. PMID:22120903

  19. A vital role for complement in heart disease.

    PubMed

    Lappegård, Knut T; Garred, Peter; Jonasson, Lena; Espevik, Terje; Aukrust, Pål; Yndestad, Arne; Mollnes, Tom E; Hovland, Anders

    2014-10-01

    Heart diseases are common and significant contributors to worldwide mortality and morbidity. During recent years complement mediated inflammation has been shown to be an important player in a variety of heart diseases. Despite some negative results from clinical trials using complement inhibitors, emerging evidence points to an association between the complement system and heart diseases. Thus, complement seems to be important in coronary heart disease as well as in heart failure, where several studies underscore the prognostic importance of complement activation. Furthermore, patients with atrial fibrillation often share risk factors both with coronary heart disease and heart failure, and there is some evidence implicating complement activation in atrial fibrillation. Moreover, Chagas heart disease, a protozoal infection, is an important cause of heart failure in Latin America, and the complement system is crucial for the protozoa-host interaction. Thus, complement activation appears to be involved in the pathophysiology of a diverse range of cardiac conditions. Determination of the exact role of complement in the various heart diseases will hopefully help to identify patients that might benefit from therapeutic complement intervention.

  20. The complement system in systemic lupus erythematosus: an update.

    PubMed

    Leffler, Jonatan; Bengtsson, Anders A; Blom, Anna M

    2014-09-01

    The complement system plays a major role in the autoimmune disease, systemic lupus erythematosus (SLE). However, the role of complement in SLE is complex since it may both prevent and exacerbate the disease. In this review, we explore the latest findings in complement-focused research in SLE. C1q deficiency is the strongest genetic risk factor for SLE, although such deficiency is very rare. Various recently discovered genetic associations include mutations in the complement receptors 2 and 3 as well as complement inhibitors, the latter related to earlier onset of nephritis. Further, autoantibodies are a distinct feature of SLE that are produced as the result of an adaptive immune response and how complement can affect that response is also being reviewed. SLE generates numerous disease manifestations involving contributions from complement such as glomerulonephritis and the increased risk of thrombosis. Furthermore, since most of the complement system is present in plasma, complement is very accessible and may be suitable as biomarker for diagnosis or monitoring of disease activity. This review highlights the many roles of complement for SLE pathogenesis and how research has progressed during recent years.

  1. Social factors modulate restraint stress induced hyperthermia in mice.

    PubMed

    Watanabe, Shigeru

    2015-10-22

    Stress-induced hyperthermia (SIH) was examined in three different social conditions in mice by thermographic measurement of the body surface temperature. Placing animals in cylindrical holders induced restraint stress. I examined the effect of the social factors in SIH using the thermograph (body surface temperature). Mice restrained in the holders alone showed SIH. Mice restrained in the holders at the same time as other similarly restrained cage mates (social equality condition) showed less hyperthermia. Interestingly, restrained mice with free moving cage mates (social inequality condition) showed the highest hyperthermia. These results are consistent with a previous experiment measuring the memory-enhancing effects of stress and the stress-induced elevation of corticosterone, and suggest that social inequality enhances stress.

  2. Social factors modulate restraint stress induced hyperthermia in mice.

    PubMed

    Watanabe, Shigeru

    2015-10-22

    Stress-induced hyperthermia (SIH) was examined in three different social conditions in mice by thermographic measurement of the body surface temperature. Placing animals in cylindrical holders induced restraint stress. I examined the effect of the social factors in SIH using the thermograph (body surface temperature). Mice restrained in the holders alone showed SIH. Mice restrained in the holders at the same time as other similarly restrained cage mates (social equality condition) showed less hyperthermia. Interestingly, restrained mice with free moving cage mates (social inequality condition) showed the highest hyperthermia. These results are consistent with a previous experiment measuring the memory-enhancing effects of stress and the stress-induced elevation of corticosterone, and suggest that social inequality enhances stress. PMID:26232073

  3. Complement--tapping into new sites and effector systems.

    PubMed

    Kolev, Martin; Le Friec, Gaelle; Kemper, Claudia

    2014-12-01

    Complement is traditionally known to be a system of serum proteins that provide protection against pathogens through direct cell lysis and the mobilization of innate and adaptive immunity. However, recent work indicates that the complement system has additional physiological roles beyond those in host defence. In this Opinion article, we describe the new modes and locations of complement activation that enable it to interact with other cell effector systems, such as growth factor receptors, inflammasomes and metabolic pathways. We propose that the location of complement activation dictates its function.

  4. Dexamethasone impairs hypoxia-inducible factor-1 function

    SciTech Connect

    Wagner, A.E.; Huck, G.; Stiehl, D.P.; Jelkmann, W.; Hellwig-Buergel, T.

    2008-07-25

    Hypoxia-inducible factor-1 (HIF-1) is a heterodimeric transcription-factor composed of {alpha}- and {beta}-subunits. HIF-1 is not only necessary for the cellular adaptation to hypoxia, but it is also involved in inflammatory processes and wound healing. Glucocorticoids (GC) are therapeutically used to suppress inflammatory responses. Herein, we investigated whether GC modulate HIF-1 function using GC receptor (GR) possessing (HepG2) and GR deficient (Hep3B) human hepatoma cell cultures as model systems. Dexamethasone (DEX) treatment increased HIF-1{alpha} levels in the cytosol of HepG2 cells, while nuclear HIF-1{alpha} levels and HIF-1 DNA-binding was reduced. In addition, DEX dose-dependently lowered the hypoxia-induced luciferase activity in a reporter gene system. DEX suppressed the hypoxic stimulation of the expression of the HIF-1 target gene VEGF (vascular endothelial growth factor) in HepG2 cultures. DEX did not reduce hypoxically induced luciferase activity in HRB5 cells, a Hep3B derivative lacking GR. Transient expression of the GR in HRB5 cells restored the susceptibility to DEX. Our study discloses the inhibitory action of GC on HIF-1 dependent gene expression, which may be important with respect to the impaired wound healing in DEX-treated patients.

  5. Complement-Mediated Regulation of Metabolism and Basic Cellular Processes.

    PubMed

    Hess, Christoph; Kemper, Claudia

    2016-08-16

    Complement is well appreciated as a critical arm of innate immunity. It is required for the removal of invading pathogens and works by directly destroying them through the activation of innate and adaptive immune cells. However, complement activation and function is not confined to the extracellular space but also occurs within cells. Recent work indicates that complement activation regulates key metabolic pathways and thus can impact fundamental cellular processes, such as survival, proliferation, and autophagy. Newly identified functions of complement include a key role in shaping metabolic reprogramming, which underlies T cell effector differentiation, and a role as a nexus for interactions with other effector systems, in particular the inflammasome and Notch transcription-factor networks. This review focuses on the contributions of complement to basic processes of the cell, in particular the integration of complement with cellular metabolism and the potential implications in infection and other disease settings. PMID:27533012

  6. Divergent functions of three Candida albicans zinc-cluster transcription factors (CTA4, ASG1 and CTF1) complementing pleiotropic drug resistance in Saccharomyces cerevisiae.

    PubMed

    Coste, Alix T; Ramsdale, Mark; Ischer, Françoise; Sanglard, Dominique

    2008-05-01

    One of the mediators of pleiotropic drug resistance in Saccharomyces cerevisiae is the ABC-transporter gene PDR5. This gene is regulated by at least two transcription factors with Zn(2)-Cys(6) finger DNA-binding motifs, Pdr1p and Pdr3p. In this work, we searched for functional homologues of these transcription factors in Candida albicans. A C. albicans gene library was screened in a S. cerevisiae mutant lacking PDR1 and PDR3 and clones resistant to azole antifungals were isolated. From these clones, three genes responsible for azole resistance were identified. These genes (CTA4, ASG1 and CTF1) encode proteins with Zn(2)-Cys(6)-type zinc finger motifs in their N-terminal domains. The C. albicans genes expressed in S. cerevisiae could activate the transcription of a PDR5-lacZ reporter system and this reporter activity was PDRE-dependent. They could also confer resistance to azoles in a S. cerevisiae strain lacking PDR1, PDR3 and PDR5, suggesting that CTA4-, ASG1- and CTF1-dependent azole resistance can be caused by genes other than PDR5 in S. cerevisiae. Deletion of CTA4, ASG1 and CTF1 in C. albicans had no effect on fluconazole susceptibility and did not alter the expression of the ABC-transporter genes CDR1 and CDR2 or the major facilitator gene MDR1, which encode multidrug transporters known as mediators of azole resistance in C. albicans. However, additional phenotypic screening tests on the C. albicans mutants revealed that the presence of ASG1 was necessary to sustain growth on non-fermentative carbon sources (sodium acetate, acetic acid, ethanol). In conclusion, C. albicans possesses functional homologues of the S. cerevisiae Pdr1p and Pdr3p transcription factors; however, their properties in C. albicans have been rewired to other functions. PMID:18451058

  7. Maximality and Idealized Cognitive Models: The Complementation of Spanish "Tener."

    ERIC Educational Resources Information Center

    Hilferty, Joseph; Valenzuela, Javier

    2001-01-01

    Discusses the bare-noun phrase (NP) complementation pattern of the Spanish verb "tener" (have). Shows that the maximality of the complement NP is dependent upon three factors: (1) idiosyncratic valence requirements; (2) encyclopedic knowledge related to possession; and (3) contextualized semantic construal. (Author/VWL)

  8. Drug-induced lupus: Including anti-tumour necrosis factor and interferon induced.

    PubMed

    Araújo-Fernández, S; Ahijón-Lana, M; Isenberg, D A

    2014-05-01

    Drug-induced lupus erythematosus is defined as a syndrome with clinical and serological features similar to systemic lupus erythematosus that is temporally related to continuous drug exposure and which resolves after discontinuation of this drug. More than 90 drugs, including biological modulators such as tumour necrosis factor-α inhibitors and interferons, have been identified as likely 'culprits'. While there are no standard diagnostic criteria for drug-induced lupus erythematosus, guidelines that can help to distinguish drug-induced lupus erythematosus from systemic lupus erythematosus have been proposed and several different patterns of drug-induced lupus erythematosus are emerging. Distinguishing drug-induced lupus erythematosus from systemic lupus erythematosus is important because the prognosis of drug-induced lupus erythematosus is usually good when the drug is withdrawn. This review discusses the differences between drug-induced lupus erythematosus and systemic lupus erythematosus, the mechanisms of action of drug-induced lupus erythematosus and drugs that are usually associated with drug-induced lupus erythematosus, with particular focus on the biological treatments.

  9. Nuclear Factor of Activated T Cells Transcription Factor Nfatp Controls Superantigen-Induced Lethal Shock

    PubMed Central

    Tsytsykova, Alla V.; Goldfeld, Anne E.

    2000-01-01

    Tumor necrosis factor α (TNF-α) is the key mediator of superantigen-induced T cell lethal shock. Here, we show that nuclear factor of activated T cells transcription factor, NFATp, controls susceptibility to superantigen-induced lethal shock in mice through its activation of TNF-α gene transcription. In NFATp-deficient mice, T cell stimulation leads to delayed induction and attenuation of TNF-α mRNA levels, decreased TNF-α serum levels, and resistance to superantigen-induced lethal shock. By contrast, after lipopolysaccharide (LPS) challenge, serum levels of TNF-α and susceptibility to shock are unaffected. These results demonstrate that NFATp is an essential activator of immediate early TNF-α gene expression in T cells and they present in vivo evidence of the inducer- and cell type–specific regulation of TNF-α gene expression. Furthermore, they suggest NFATp as a potential selective target in the treatment of superantigen-induced lethal shock. PMID:10952728

  10. Increased Autoreactivity of the Complement-Activating Molecule Mannan-Binding Lectin in a Type 1 Diabetes Model

    PubMed Central

    Østergaard, Jakob Appel; Ruseva, Marieta Milkova; Malik, Talat Habib; Hoffmann-Petersen, Ingeborg Torp; Pickering, Matthew Caleb; Thiel, Steffen; Hansen, Troels Krarup

    2016-01-01

    Background. Diabetic kidney disease is the leading cause of end-stage renal failure despite intensive treatment of modifiable risk factors. Identification of new drug targets is therefore of paramount importance. The complement system is emerging as a potential new target. The lectin pathway of the complement system, initiated by the carbohydrate-recognition molecule mannan-binding lectin (MBL), is linked to poor kidney prognosis in diabetes. We hypothesized that MBL activates complement upon binding within the diabetic glomerulus. Methods. We investigated this by comparing complement deposition and activation in kidneys from streptozotocin-induced diabetic mice and healthy control mice. Results. After 20 weeks of diabetes, glomerular deposition of MBL was significantly increased. Diabetic animals had 2.0-fold higher (95% CI 1.6–2.5) immunofluorescence intensity from anti-MBL antibodies compared with controls (P < 0.001). Diabetes and control groups did not differ in glomerular immunofluorescence intensity obtained by antibodies against complement factors C4, C3, and C9. However, the circulating complement activation product C3a was increased in diabetes as compared to control mice (P = 0.04). Conclusion. 20 weeks of diabetes increased MBL autoreactivity in the kidney and circulating C3a concentration. Together with previous findings, these results indicate direct effects of MBL within the kidney in diabetes. PMID:26977416

  11. Gallium induces the production of virulence factors in Pseudomonas aeruginosa.

    PubMed

    García-Contreras, Rodolfo; Pérez-Eretza, Berenice; Lira-Silva, Elizabeth; Jasso-Chávez, Ricardo; Coria-Jiménez, Rafael; Rangel-Vega, Adrián; Maeda, Toshinari; Wood, Thomas K

    2014-02-01

    The novel antimicrobial gallium is a nonredox iron III analogue with bacteriostatic and bactericidal properties, effective for the treatment of Pseudomonas aeruginosa in vitro and in vivo in mouse and rabbit infection models. It interferes with iron metabolism, transport, and presumably its homeostasis. As gallium exerts its antimicrobial effects by competing with iron, we hypothesized that it ultimately will lead cells to an iron deficiency status. As iron deficiency promotes the expression of virulence factors in vitro and promotes the pathogenicity of P. aeruginosa in animal models, it is anticipated that treatment with gallium will also promote the production of virulence factors. To test this hypothesis, the reference strain PA14 and two clinical isolates from patients with cystic fibrosis were exposed to gallium, and their production of pyocyanin, rhamnolipids, elastase, alkaline protease, alginate, pyoverdine, and biofilm was determined. Gallium treatment induced the production of all the virulence factors tested in the three strains except for pyoverdine. In addition, as the Ga-induced virulence factors are quorum sensing controlled, co-administration of Ga and the quorum quencher brominated furanone C-30 was assayed, and it was found that C-30 alleviated growth inhibition from gallium. Hence, adding both C-30 and gallium may be more effective in the treatment of P. aeruginosa infections.

  12. A metalloproteinase mirolysin of Tannerella forsythia inhibits all pathways of the complement system

    PubMed Central

    Jusko, Monika; Potempa, Jan; Mizgalska, Danuta; Bielecka, Ewa; Ksiazek, Miroslaw; Riesbeck, Kristian; Garred, Peter; Eick, Sigrun; Blom, Anna M.

    2015-01-01

    Recent reports focusing on virulence factors of periodontal pathogens implicated proteinases as major determinants of remarkable pathogenicity of these species, with special emphasis on their capacity to modulate complement activity. In particular, bacteria-mediated cleavage of C5 and subsequent release of C5a seems to be an important phenomenon in the manipulation of the local inflammatory response in periodontitis. Here we present mirolysin, a novel metalloproteinase secreted by Tannerella forsythia, a well-recognized pathogen strongly associated with periodontitis. Mirolysin exhibited a strong effect on all complement pathways. It inhibited the classical and lectin complement pathways due to efficient degradation of mannose-binding lectin, ficolin-2, ficolin-3 and C4, while inhibition of the alternative pathway was caused by degradation of C5. This specificity toward complement largely resembled the activity of a previously characterized metalloproteinase of T. forsythia, karilysin. Interestingly, mirolysin released the biologically active C5a peptide in human plasma and induced migration of neutrophils. Importantly, we demonstrated that combination of mirolysin with karilysin, as well as a cysteine proteinase of another periodontal pathogen, Prevotella intermedia, resulted in a strong synergistic effect on complement. Furthermore, mutant strains of T. forsythia, devoid of either mirolysin or karilysin, showed diminished survival in human serum, providing further evidence for the synergistic inactivation of complement by these metalloproteinases. Taken together, our findings on interactions of mirolysin with complement significantly add to the understanding of immune evasion strategies of T. forsythia, and expand the knowledge on molecular mechanisms driving pathogenic events in the infected periodontium. PMID:26209620

  13. A Metalloproteinase Mirolysin of Tannerella forsythia Inhibits All Pathways of the Complement System.

    PubMed

    Jusko, Monika; Potempa, Jan; Mizgalska, Danuta; Bielecka, Ewa; Ksiazek, Miroslaw; Riesbeck, Kristian; Garred, Peter; Eick, Sigrun; Blom, Anna M

    2015-09-01

    Recent reports focusing on virulence factors of periodontal pathogens implicated proteinases as major determinants of remarkable pathogenicity of these species, with special emphasis on their capacity to modulate complement activity. In particular, bacteria-mediated cleavage of C5 and subsequent release of C5a seems to be an important phenomenon in the manipulation of the local inflammatory response in periodontitis. In this study, we present mirolysin, a novel metalloproteinase secreted by Tannerella forsythia, a well-recognized pathogen strongly associated with periodontitis. Mirolysin exhibited a strong effect on all complement pathways. It inhibited the classical and lectin complement pathways due to efficient degradation of mannose-binding lectin, ficolin-2, ficolin-3, and C4, whereas inhibition of the alternative pathway was caused by degradation of C5. This specificity toward complement largely resembled the activity of a previously characterized metalloproteinase of T. forsythia, karilysin. Interestingly, mirolysin released the biologically active C5a peptide in human plasma and induced migration of neutrophils. Importantly, we demonstrated that combination of mirolysin with karilysin, as well as a cysteine proteinase of another periodontal pathogen, Prevotella intermedia, resulted in a strong synergistic effect on complement. Furthermore, mutant strains of T. forsythia, devoid of either mirolysin or karilysin, showed diminished survival in human serum, providing further evidence for the synergistic inactivation of complement by these metalloproteinases. Taken together, our findings on interactions of mirolysin with complement significantly add to the understanding of immune evasion strategies of T. forsythia and expand the knowledge on molecular mechanisms driving pathogenic events in the infected periodontium.

  14. Evolution of the complement system.

    PubMed

    Nonaka, Masaru

    2014-01-01

    The mammalian complement system constitutes a highly sophisticated body defense machinery comprising more than 30 components. Research into the evolutionary origin of the complement system has identified a primitive version composed of the central component C3 and two activation proteases Bf and MASP in cnidaria. This suggests that the complement system was established in the common ancestor of eumetazoa more than 500 million years ago. The original activation mechanism of the original complement system is believed to be close to the mammalian lectin and alternative activation pathways, and its main role seems to be opsonization and induction of inflammation. This primitive complement system has been retained by most deuterostomes without major change until the appearance of jawed vertebrates. At this stage, duplication of the C3, Bf and MASP genes as well as recruitment of membrane attack components added the classical and lytic pathways to the primitive complement system, converting it to the modern complement system. In contrast, the complement system was lost multiple times independently in the protostome lineage.

  15. Hematopoietic growth factors in drug-induced agranulocytosis.

    PubMed

    Pavithran, K; Thomas, M

    2002-05-01

    Drug-induced agranulocytosis (DIA) is a potentially fatal disorder. Hematopoietic growth factors have been used in the treatment of DIA. We report nine cases of DIA treated with granulocyte macrophage - colony stimulating factor (GM-CSF) in a dose of 300 microg/day. All the patients had evidence of systemic infection. Mean time to reach an absolute neutrophil count of 0.5 x 10(9)/L was three days. One patient succumbed to the disease. The cause of death was multiorgan failure. No adverse events were observed with GM-CSF. We conclude that hematopoietic growth factors are useful in shortening the period of neutropenia and reducing morbidity and mortality in these patients.

  16. Generation of three different fragments of bound C3 with purified factor I or serum. II. Location of binding sites in the C3 Fragments for Factors B and H, complement receptors , and bovine conglutinin

    PubMed Central

    Ross, GD; Newman, SL; Lambris, JD; Devery-Pocius, JE; Cain, JA; Lackmann, PJ

    1983-01-01

    The many different recognized functions of C3 are dependent upon the ability of the activated C3 molecule both to bind covalently to protein and carbohydrate surfaces and to provide binding sites for as many as eleven different proteins. The location of the binding sites for six of these different proteins (factors B and H, complement receptors CR(1), CR(2) and CR(3) and conglutinin) was examined in the naturally occurring C3-fragments generated by C3 activation (C3b) and degradation by Factor I (iC3b, C3c, C3d,g) and trypsin (C3d). Evidence was obtained for at least four distinct binding sites in C3 for these six different C3 ligands. One binding site for B was detectable only in C3b, whereas a second binding site for H and CR(1) was detectable in both C3b and iC3b. The affinity of the binding site for H and CR(1) was charge dependent and considerably reduced in iC3b as compared to C3b. H binding to iC3b-coated sheep erythrocytes (EC3bi) was measurable only in low ionic strength buffer (4 mS). The finding that C3c-coated microspheres bound to CR(1), indicated that this second binding site was still intact in the C3c fragment. However, H binding to C3c was not examined. A third binding site in C3 for CR(2) was exposed in the d region by factor I cleavage of C3b into iC3b, and the activity of this site was unaffected by the further I cleavage of iC3b into C3d,g. Removal of the 8,000-dalton C3g fragment from C3d,g with trypsin forming C3d, resulted in reduced CR2 activity. However, because saturating amounts of monoclonal anti-C3g did not block the CR(2)-binding activity of EC3d,g, it appears unlikely that the g region of C3d,g or iC3b forms a part of the CR(2)-binding site. In addition, detergent-solubilized EC3d (C3d-OR) inhibited the CR(2)-binding activity of EC3d,g. Monocytes and neutrophils, that had been previously thought to lack CR(2) because of their inability to form EC3d rosettes, did bind EC3d,g containing greater than 5 × 10(4) C3d,g molecules per E

  17. Structural polymorphism in the major groove of a 5S RNA gene complements the zinc finger domains of transcription factor IIIA.

    PubMed Central

    Huber, P W; Morii, T; Mei, H Y; Barton, J K

    1991-01-01

    Metal complexes that bind to DNA on the basis of shape-selection have been used to map the conformational features of the DNA binding site for transcription factor IIIA. Conformationally distinct segments are detected on the 5S rRNA gene that correspond closely to the binding sites identified for the individual zinc finger domains of the protein. The local conformations are characterized by a major groove opened because of a change in base pair inclination and/or displacement at a central 5'-pyrimidine-purine-3' step, flanked by a widened minor groove, as would arise at the junctions between alternating B- and A-like DNA segments. Docking experiments with a consensus structure of a zinc finger reveal that the mixed A-B binding site accommodates the peptide domain better than either canonical B- or A-DNA helices. The close structural matching of the conformational variations in the 5S rDNA both to the proposed sites of zinc finger binding and to the shape of an individual zinc finger domain points to DNA structural polymorphism as providing an important determinant in recognition. In particular, shape selection in the 5' half of the internal control region may orient the multiple finger domains. Images PMID:1961749

  18. Activated Ki-Ras complements erythropoietin signaling in CTLL-2 cells, inducing tyrosine phosphorylation of a 160-kDa protein.

    PubMed Central

    Yamamura, Y; Noda, M; Ikawa, Y

    1994-01-01

    We have previously shown that expression of erythropoietin (EPO) receptor (EPOR) alone failed to confer EPO responsiveness on the interleukin 2-dependent T-cell line CTLL-2, whereas the introduction of the EPOR into interleukin 3-dependent pro-B-cell lines, such as BAF-B03, allowed the cells to proliferate in response to EPO. Here, we report that additional expression of v-Ki-Ras conferred EPO-dependent growth on CTLL-2 cells expressing the EPOR, with additional formation of a high-affinity EPOR. To investigate possible mechanisms of EPOR downstream signaling induced by v-Ki-Ras expression in these CTLL-2-derived cells, we carried out anti-phosphotyrosine immunoblot analysis of the EPOR complex immunoprecipitated with anti-EPOR antibody from lysates of cells with and without cytokine stimulation, revealing two 160-kDa and 130-kDa phosphotyrosyl proteins. An anti-JAK2 antibody did not react with these proteins, suggesting that they may represent cellular components involved in an EPO-EPOR signaling pathway induced by v-Ki-Ras. Similar phosphotyrosyl proteins were present among Friend erythroleukemia cell lines, in which the Friend virus gp55/EPOR complex on the cell surface constitutively sends signals for cell growth. Images PMID:7522324

  19. Antibody directs properdin-dependent activation of the complement alternative pathway in a mouse model of abdominal aortic aneurysm.

    PubMed

    Zhou, Hui-Fang; Yan, Huimin; Stover, Cordula M; Fernandez, Tamara Montes; Rodriguez de Cordoba, Santiago; Song, Wen-Chao; Wu, Xiaobo; Thompson, Robert W; Schwaeble, Wilhelm J; Atkinson, John P; Hourcade, Dennis E; Pham, Christine T N

    2012-02-14

    Abdominal aortic aneurysm (AAA) is a complex inflammatory vascular disease. There are currently limited treatment options for AAA when surgery is inapplicable. Therefore, insights into molecular mechanisms underlying AAA pathogenesis may reveal therapeutic targets that could be manipulated pharmacologically or biologically to halt disease progression. Using an elastase-induced AAA mouse model, we previously established that the complement alternative pathway (AP) plays a critical role in the development of AAA. However, the mechanism by which complement AP is initiated remains undefined. The complement protein properdin, traditionally viewed as a positive regulator of the AP, may also initiate complement activation by binding directly to target surfaces. In this study, we sought to determine whether properdin serves as a focal point for the initiation of the AP complement activation in AAA. Using a properdin loss of function mutation in mice and a mutant form of the complement factor B protein that produces a stable, properdin-free AP C3 convertase, we show that properdin is required for the development of elastase-induced AAA in its primary role as a convertase stabilizer. Unexpectedly, we find that, in AAA, natural IgG antibodies direct AP-mediated complement activation. The absence of IgG abrogates C3 deposition in elastase-perfused aortic wall and protects animals from AAA development. We also determine that blockade of properdin activity prevents aneurysm formation. These results indicate that an innate immune response to self-antigens activates the complement system and initiates the inflammatory cascade in AAA. Moreover, the study suggests that properdin-targeting strategies may halt aneurysmal growth.

  20. Bimolecular fluorescence complementation.

    PubMed

    Wong, Katy A; O'Bryan, John P

    2011-01-01

    Defining the subcellular distribution of signaling complexes is imperative to understanding the output from that complex. Conventional methods such as immunoprecipitation do not provide information on the spatial localization of complexes. In contrast, BiFC monitors the interaction and subcellular compartmentalization of protein complexes. In this method, a fluororescent protein is split into amino- and carboxy-terminal non-fluorescent fragments which are then fused to two proteins of interest. Interaction of the proteins results in reconstitution of the fluorophore (Figure 1). A limitation of BiFC is that once the fragmented fluorophore is reconstituted the complex is irreversible. This limitation is advantageous in detecting transient or weak interactions, but precludes a kinetic analysis of complex dynamics. An additional caveat is that the reconstituted flourophore requires 30min to mature and fluoresce, again precluding the observation of real time interactions. BiFC is a specific example of the protein fragment complementation assay (PCA) which employs reporter proteins such as green fluorescent protein variants (BiFC), dihydrofolate reductase, b-lactamase, and luciferase to measure protein:protein interactions. Alternative methods to study protein:protein interactions in cells include fluorescence co-localization and Förster resonance energy transfer (FRET). For co-localization, two proteins are individually tagged either directly with a fluorophore or by indirect immunofluorescence. However, this approach leads to high background of non-interacting proteins making it difficult to interpret co-localization data. In addition, due to the limits of resolution of confocal microscopy, two proteins may appear co-localized without necessarily interacting. With BiFC, fluorescence is only observed when the two proteins of interest interact. FRET is another excellent method for studying protein:protein interactions, but can be technically challenging. FRET

  1. Factor D of the alternative pathway of human complement. Purification, alignment and N-terminal amino acid sequences of the major cyanogen bromide fragments, and localization of the serine residue at the active site.

    PubMed Central

    Johnson, D M; Gagnon, J; Reid, K B

    1980-01-01

    The serine esterase factor D of the complement system was purified from outdated human plasma with a yield of 20% of the initial haemolytic activity found in serum. This represented an approx. 60 000-fold purification. The final product was homogeneous as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis (with an apparent mol.wt. of 24 000), its migration as a single component in a variety of fractionation procedures based on size and charge, and its N-terminal amino-acid-sequence analysis. The N-terminal amino acid sequence of the first 36 residues of the intact molecule was found to be homologous with the N-terminal amino acid sequences of the catalytic chains of other serine esterases. Factor D showed an especially strong homology (greater than 60% identity) with rat 'group-specific protease' [Woodbury, Katunuma, Kobayashi, Titani, & Neurath (1978) Biochemistry 17, 811-819] over the first 16 amino acid residues. This similarity is of interest since it is considered that both enzymes may be synthesized in their active, rather than zymogen, forms. The three major CNBr fragments of factor D, which had apparent mol.wts. of 15 800, 6600 and 1700, were purified and then aligned by N-terminal amino acid sequence analysis and amino acid analysis. By using factor D labelled with di-[1,3-14C]isopropylphosphofluoridate it was shown that the CNBr fragment of apparent mol.wt. 6600, which is located in the C-terminal region of factor D, contained the active serine residue. The amino acid sequence around this residue was determined. Images Fig. 1. Fig. 2. PMID:6821372

  2. Factors influencing fluoxetine-induced sexual dysfunction in female rats.

    PubMed

    Adams, Sarah; Heckard, Danyeal; Hassell, James; Uphouse, Lynda

    2012-11-01

    Treatment with selective serotonin reuptake inhibitors, such as fluoxetine, produces sexual side effects with low sexual desire being the most prevalent effect in females. In few studies have preclinical models for such antidepressant-induced sexual dysfunction been fruitful. In the current manuscript, the effects of fluoxetine on multiple measures of female sexual motivation and sexual receptivity were examined. Ovariectomized, Fischer rats were primed with 10 μg estradiol benzoate and 500 μg progesterone. Partner preference, active investigation of the male, and measures of sexual behavior were examined after injection with 15 mg/kg fluoxetine. Factors (pretesting for sexual behavior, size of the test arena, non-contact time with a male) that differ among experiments designed to study antidepressant-induced female rat sexual dysfunction were studied. The male preference ratio was not affected by fluoxetine treatment but active investigation of the male was reduced; lordosis behavior was inhibited and pretesting for sexual receptivity amplified fluoxetine's inhibition; size of the testing arena or non-contact experience with the male had no effect. Regardless of test condition, when given the opportunity to escape from the male, fluoxetine-treated females displayed escape behavior. Measures of male preference and active investigation, but not lordosis behavior, appeared to be affected by fluoxetine's impact on activity. The collective data provided a behavioral profile of fluoxetine-induced sexual dysfunction. These findings reinforce the value of multiple measures when attempting to model antidepressant-induced female sexual dysfunction.

  3. Activated Factor X Induces Endothelial Cell Senescence Through IGFBP-5

    PubMed Central

    Sanada, Fumihiro; Taniyama, Yoshiaki; Muratsu, Jun; Otsu, Rei; Iwabayashi, Masaaki; Carracedo, Miguel; Rakugi, Hiromi; Morishita, Ryuichi

    2016-01-01

    Uncontrolled coagulation contributes to the pathophysiology of several chronic inflammatory diseases. In these conditions, senescent cells are often observed and is involved in the generation of inflammation. The coincidence of hyper-coagulation, cell senescence, and inflammation suggests the existence of a common underlying mechanism. Recent evidence indicates that activated coagulation factor X (FXa) plays a role in the processes beyond blood coagulation. This non-hematologic function entails the mediation of inflammation and tissue remodeling. We therefore tested the hypothesis that FXa induces cell senescence resulting in tissue inflammation and impaired tissue regeneration. Human umbilical vein endothelial cells were stimulated with FXa for 14 days. The proliferation of cells treated with FXa was significantly smaller, and the fraction of senescence-associated β-galactosidase-positive cells was increased as compared to the control group. RT-qPCR array revealed that FXa increased the expression of IGFBP-5, EGR-1, p53, and p16INK4a. Inhibition of FXa by a direct FXa inhibitor, rivaroxaban, or IGFBP-5 by siRNA decreased FXa-induced cell senescence, restoring cell proliferation. Moreover, in an ischemic hind limb mouse model, FXa inhibited neovascularization by endothelial progenitor cell. However, rivaroxaban significantly restored FXa-induced impaired angiogenesis. In summary, FXa induced endothelial cell senescence through IGFBP-5, resulting in impaired angiogenesis. PMID:27752126

  4. The neurohormone orexin stimulates hypoxia-inducible factor-1 activity.

    PubMed

    Sikder, Devanjan; Kodadek, Thomas

    2007-11-15

    Orexin A and Orexin B (also known as hypocretins) are neuropeptides that bind two related G-coupled protein receptors (OXR1 and OXR2) and thus induce wakefulness, food consumption, and locomotion. Conversely, deletion of the orexin gene in mice produces a condition similar to canine and human narcolepsy. Despite the central importance of the orexin system in regulating wakefulness and feeding behavior, little is known about the downstream signaling mechanisms that achieve these effects. In this study, genomics techniques are used to probe this question and reveal that orexin activates the hypoxia-inducible factor 1 (HIF-1), a heterodimeric transcription factor whose pathogenic role in stimulating angiogenesis in hypoxic tumors has been the focus of intense investigation. Orexin-stimulated HIF-1 activity is due to both increased HIF-1alpha gene transcription and a down-regulation of von Hippel-Lindau (VHL), the E3 ubiquitin ligase that mediates the turnover of HIF-1 via the ubiquitin-proteasome pathway. Orexin-mediated activation of HIF-1 results in increased glucose uptake and higher glycolytic activity, as expected from studies of hypoxic cells. However, orexin receptor-expressing cells somehow override the HIF-1-mediated preference for funneling pyruvate into anaerobic glycolysis and instead favor ATP production through the tricarboxylic acid cycle and oxidative phosphorylation. These findings implicate HIF-1 as an important transcription factor in the hormone-mediated regulation of hunger and wakefulness.

  5. Tumor necrosis factor-alpha antagonist-induced sarcoidosis.

    PubMed

    Clementine, Rochelle Robicheaux; Lyman, Justin; Zakem, Jerald; Mallepalli, Jyothi; Lindsey, Stephen; Quinet, Robert

    2010-09-01

    Sarcoidosis is a multisystem granulomatous disease of unknown etiology. Tumor necrosis factor (TNF)-alpha is an important player in granuloma formation, and recent clinical trials have investigated the efficacy of TNF-alpha inhibitors in sarcoidosis. Paradoxically, there are several case reports in the medical literature describing the development of sarcoidosis in patients treated with TNF-alpha inhibitors. We describe 3 cases of TNF-alpha antagonist-induced sarcoidosis: 1 case of pulmonary, ocular and cutaneous sarcoidosis developing in a patient receiving infliximab for erosive rheumatoid arthritis, 1 case of etanercept-induced sarcoidosis in a patient with seronegative rheumatoid arthritis, and 1 case of sarcoidosis developing in a patient receiving etanercept for erosive rheumatoid arthritis. We also provide a brief discussion on the role of TNF alpha in granuloma formation and implications in the use of TNF-alpha antagonists in autoimmune disease.

  6. Phosphate starvation induces the sporulation killing factor of Bacillus subtilis.

    PubMed

    Allenby, Nicholas E E; Watts, Carys A; Homuth, Georg; Prágai, Zoltán; Wipat, Anil; Ward, Alan C; Harwood, Colin R

    2006-07-01

    Bacillus subtilis produces and exports a peptide sporulation killing factor (SkfA) that induces lysis of sibling cells. skfA is part of the skf operon (skfA-H), which is responsible for immunity to SkfA, as well as for production and export of SkfA. Here we report that transcription of skfA is markedly induced when cells of B. subtilis are subjected to phosphate starvation. The role of PhoP in regulation of the skf operon was confirmed by in vitro gel shift assays, which showed that this operon is a new member of the PhoP regulon. A putative stem-loop structure in the skfA-skfB intergenic region is proposed to act as a stabilizer of an skfA-specific transcript. PMID:16816204

  7. Phosphate Starvation Induces the Sporulation Killing Factor of Bacillus subtilis

    PubMed Central

    Allenby, Nicholas E. E.; Watts, Carys A.; Homuth, Georg; Prágai, Zoltán; Wipat, Anil; Ward, Alan C.; Harwood, Colin R.

    2006-01-01

    Bacillus subtilis produces and exports a peptide sporulation killing factor (SkfA) that induces lysis of sibling cells. skfA is part of the skf operon (skfA-H), which is responsible for immunity to SkfA, as well as for production and export of SkfA. Here we report that transcription of skfA is markedly induced when cells of B. subtilis are subjected to phosphate starvation. The role of PhoP in regulation of the skf operon was confirmed by in vitro gel shift assays, which showed that this operon is a new member of the PhoP regulon. A putative stem-loop structure in the skfA-skfB intergenic region is proposed to act as a stabilizer of an skfA-specific transcript. PMID:16816204

  8. Phosphate starvation induces the sporulation killing factor of Bacillus subtilis.

    PubMed

    Allenby, Nicholas E E; Watts, Carys A; Homuth, Georg; Prágai, Zoltán; Wipat, Anil; Ward, Alan C; Harwood, Colin R

    2006-07-01

    Bacillus subtilis produces and exports a peptide sporulation killing factor (SkfA) that induces lysis of sibling cells. skfA is part of the skf operon (skfA-H), which is responsible for immunity to SkfA, as well as for production and export of SkfA. Here we report that transcription of skfA is markedly induced when cells of B. subtilis are subjected to phosphate starvation. The role of PhoP in regulation of the skf operon was confirmed by in vitro gel shift assays, which showed that this operon is a new member of the PhoP regulon. A putative stem-loop structure in the skfA-skfB intergenic region is proposed to act as a stabilizer of an skfA-specific transcript.

  9. The low-temperature- and salt-induced RCI2A gene of Arabidopsis complements the sodium sensitivity caused by a deletion of the homologous yeast gene SNA1.

    PubMed

    Nylander, M; Heino, P; Helenius, E; Palva, E T; Ronne, H; Welin, B V

    2001-02-01

    Two closely related, tandemly arranged, low-temperature- and salt-induced Arabidopsis genes, corresponding to the previously isolated cDNAs RCI2A and RCI2B, were isolated and characterized. The RCI2A transcript accumulated primarily in response to low temperature or high salinity, and to a lesser extent in response to ABA treatment or water deficit stress. The RCI2B transcript was present at much lower levels than RCI2A, and could only be detected by reverse transcription-PCR amplification. The predicted 6 kDa RCI2 proteins are highly hydrophobic and contain two putative membrane-spanning regions. The polypeptides exhibit extensive similarity to deduced low-temperature- and/or salt-induced proteins from barley, wheat grass and strawberry, and to predicted proteins from bacteria, fungi, nematodes and yeast. Interestingly, we found that a deletion of the RCI2 homologous gene, SNA1 (YRD276c), in yeast causes a salt-sensitive phenotype. This effect is specific for sodium, since no growth defect was observed for the sna1 mutant on 1.7 M sorbitol, 1 M KCl or 0.6 M LiCl. Finally, we found that the Arabidopsis RCI2A cDNA can complement the sna1 mutant when expressed in yeast, indicating that the plant and yeast proteins have similar functions during high salt stress.

  10. Selectivity of C3-opsonin targeted complement inhibitors: A distinct advantage in the protection of erythrocytes from paroxysmal nocturnal hemoglobinuria patients.

    PubMed

    Schmidt, Christoph Q; Harder, Markus J; Nichols, Eva-Maria; Hebecker, Mario; Anliker, Markus; Höchsmann, Britta; Simmet, Thomas; Csincsi, Ádám I; Uzonyi, Barbara; Pappworth, Isabel Y; Ricklin, Daniel; Lambris, John D; Schrezenmeier, Hubert; Józsi, Mihály; Marchbank, Kevin J

    2016-04-01

    Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by complement-mediated cell lysis due to deficiency of GPI-anchored complement regulators. Blockage of the lytic pathway by eculizumab is the only available therapy for PNH patients and shows remarkable benefits, but regularly yields PNH erythrocytes opsonized with fragments of complement protein C3, rendering such erythrocytes prone to extravascular hemolysis. This effect is associated with insufficient responsiveness seen in a subgroup of PNH patients. Novel C3-opsonin targeted complement inhibitors act earlier in the cascade, at the level of activated C3 and are engineered from parts of the natural complement regulator Factor H (FH) or complement receptor 2 (CR2). This inhibitor class comprises three variants of "miniFH" and the clinically developed "FH-CR2" fusion-protein (TT30). We show that the approach of FH-CR2 to target C3-opsonins was more efficient in preventing complement activation induced by foreign surfaces, whereas the miniFH variants were substantially more active in controlling complement on PNH erythrocytes. Subtle differences were noted in the ability of each version of miniFH to protect human PNH cells. Importantly, miniFH and FH-CR2 interfered only minimally with complement-mediated serum killing of bacteria when compared to untargeted inhibition of all complement pathways by eculizumab. Thus, the molecular design of each C3-opsonin targeted complement inhibitor determines its potency in respect to the nature of the activator/surface providing potential functionality in PNH. PMID:26792457

  11. Growth factor involvement in tension-induced skeletal muscle growth

    NASA Technical Reports Server (NTRS)

    Vandenburgh, H. H.

    1987-01-01

    Muscle tissue culture techniques were developed to grow skeletal myofibers which differentiate into more adult-like myofibers. Mechanical simulation studies of these muscle cells in a newly developed mechanical cell simulator can now be performed to study growth processes in skeletal muscle. Conditions in the mechanical cell simulator were defined where mechanical activity can either prevent muscle wasting or stimulate muscle growth. The role of endogenous and exogenous growth factors in tension-induced muscle growth is being investigated under the defined conditions of tissue culture.

  12. Complement-dependent cytotoxicity crossmatch.

    PubMed

    Peña, Jeremy Ryan; Fitzpatrick, Donna; Saidman, Susan L

    2013-01-01

    The complement-dependent cytotoxic crossmatch is an informative test that detects alloantibodies in pre- and post-transplant patients, which may dictate clinical management of transplant patients. While challenging to perform, the cytotoxic crossmatch represents the only assay that provides direct evidence for the presence of potentially pathologic (i.e., cytotoxic) alloantibodies. The cytotoxic crossmatch combines patient (recipient) serum and donor cells. If donor-reactive alloantibodies are present in patient serum, these antibodies can bind donor cells. Antibody-antigen complexes, in turn, can activate the complement cascade, leading to complement-mediated cytotoxicity. Two commonly performed cytotoxic crossmatches, using donor lymphocytes as target cells, are described.

  13. Hypoxia-Inducible Factor-1α and Autoimmune Lupus, Arthritis.

    PubMed

    Yang, Zu-Cheng; Liu, Yi

    2016-06-01

    Hypoxia elicits an orchestrated response in cells, tissues, and entire organisms to survive a hypoxic challenge. On a molecular level, this response can be controlled by oxygen-dependent stabilization of the transcription factor hypoxia-inducible factor (HIF)-1α. Recently, studies have shown that HIF-1α plays an important role in the development and function of T helper (Th) cells, regulatory T (Treg) cells, and dendritic cells (DCs). Because these cells are critical in the pathogenesis of autoimmune diseases, such as systemic lupus erythematosus and rheumatoid arthritis, the roles of HIF-1α in these autoimmune disorders cannot be neglected. In this review, we discuss recent findings on the important roles of HIF-1α in immune cells and the possible pathologic roles of HIF-1α in autoimmune diseases. The obtained information may lead to deeper insights into the roles of HIF-1α in these disorders. PMID:27032396

  14. Three-phase power factor controller with induced EMF sensing

    NASA Technical Reports Server (NTRS)

    Nola, F. J. (Inventor)

    1984-01-01

    A power factor controller for an ac induction motor is provided which is of the type comprising thyristor switches connected in series with the motor, phase detectors for sensing the motor current and voltage and providing an output proportional to the phase difference between the motor voltage and current, and a control circuit, responsive to the output of the phase detector and to a power factor command signal, for controlling switching of the thyristor. The invention involves sensing the induced emf produced by the motor during the time interval when the thyristor is off and for producing a corresponding feedback signal for controlling switching of the thyristor. The sensed emf is also used to enhance soft starting of the motor.

  15. Vectorology and Factor Delivery in Induced Pluripotent Stem Cell Reprogramming

    PubMed Central

    2014-01-01

    Induced pluripotent stem cell (iPSC) reprogramming requires sustained expression of multiple reprogramming factors for a limited period of time (10–30 days). Conventional iPSC reprogramming was achieved using lentiviral or simple retroviral vectors. Retroviral reprogramming has flaws of insertional mutagenesis, uncontrolled silencing, residual expression and re-activation of transgenes, and immunogenicity. To overcome these issues, various technologies were explored, including adenoviral vectors, protein transduction, RNA transfection, minicircle DNA, excisable PiggyBac (PB) transposon, Cre-lox excision system, negative-sense RNA replicon, positive-sense RNA replicon, Epstein-Barr virus-based episomal plasmids, and repeated transfections of plasmids. This review provides summaries of the main vectorologies and factor delivery systems used in current reprogramming protocols. PMID:24625220

  16. Activation of the alternative complement pathway by exposure of phosphatidylethanolamine and phosphatidylserine on erythrocytes from sickle cell disease patients.

    PubMed Central

    Wang, R H; Phillips, G; Medof, M E; Mold, C

    1993-01-01

    Deoxygenation of erythrocytes from sickle cell anemia (SCA) patients alters membrane phospholipid distribution with increased exposure of phosphatidylethanolamine (PE) and phosphatidylserine (PS) on the outer leaflet. This study investigated whether altered membrane phospholipid exposure on sickle erythrocytes results in complement activation. In vitro deoxygenation of sickle but not normal erythrocytes resulted in complement activation measured by C3 binding. Additional evidence indicated that this activation was the result of the alterations in membrane phospholipids. First, complement was activated by normal erythrocytes after incubation with sodium tetrathionate, which produces similar phospholipid changes. Second, antibody was not required for complement activation by sickle or tetrathionate-treated erythrocytes. Third, the membrane regulatory proteins, decay-accelerating factor (CD55) and the C3b/C4b receptor (CD35), were normal on sickle and tetrathionate-treated erythrocytes. Finally, insertion of PE or PS into normal erythrocytes induced alternative pathway activation. SCA patients in crisis exhibited increased plasma factor Bb levels compared with baseline, and erythrocytes isolated from hospitalized SCA patients had increased levels of bound C3, indicating that alternative pathway activation occurs in vivo. Activation of complement may be a contributing factor in sickle crisis episodes, shortening the life span of erythrocytes and decreasing host defense against infections. Images PMID:7690777

  17. Macrophage-secreted factors induce adipocyte inflammation and insulin resistance

    SciTech Connect

    Permana, Paska A. . E-mail: Paska.Permana@med.va.gov; Menge, Christopher; Reaven, Peter D.

    2006-03-10

    Macrophage infiltration into adipose tissue increases with obesity, a condition associated with low-grade inflammation and insulin resistance. We investigated the direct effects of macrophage-secreted factors on adipocyte inflammation and insulin resistance. 3T3-L1 adipocytes incubated with media conditioned by RAW264.7 macrophages (RAW-CM) showed dramatically increased transcription of several inflammation-related genes, greater nuclear factor kappa B (NF-{kappa}B) activity, and enhanced binding of U937 monocytes. All of these effects were prevented by co-incubation with pyrrolidinedithiocarbamate, an NF-{kappa}B inhibitor. Adipocytes incubated with RAW-CM also released more non-esterified fatty acids and this increased lipolysis was not suppressed by insulin. In addition, RAW-CM treatment decreased insulin-stimulated glucose uptake in adipocytes. Taken together, these results indicate that macrophage-secreted factors induce inflammatory responses and reduce insulin responsiveness in adipocytes. These effects of macrophage-secreted factors on adipocytes may contribute significantly to the systemic inflammation and insulin resistance associated with obesity.

  18. Hide and Seek: How Lyme Disease Spirochetes Overcome Complement Attack

    PubMed Central

    Kraiczy, Peter

    2016-01-01

    Overcoming the first line of the innate immune system is a general hallmark of pathogenic microbes to avoid recognition and to enter the human host. In particular, spirochetes belonging to the Borrelia burgdorferi sensu lato complex have developed various means to counter the immune response and to successfully survive in diverse host environments for a prolonged period of time. In regard to complement resistance, Borrelia utilize a plethora of immune evasion strategies involves capturing of host-derived complement regulators, terminating complement activation as well as shedding of cell-destroying complement complexes to manipulate and to expeditiously inhibit human complement. Owing to their mode of action, the interacting surface-exposed proteins identified among B. burgdorferi sensu stricto (s.s.), Borrelia afzelii, Borrelia spielmanii, and Borrelia bavariensis can be classified into at least two major categories, namely, molecules that directly interfere with distinct complement components including BBK32, CspA, BGA66, BGA71, and a CD59-like protein or molecules, which indirectly counteract complement activation by binding various complement regulators such as Factor H, Factor H-like protein 1 (FHL-1), Factor H-related proteins FHR-1, FHR-2, or C4Bp. The latter group of genetically and structurally unrelated proteins has been collectively referred to as “complement regulator-acquiring surface proteins” and consists of CspA, CspZ, ErpA, ErpC, ErpP, and the as yet unidentified protein p43. This review focuses on the current knowledge of immune evasion mechanisms exhibited by Lyme disease spirochetes and highlights the role of complement-interfering, infection-associated molecules playing an important part in these processes. Deciphering the immune evasion strategies may provide novel avenues for improved diagnostic approaches and therapeutic interventions. PMID:27725820

  19. The hypoxia-inducible factor-1α activates ectopic production of fibroblast growth factor 23 in tumor-induced osteomalacia

    PubMed Central

    Zhang, Qian; Doucet, Michele; Tomlinson, Ryan E; Han, Xiaobin; Quarles, L Darryl; Collins, Michael T; Clemens, Thomas L

    2016-01-01

    Tumor-induced osteomalacia (TIO) is a rare paraneoplastic syndrome in which ectopic production of fibroblast growth factor 23 (FGF23) by non-malignant mesenchymal tumors causes phosphate wasting and bone fractures. Recent studies have implicated the hypoxia-inducible factor-1α (HIF-1α) in other phosphate wasting disorders caused by elevated FGF23, including X-linked hypophosphatemic rickets and autosomal dominant hypophosphatemia. Here we provide evidence that HIF-1α mediates aberrant FGF23 in TIO by transcriptionally activating its promoter. Immunohistochemical studies in phosphaturic mesenchymal tumors resected from patients with documented TIO showed that HIF-1α and FGF23 were co-localized in spindle-shaped cells adjacent to blood vessels. Cultured tumor tissue produced high levels of intact FGF23 and demonstrated increased expression of HIF-1α protein. Transfection of MC3T3-E1 and Saos-2 cells with a HIF-1α expression construct induced the activity of a FGF23 reporter construct. Prior treatment of tumor organ cultures with HIF-1α inhibitors decreased HIF-1α and FGF23 protein accumulation and inhibited HIF-1α-induced luciferase reporter activity in transfected cells. Chromatin immunoprecipitation assays confirmed binding to a HIF-1α consensus sequence within the proximal FGF23 promoter, which was eliminated by treatment with a HIF-1α inhibitor. These results show for the first time that HIF-1α is a direct transcriptional activator of FGF23 and suggest that upregulation of HIF-1α activity in TIO contributes to the aberrant FGF23 production in these patients. PMID:27468359

  20. Placental Induced Growth Factor (PIGf) in Coronary Artery Disease

    NASA Technical Reports Server (NTRS)

    Sundaresan, Alamelu; Carabello, Blaise; Mehta, Satish; Schlegel, Todd; Pellis, Neal; Ott, Mark; Pierson, Duane

    2010-01-01

    Our previous studies on normal human lymphocytes have shown a five-fold increase (p less than 0.001) in angiogenic inducers such as Placental Induced Growth Factor (PIGf) in physiologically stressful environments such as modeled microgravity, a space analog. This suggests de-regulation of cardiovascular signalling pathways indicated by upregulation of PIGf. In the current study, we measured PIGf in the plasma of 33 patients with and without coronary artery disease (CAD) to investigate whether such disease is associated with increased levels of PIGf. A control consisting of 31 sex matched apparently healthy subjects was also included in the study. We observed that the levels of PIGf in CAD patients were significantly increased compared to those in healthy control subjects (p less than 0.001) and usually increased beyond the clinical threshold level (greater than 27ng/L). The mechanisms leading to up-regulation of angiogenic factors and the adaptation of organisms to stressful environments such as isolation, high altitude, hypoxia, ischemia, microgravity, increased radiation, etc are presently unknown and require further investigation in spaceflight and these other physiologically stressed environments.

  1. Regulation of erythropoiesis by hypoxia-inducible factors.

    PubMed

    Haase, Volker H

    2013-01-01

    A classic physiologic response to systemic hypoxia is the increase in red blood cell production. Hypoxia-inducible factors (HIFs) orchestrate this response by inducing cell-type specific gene expression changes that result in increased erythropoietin (EPO) production in kidney and liver, in enhanced iron uptake and utilization and in adjustments of the bone marrow microenvironment that facilitate erythroid progenitor maturation and proliferation. In particular HIF-2 has emerged as the transcription factor that regulates EPO synthesis in the kidney and liver and plays a critical role in the regulation of intestinal iron uptake. Its key function in the hypoxic regulation of erythropoiesis is underscored by genetic studies in human populations that live at high-altitude and by mutational analysis of patients with familial erythrocytosis. This review provides a perspective on recent insights into HIF-controlled erythropoiesis and iron metabolism, and examines cell types that have EPO-producing capability. Furthermore, the review summarizes clinical syndromes associated with mutations in the O(2)-sensing pathway and the genetic changes that occur in high altitude natives. The therapeutic potential of pharmacologic HIF activation for the treatment of anemia is discussed. PMID:23291219

  2. Minor Role of Plasminogen in Complement Activation on Cell Surfaces

    PubMed Central

    Hyvärinen, Satu; Jokiranta, T. Sakari

    2015-01-01

    Atypical hemolytic uremic syndrome (aHUS) is a rare, but severe thrombotic microangiopathy. In roughly two thirds of the patients, mutations in complement genes lead to uncontrolled activation of the complement system against self cells. Recently, aHUS patients were described with deficiency of the fibrinolytic protein plasminogen. This zymogen and its protease form plasmin have both been shown to interact with complement proteins in the fluid phase. In this work we studied the potential of plasminogen to restrict complement propagation. In hemolytic assays, plasminogen inhibited complement activation, but only when it had been exogenously activated to plasmin and when it was used at disproportionately high concentrations compared to serum. Addition of only the zymogen plasminogen into serum did not hinder complement-mediated lysis of erythrocytes. Plasminogen could not restrict deposition of complement activation products on endothelial cells either, as was shown with flow cytometry. With platelets, a very weak inhibitory effect on deposition of C3 fragments was observed, but it was considered too weak to be significant for disease pathogenesis. Thus it was concluded that plasminogen is not an important regulator of complement on self cells. Instead, addition of plasminogen was shown to clearly hinder platelet aggregation in serum. This was attributed to plasmin causing disintegration of formed platelet aggregates. We propose that reduced proteolytic activity of plasmin on structures of growing thrombi, rather than on complement activation fragments, explains the association of plasminogen deficiency with aHUS. This adds to the emerging view that factors unrelated to the complement system can also be central to aHUS pathogenesis and suggests that future research on the mechanism of the disease should expand beyond complement dysregulation. PMID:26637181

  3. Growth Factors and Tension-Induced Skeletal Muscle Growth

    NASA Technical Reports Server (NTRS)

    Vandenburgh, Herman H.

    1994-01-01

    The project investigated biochemical mechanisms to enhance skeletal muscle growth, and developed a computer based mechanical cell stimulator system. The biochemicals investigated in this study were insulin/(Insulin like Growth Factor) IGF-1 and Steroids. In order to analyze which growth factors are essential for stretch-induced muscle growth in vitro, we developed a defined, serum-free medium in which the differentiated, cultured avian muscle fibers could be maintained for extended periods of time. The defined medium (muscle maintenance medium, MM medium) maintains the nitrogen balance of the myofibers for 3 to 7 days, based on myofiber diameter measurements and myosin heavy chain content. Insulin and IGF-1, but not IGF-2, induced pronounced myofiber hypertrophy when added to this medium. In 5 to 7 days, muscle fiber diameters increase by 71 % to 98% compared to untreated controls. Mechanical stimulation of the avian muscle fibers in MM medium increased the sensitivity of the cells to insulin and IGF-1, based on a leftward shift of the insulin dose/response curve for protein synthesis rates. (54). We developed a ligand binding assay for IGF-1 binding proteins and found that the avian skeletal muscle cultures produced three major species of 31, 36 and 43 kD molecular weight (54) Stretch of the myofibers was found to have no significant effect on the efflux of IGF-1 binding proteins, but addition of exogenous collagen stimulated IGF-1 binding protein production 1.5 to 5 fold. Steroid hormones have a profound effect on muscle protein turnover rates in vivo, with the stress-related glucocorticoids inducing rapid skeletal muscle atrophy while androgenic steroids induce skeletal muscle growth. Exercise in humans and animals reduces the catabolic effects of glucocorticoids and may enhance the anabolic effects of androgenic steroids on skeletal muscle. In our continuing work on the involvement of exogenrus growth factors in stretch-induced avian skeletal muscle growth, we

  4. Developmental Expression and Hypoxic Induction of Hypoxia Inducible Transcription Factors in the Zebrafish.

    PubMed

    Köblitz, Louise; Fiechtner, Birgit; Baus, Katharina; Lussnig, Rebecca; Pelster, Bernd

    2015-01-01

    The hypoxia inducible transcription factor (HIF) has been shown to coordinate the hypoxic response of vertebrates and is expressed in three different isoforms, HIF-1α, HIF-2α and HIF-3α. Knock down of either Hif-1α or Hif-2α in mice results in lethality in embryonic or perinatal stages, suggesting that this transcription factor is not only controlling the hypoxic response, but is also involved in developmental phenomena. In the translucent zebrafish embryo the performance of the cardiovascular system is not essential for early development, therefore this study was designed to analyze the expression of the three Hif-isoforms during zebrafish development and to test the hypoxic inducibility of these transcription factors. To complement the existing zfHif-1α antibody we expressed the whole zfHif-2α protein and used it for immunization and antibody generation. Similarly, fragments of the zfHif-3α protein were used for immunization and generation of a zfHif-3α specific antibody. To demonstrate presence of the Hif-isoforms during development [between 1 day post fertilization (1 dpf) and 9 dpf] affinity-purified antibodies were used. Hif-1α protein was present under normoxic conditions in all developmental stages, but no significant differences between the different developmental stages could be detected. Hif-2α was also present from 1 dpf onwards, but in post hatching stages (between 5 and 9 dpf) the expression level was significantly higher than prior to hatching. Similarly, Hif-3α was expressed from 1 dpf onwards, and the expression level significantly increased until 5 dpf, suggesting that Hif-2α and Hif-3α play a particular role in early development. Hypoxic exposure (oxygen partial pressure = 5 kPa) in turn caused a significant increase in the level of Hif-1α protein even at 1 dpf and in later stages, while neither Hif-2α nor Hif-3α protein level were affected. In these early developmental stages Hif-1α therefore appears to be more important for

  5. How antibodies use complement to regulate antibody responses.

    PubMed

    Sörman, Anna; Zhang, Lu; Ding, Zhoujie; Heyman, Birgitta

    2014-10-01

    Antibodies, forming immune complexes with their specific antigen, can cause complete suppression or several 100-fold enhancement of the antibody response. Immune complexes containing IgG and IgM may activate complement and in such situations also complement components will be part of the immune complex. Here, we review experimental data on how antibodies via the complement system upregulate specific antibody responses. Current data suggest that murine IgG1, IgG2a, and IgG2b upregulate antibody responses primarily via Fc-receptors and not via complement. In contrast, IgM and IgG3 act via complement and require the presence of complement receptors 1 and 2 (CR1/2) expressed on both B cells and follicular dendritic cells. Complement plays a crucial role for antibody responses not only to antigen complexed to antibodies, but also to antigen administered alone. Lack of C1q, but not of Factor B or MBL, severely impairs antibody responses suggesting involvement of the classical pathway. In spite of this, normal antibody responses are found in mice lacking several activators of the classical pathway (complement activating natural IgM, serum amyloid P component (SAP), specific intracellular adhesion molecule-grabbing non-integrin R1 (SIGN-R1) or C-reactive protein. Possible explanations to these observations will be discussed.

  6. The emerging role of complement inhibitors in transplantation.

    PubMed

    Frémeaux-Bacchi, Véronique; Legendre, Christophe M

    2015-11-01

    The role of complement in the biology of kidney transplantation is becoming more and more significant, especially but not only because we now have access to drugs inhibiting complement. After describing the main characteristics of complement biology, both activation of the complement cascade and the many regulatory factors, we will review the precise role of complement in kidney transplant biology. Complement activation has been involved in ischemia-reperfusion injury, in the recurrence of several diseases such as atypical hemolytic uremic syndrome, C3 glomerulopathies, and antiphospholipid syndrome, as well as the process of antibody-mediated rejection, either acute or chronic. There are many potentially interesting drugs interfering with complement inhibition that have been or may be studied in kidney transplantation. Currently, the bulk of data concerns eculizumab, a monoclonal antibody blocking the complement cascade at the C5. Its efficacy has been demonstrated in the treatment and prevention of recurrence of atypical hemolytic uremic syndrome with an overall good safety profile. Although it has been reported to be efficacious to prevent antibody-mediated rejection, properly designed trials are currently being performed to state this efficacy. In addition, randomized trials are, in the process, regarding the prevention of ischemia-reperfusion injury after kidney transplantation.

  7. The hypoxia-inducible factor renders cancer cells more sensitive to vitamin C-induced toxicity.

    PubMed

    Tian, Weihua; Wang, Yu; Xu, Yan; Guo, Xiangpeng; Wang, Bo; Sun, Li; Liu, Longqi; Cui, Fenggong; Zhuang, Qiang; Bao, Xichen; Schley, Gunnar; Chung, Tung-Liang; Laslett, Andrew L; Willam, Carsten; Qin, Baoming; Maxwell, Patrick H; Esteban, Miguel A

    2014-02-01

    Megadose vitamin C (Vc) is one of the most enduring alternative treatments for diverse human diseases and is deeply engrafted in popular culture. Preliminary studies in the 1970s described potent effects of Vc on prolonging the survival of patients with terminal cancer, but these claims were later criticized. An improved knowledge of the pharmacokinetics of Vc and recent reports using cancer cell lines have renewed the interest in this subject. Despite these findings, using Vc as an adjuvant for anticancer therapy remains questionable, among other things because there is no proper mechanistic understanding. Here, we show that a Warburg effect triggered by activation of the hypoxia-inducible factor (HIF) pathway greatly enhances Vc-induced toxicity in multiple cancer cell lines, including von Hippel-Lindau (VHL)-defective renal cancer cells. HIF increases the intracellular uptake of oxidized Vc through its transcriptional target glucose transporter 1 (GLUT1), synergizing with the uptake of its reduced form through sodium-dependent Vc transporters. The resulting high levels of intracellular Vc induce oxidative stress and massive DNA damage, which then causes metabolic exhaustion by depleting cellular ATP reserves. HIF-positive cells are particularly sensitive to Vc-induced ATP reduction because they mostly rely on the rather inefficient glycolytic pathway for energy production. Thus, our experiments link Vc-induced toxicity and cancer metabolism, providing a new explanation for the preferential effect of Vc on cancer cells. PMID:24371136

  8. Erythropoietin is a hypoxia inducible factor-induced protective molecule in experimental autoimmune neuritis.

    PubMed

    Luo, Bangwei; Jiang, Man; Yang, Xiaofeng; Zhang, Zhiyuan; Xiong, Jian; Schluesener, Hermann J; Zhang, Zhiren; Wu, Yuzhang

    2013-08-01

    Experimental autoimmune neuritis (EAN), an autoantigen-specific T-cell-mediated disease model for human demyelinating inflammatory disease of the peripheral nervous system, is characterized by self-limitation. Here we investigated the regulation and contribution of erythropoietin (EPO) in EAN self-limitation. In EAN sciatic nerves, hypoxia, and protein and mRNA levels of hypoxia-inducible factor 1α (HIF-1α), HIF-2α, EPO and EPO receptor (EPOR) were induced in parallel at disease peak phase but reduced at recovery periods. Further, the deactivation of HIF reduced EAN-induced EPO/EPOR upregulation in EAN, suggesting the central contribution of HIF to EPO/EPOR induction. The deactivation of EPOR signalling exacerbated EAN progression, implying that endogenous EPO contributed to EAN recovery. Exogenous EPO treatment greatly improved EAN recovery. In addition, EPO was shown to promote Schwann cell survival and myelin production. In EAN, EPO treatment inhibited lymphocyte proliferation and altered helper T cell differentiation by inducing increase of Foxp3(+)/CD4(+) regulatory T cells and decrease of IFN-γ(+)/CD4(+) Th1 cells. Furthermore, EPO inhibited inflammatory macrophage activation and promoted its phagocytic activity. In summary, our data demonstrated that EPO was induced in EAN by HIF and contributed to EAN recovery, and endogenous and exogenous EPO could effectively suppress EAN by attenuating inflammation and exerting direct cell protection, indicating that EPO contributes to the self-recovery of EAN and could be a potent candidate for treatment of autoimmune neuropathies. PMID:23603807

  9. Multi-Faceted Proteomic Characterization of Host Protein Complement of Rift Valley Fever Virus Virions and Identification of Specific Heat Shock Proteins, Including HSP90, as Important Viral Host Factors

    PubMed Central

    Nuss, Jonathan E.; Kehn-Hall, Kylene; Benedict, Ashwini; Costantino, Julie; Ward, Michael; Peyser, Brian D.; Retterer, Cary J.; Tressler, Lyal E.; Wanner, Laura M.; McGovern, Hugh F.; Zaidi, Anum; Anthony, Scott M.; Kota, Krishna P.; Bavari, Sina; Hakami, Ramin M.

    2014-01-01

    Rift Valley fever is a potentially fatal disease of humans and domestic animals caused by Rift Valley fever virus (RVFV). Infection with RVFV in ruminants can cause near 100% abortion rates and recent outbreaks in naïve human populations have suggested case fatality rates of greater than thirty percent. To elucidate the roles that host proteins play during RVFV infection, proteomic analysis of RVFV virions was conducted using complementary analytical approaches, followed by functional validation studies of select identified host factors. Coupling the more traditional Gel LC/MS/MS approach (SDS PAGE followed by liquid chromatography tandem mass spectrometry) with an alternative technique that preserves protein complexes allowed the protein complement of these viral particles to be thoroughly examined. In addition to viral proteins present within the virions and virion-associated host proteins, multiple macromolecular complexes were identified. Bioinformatic analysis showed that host chaperones were among over-represented protein families associated with virions, and functional experiments using siRNA gene silencing and small molecule inhibitors identified several of these heat shock proteins, including heat shock protein 90 (HSP90), as important viral host factors. Further analysis indicated that HSP inhibition effects occur during the replication/transcription phase of the virus life cycle, leading to significant lowering of viral titers without compromising the functional capacity of released virions. Overall, these studies provide much needed further insight into interactions between RVFV and host cells, increasing our understanding of the infection process and suggesting novel strategies for anti-viral development. In particular, considering that several HSP90 inhibitors have been advancing through clinical trials for cancer treatment, these results also highlight the exciting potential of repurposing HSP90 inhibitors to treat RVF. PMID:24809507

  10. Multi-faceted proteomic characterization of host protein complement of Rift Valley fever virus virions and identification of specific heat shock proteins, including HSP90, as important viral host factors.

    PubMed

    Nuss, Jonathan E; Kehn-Hall, Kylene; Benedict, Ashwini; Costantino, Julie; Ward, Michael; Peyser, Brian D; Retterer, Cary J; Tressler, Lyal E; Wanner, Laura M; McGovern, Hugh F; Zaidi, Anum; Anthony, Scott M; Kota, Krishna P; Bavari, Sina; Hakami, Ramin M

    2014-01-01

    Rift Valley fever is a potentially fatal disease of humans and domestic animals caused by Rift Valley fever virus (RVFV). Infection with RVFV in ruminants can cause near 100% abortion rates and recent outbreaks in naïve human populations have suggested case fatality rates of greater than thirty percent. To elucidate the roles that host proteins play during RVFV infection, proteomic analysis of RVFV virions was conducted using complementary analytical approaches, followed by functional validation studies of select identified host factors. Coupling the more traditional Gel LC/MS/MS approach (SDS PAGE followed by liquid chromatography tandem mass spectrometry) with an alternative technique that preserves protein complexes allowed the protein complement of these viral particles to be thoroughly examined. In addition to viral proteins present within the virions and virion-associated host proteins, multiple macromolecular complexes were identified. Bioinformatic analysis showed that host chaperones were among over-represented protein families associated with virions, and functional experiments using siRNA gene silencing and small molecule inhibitors identified several of these heat shock proteins, including heat shock protein 90 (HSP90), as important viral host factors. Further analysis indicated that HSP inhibition effects occur during the replication/transcription phase of the virus life cycle, leading to significant lowering of viral titers without compromising the functional capacity of released virions. Overall, these studies provide much needed further insight into interactions between RVFV and host cells, increasing our understanding of the infection process and suggesting novel strategies for anti-viral development. In particular, considering that several HSP90 inhibitors have been advancing through clinical trials for cancer treatment, these results also highlight the exciting potential of repurposing HSP90 inhibitors to treat RVF.

  11. Moisture-Induced Alumina Scale Spallation: The Hydrogen Factor

    NASA Technical Reports Server (NTRS)

    Smialek, James L.

    2010-01-01

    For some time the oxidation community has been concerned with interfacial spallation of protective alumina scales, not just upon immediate cool down, but as a time-delayed phenomenon. Moisture-induced delayed spallation (MIDS) and desktop spallation (DTS) of thermal barrier coatings (TBCs) refer to this process. It is most apparent for relatively adherent alumina scales that have survived initial cool down in a dry environment, have built up considerable thickness and strain energy, and have been somewhat damaged, such as by cyclic oxidation cracking. Indeed, a "sensitive zone" can be described that maximizes the observed effect as a function of all the relevant factors. Moisture has been postulated to serve as a source of interfacial hydrogen embrittlement. Hydrogen is derived from reaction with aluminum in the alloy at an exposed interface. The purpose of this monograph is to trace the close analogy of this phenomenon to other hydrogen-induced effects, such as embrittlement of aluminides and blistering of alloys and anodic alumina films. A formalized, top-down, logic-tree structure is presented as a guide to this discussion. A theoretical basis for interfacial weakening by hydrogen is first cited, as are demonstrations of hydrogen detection as a reaction product or interfacial species. Further support is provided by critical experiments that recreate the moisture effect, but by isolating hydrogen from other potential causative factors. These experiments include tests in H 2-containing atmospheres or cathodic hydrogen charging. Accordingly, they strongly indicate that interfacial hydrogen, derived from moisture, is the key chemical species accounting for delayed alumina scale spallation.

  12. Factors associated with induced abortion among women in Hohoe, Ghana.

    PubMed

    Mote, Charity V; Otupiri, Easmon; Hindin, Michelle J

    2010-12-01

    In Hohoe, Ghana, induced abortion is the second highest cause of hospital admissions. We aimed to describe factors influencing induced abortion among 408 randomly selected women aged 15-49 years. 21% of the women had had an abortion; of those, 36% said they did not want to disrupt their education or employment; 66% of the abortions were performed by doctors. Bivariate logistic regression showed that compared with women with secondary education, women with basic education (OR = 0.31, 95% CI: 0.18-0.54) and uneducated women (OR = 0.24, 95% CI: 0.07-0.70) were significantly less likely to have had an abortion. Women who were married (OR = 1.83, 95% CI: 1.10-3.04), peri-urban residents (OR = 1.88, 95% CI: 0.95-3.94), and women with formal employment (OR = 2.22, 95% CI: 0.86-5.45) were more likely to have had an abortion. Stakeholders should improve access to effective contraception to lower the chance of needing an abortion and target education programmes at those with unmet need for contraception.

  13. Platelet-activating factor-induced increases in glucose kinetics

    SciTech Connect

    Lang, C.H.; Dobrescu, C.; Hargrove, D.M.; Bagby, G.J.; Spitzer, J.J. )

    1988-02-01

    Platelet-activating factor (PAF) is a postulated mediator of many of the early hemodynamic effects of endotoxin. The aim of the present study was to determine whether in vivo administration of PAF could produce alterations in whole-body glucose metabolism that would mimic those seen during endotoxemia. Glucose kinetics were assessed in chronically catheterized conscious rats by the constant infusion of (6-{sup 3}H)- and (U-{sup 14}C)glucose before and for 4 h after either a bolus injection or a constant infusion of PAF. The bolus injection of PAF elevated the rate of glucose appearance (R{sub a}; 44%) for 1.5 h. The lower PAF infusion rate decreased blood pressure 11% to 104 mmHg, whereas the higher infusion rate decreased pressure 34% to 77 mmHg. Both PAF infusion rates produced elevations in plasma glucose and glucose R{sub a} throughout the 4-h infusion period in a dose-related manner. The PAF infusions also induced dose-related increases in plasma glucagon and catecholamine levels throughout the infusion period. Because the constant infusion of PAF did stimulate many of the hemodynamic and metabolic alterations produced by endotoxin, this study provides additional support for the potential importance of PAF as a mediator of the early hemodynamic and metabolic sequela of endotoxin shock. Furthermore, the PAF-induced changes in glucose metabolism appear to be mediated by the resultant elevation in plasma catecholamines.

  14. Tumor necrosis factor alpha-induced pulmonary vascular endothelial injury.

    PubMed Central

    Goldblum, S E; Hennig, B; Jay, M; Yoneda, K; McClain, C J

    1989-01-01

    Tumor necrosis factor alpha (TNF-alpha) mediates components of the acute-phase response, stimulates granulocyte metabolism, and induces endothelial cell surface changes. We studied whether human recombinant TNF-alpha (rTNF-alpha) could increase pulmonary edema formation and pulmonary vascular permeability. Rabbits preinfused with 125I-albumin were administered rTNF-alpha or saline. Animals were sacrificed, and lung wet/dry weight ratios as well as bronchoalveolar lavage fluid and plasma 125I activities were determined. rTNF-alpha increased lung wet/dry weight ratios by 151% (P less than 0.02) and bronchoalveolar lavage fluid/plasma 125I activity ratios by 376% (P less than 0.01) compared with values for saline controls. Electron microscopy of lung sections demonstrated endothelial injury, perivascular edema, and extravasation of an ultrastructural permeability tracer. To demonstrate that rTNF-alpha could directly increase pulmonary vascular endothelial permeability in vitro, we studied albumin transfer across cultured porcine pulmonary artery endothelial cell monolayers. rTNF-alpha induced time-dependent dose-response increments in transendothelial albumin flux in the absence of granulocyte effector cells. These observations suggest that rTNF-alpha can provoke acute pulmonary vascular endothelial injury in vivo as well as in vitro. Images PMID:2925247

  15. Hypoxia‐inducible factor expression in human RPE cells

    PubMed Central

    Forooghian, Farzin; Razavi, Rozita; Timms, Lee

    2007-01-01

    Background Hypoxia‐inducible factor (HIF) is a common transcription factor for many angiogenic proteins. Retinal pigment epithelial (RPE) cells are an important source of angiogenic factors in the retina. The expression of HIF, its regulation by proline hydroxylase (PHD) enzymes, and its downstream regulation of angiogenic factors like vascular endothelial growth factor (VEGF) and erythropoietin (EPO) was studied in RPE cells in order to determine some of the molecular mechanisms underlying ischaemic retinal disease. Methods ARPE‐19 cells were cultured for various times under hypoxic conditions. Cellular HIF and PHD isoforms were analysed and quantified using western blot and densitometry. VEGF and EPO secreted into the media were assayed using enzyme‐linked immunosorbent assay (ELISA). Messenger RNA (mRNA) was quantified using real‐time quantitative reverse transcriptase polymerase chain reaction (qPCR). RNA interference was achieved using siRNA techniques. Results HIF‐1α was readily produced by ARPE‐19 cells under hypoxia, but HIF‐2α and HIF‐3α could not be detected even after HIF‐1α silencing. HIF‐1α protein levels showed an increasing trend for the first 24 h while HIF‐1α mRNA levels fluctuated during this time. After 36 h HIF‐1α protein levels declined to baseline levels, a change that was coincident with a rise in both PHD2 and PHD3. Silencing HIF‐1α significantly decreased VEGF secretion. Significant production of EPO could not be detected at the protein or mRNA level. Conclusions HIF‐1α appears to be the main isoform of HIF functioning in ARPE‐19 cells. Under hypoxia, HIF‐1α levels are likely self‐regulated by a feedback loop that involves both transcriptional and post‐translational mechanisms. VEGF production by human RPE cells is regulated by HIF‐1α. EPO was not produced in significant amounts by RPE cells under hypoxic conditions, suggesting that other cells and/or transcription factors in the retina

  16. Complement activation promotes muscle inflammation during modified muscle use

    NASA Technical Reports Server (NTRS)

    Frenette, J.; Cai, B.; Tidball, J. G.

    2000-01-01

    Modified muscle use can result in muscle inflammation that is triggered by unidentified events. In the present investigation, we tested whether the activation of the complement system is a component of muscle inflammation that results from changes in muscle loading. Modified rat hindlimb muscle loading was achieved by removing weight-bearing from the hindlimbs for 10 days followed by reloading through normal ambulation. Experimental animals were injected with the recombinant, soluble complement receptor sCR1 to inhibit complement activation. Assays for complement C4 or factor B in sera showed that sCR1 produced large reductions in the capacity for activation of the complement system through both the classical and alternative pathways. Analysis of complement C4 concentration in serum in untreated animals showed that the classical pathway was activated during the first 2 hours of reloading. Analysis of factor B concentration in untreated animals showed activation of the alternative pathway at 6 hours of reloading. Administration of sCR1 significantly attenuated the invasion of neutrophils (-49%) and ED1(+) macrophages (-52%) that occurred in nontreated animals after 6 hours of reloading. The presence of sCR1 also reduced significantly the degree of edema by 22% as compared to untreated animals. Together, these data show that increased muscle loading activated the complement system which then briefly contributes to the early recruitment of inflammatory cells during modified muscle loading.

  17. Impaired NK Cell Activation and Chemotaxis toward Dendritic Cells Exposed to Complement-Opsonized HIV-1

    PubMed Central

    Ellegård, Rada; Crisci, Elisa; Andersson, Jonas; Shankar, Esaki M.; Nyström, Sofia; Hinkula, Jorma

    2015-01-01

    Mucosa resident dendritic cells (DCs) may represent one of the first immune cells that HIV-1 encounters during sexual transmission. The virions in body fluids can be opsonized with complement factors because of HIV-mediated triggering of the complement cascade, and this appears to influence numerous aspects of the immune defense targeting the virus. One key attribute of host defense is the ability to attract immune cells to the site of infection. In this study, we investigated whether the opsonization of HIV with complement (C-HIV) or a mixture of complement and Abs (CI-HIV) affected the cytokine and chemokine responses generated by DCs, as well as their ability to attract other immune cells. We found that the expression levels of CXCL8, CXCL10, CCL3, and CCL17 were lowered after exposure to either C-HIV or CI-HIV relative to free HIV (F-HIV). DCs exposed to F-HIV induced higher cell migration, consisting mainly of NK cells, compared with opsonized virus, and the chemotaxis of NK cells was dependent on CCL3 and CXCL10. NK cell exposure to supernatants derived from HIV-exposed DCs showed that F-HIV induced phenotypic activation (e.g., increased levels of TIM3, CD69, and CD25) and effector function (e.g., production of IFNγ and killing of target cells) in NK cells, whereas C-HIV and CI-HIV did not. The impairment of NK cell recruitment by DCs exposed to complement-opsonized HIV and the lack of NK activation may contribute to the failure of innate immune responses to control HIV at the site of initial mucosa infection. PMID:26157174

  18. Impaired NK Cell Activation and Chemotaxis toward Dendritic Cells Exposed to Complement-Opsonized HIV-1.

    PubMed

    Ellegård, Rada; Crisci, Elisa; Andersson, Jonas; Shankar, Esaki M; Nyström, Sofia; Hinkula, Jorma; Larsson, Marie

    2015-08-15

    Mucosa resident dendritic cells (DCs) may represent one of the first immune cells that HIV-1 encounters during sexual transmission. The virions in body fluids can be opsonized with complement factors because of HIV-mediated triggering of the complement cascade, and this appears to influence numerous aspects of the immune defense targeting the virus. One key attribute of host defense is the ability to attract immune cells to the site of infection. In this study, we investigated whether the opsonization of HIV with complement (C-HIV) or a mixture of complement and Abs (CI-HIV) affected the cytokine and chemokine responses generated by DCs, as well as their ability to attract other immune cells. We found that the expression levels of CXCL8, CXCL10, CCL3, and CCL17 were lowered after exposure to either C-HIV or CI-HIV relative to free HIV (F-HIV). DCs exposed to F-HIV induced higher cell migration, consisting mainly of NK cells, compared with opsonized virus, and the chemotaxis of NK cells was dependent on CCL3 and CXCL10. NK cell exposure to supernatants derived from HIV-exposed DCs showed that F-HIV induced phenotypic activation (e.g., increased levels of TIM3, CD69, and CD25) and effector function (e.g., production of IFNγ and killing of target cells) in NK cells, whereas C-HIV and CI-HIV did not. The impairment of NK cell recruitment by DCs exposed to complement-opsonized HIV and the lack of NK activation may contribute to the failure of innate immune responses to control HIV at the site of initial mucosa infection. PMID:26157174

  19. Endocannabinoids participate in placental apoptosis induced by hypoxia inducible factor-1.

    PubMed

    Abán, C; Martinez, N; Carou, C; Albamonte, I; Toro, A; Seyahian, A; Franchi, A; Leguizamón, G; Trigubo, D; Damiano, A; Farina, M

    2016-10-01

    During pregnancy, apoptosis is a physiological event critical in the remodeling and aging of the placenta. Increasing evidence has pointed towards the relevance of endocannabinoids (ECs) and hypoxia as modulators of trophoblast cell death. However, the relation between these factors is still unknown. In this report, we evaluated the participation of ECs in placental apoptosis induced by cobalt chloride (CoCl2), a hypoxia mimicking agent that stabilizes the expression of hypoxia inducible factor-1 alpha (HIF-1α). We found that HIF-1α stabilization decreased FAAH mRNA and protein levels, suggesting an increase in ECs tone. Additionally, CoCl2 incubation and Met-AEA treatment reduced cell viability and increased TUNEL-positive staining in syncytiotrophoblast layer. Immunohistochemical analysis demonstrated Bax and Bcl-2 protein expression in the cytoplasm of syncytiotrophoblast. Finally, HIF-1α stabilization produced an increase in Bax/Bcl-2 ratio, activation of caspase 3 and PARP cleavage. All these changes in apoptotic parameters were reversed with AM251, a CB1 antagonist. These results demonstrate that HIF-1α may induce apoptosis in human placenta via intrinsic pathway by a mechanism that involves activation of CB1 receptor suggesting a role of the ECs in this process.

  20. Endocannabinoids participate in placental apoptosis induced by hypoxia inducible factor-1.

    PubMed

    Abán, C; Martinez, N; Carou, C; Albamonte, I; Toro, A; Seyahian, A; Franchi, A; Leguizamón, G; Trigubo, D; Damiano, A; Farina, M

    2016-10-01

    During pregnancy, apoptosis is a physiological event critical in the remodeling and aging of the placenta. Increasing evidence has pointed towards the relevance of endocannabinoids (ECs) and hypoxia as modulators of trophoblast cell death. However, the relation between these factors is still unknown. In this report, we evaluated the participation of ECs in placental apoptosis induced by cobalt chloride (CoCl2), a hypoxia mimicking agent that stabilizes the expression of hypoxia inducible factor-1 alpha (HIF-1α). We found that HIF-1α stabilization decreased FAAH mRNA and protein levels, suggesting an increase in ECs tone. Additionally, CoCl2 incubation and Met-AEA treatment reduced cell viability and increased TUNEL-positive staining in syncytiotrophoblast layer. Immunohistochemical analysis demonstrated Bax and Bcl-2 protein expression in the cytoplasm of syncytiotrophoblast. Finally, HIF-1α stabilization produced an increase in Bax/Bcl-2 ratio, activation of caspase 3 and PARP cleavage. All these changes in apoptotic parameters were reversed with AM251, a CB1 antagonist. These results demonstrate that HIF-1α may induce apoptosis in human placenta via intrinsic pathway by a mechanism that involves activation of CB1 receptor suggesting a role of the ECs in this process. PMID:27488203

  1. Moisture-Induced Alumina Scale Spallation: The Hydrogen Factor

    NASA Technical Reports Server (NTRS)

    Smialek, James L.

    2009-01-01

    For some time our community has been concerned with interfacial spallation of protective alumina scales, not just upon immediate cooldown, but as a time-delayed phenomenon. Moisture-induced delayed spallation (MIDS) and desktop spallation (DTS) of TBC's refer to this process. It is most apparent for relatively adherent alumina scales that have survived cool down in a dry environment, built up considerable thickness and strain energy, and have been somewhat damaged, such as by cyclic oxidation cracking. Indeed, a "sweet zone" can be defined that maximizes the observed effect as a function of all the relevant factors. Moisture has been postulated to serve as a source of interfacial hydrogen embrittlement derived from reaction with aluminum in the alloy at an exposed interface. The purpose of this monograph is to trace the close analogy of this phenomenon to other hydrogen effects, such as embrittlement of aluminides and blistering of alloys and anodic alumina films. A formalized, top-down, logic tree structure is presented as a guide to this discussion. A theoretical basis for interfacial weakening by hydrogen is first cited, as are demonstrations of hydrogen as a reaction product or detected interfacial species. Further support is provided by critical experiments that produce the same moisture effect, but by isolating hydrogen from other potential causative factors. These experiments include tests in H2-containing atmospheres or cathodic hydrogen charging.

  2. Connective tissue growth factor induces cardiac hypertrophy through Akt signaling

    SciTech Connect

    Hayata, Nozomi; Fujio, Yasushi; Yamamoto, Yasuhiro; Iwakura, Tomohiko; Obana, Masanori; Takai, Mika; Mohri, Tomomi; Nonen, Shinpei; Maeda, Makiko; Azuma, Junichi

    2008-05-30

    In the process of cardiac remodeling, connective tissue growth factor (CTGF/CCN2) is secreted from cardiac myocytes. Though CTGF is well known to promote fibroblast proliferation, its pathophysiological effects in cardiac myocytes remain to be elucidated. In this study, we examined the biological effects of CTGF in rat neonatal cardiomyocytes. Cardiac myocytes stimulated with full length CTGF and its C-terminal region peptide showed the increase in cell surface area. Similar to hypertrophic ligands for G-protein coupled receptors, such as endothelin-1, CTGF activated amino acid uptake; however, CTGF-induced hypertrophy is not associated with the increased expression of skeletal actin or BNP, analyzed by Northern-blotting. CTGF treatment activated ERK1/2, p38 MAPK, JNK and Akt. The inhibition of Akt by transducing dominant-negative Akt abrogated CTGF-mediated increase in cell size, while the inhibition of MAP kinases did not affect the cardiac hypertrophy. These findings indicate that CTGF is a novel hypertrophic factor in cardiac myocytes.

  3. Musashi mediates translational repression of the Drosophila hypoxia inducible factor.

    PubMed

    Bertolin, Agustina P; Katz, Maximiliano J; Yano, Masato; Pozzi, Berta; Acevedo, Julieta M; Blanco-Obregón, Dalmiro; Gándara, Lautaro; Sorianello, Eleonora; Kanda, Hiroshi; Okano, Hideyuki; Srebrow, Anabella; Wappner, Pablo

    2016-09-19

    Adaptation to hypoxia depends on a conserved α/β heterodimeric transcription factor called Hypoxia Inducible Factor (HIF), whose α-subunit is regulated by oxygen through different concurrent mechanisms. In this study, we have identified the RNA binding protein dMusashi, as a negative regulator of the fly HIF homologue Sima. Genetic interaction assays suggested that dMusashi participates of the HIF pathway, and molecular studies carried out in Drosophila cell cultures showed that dMusashi recognizes a Musashi Binding Element in the 3' UTR of the HIFα transcript, thereby mediating its translational repression in normoxia. In hypoxic conditions dMusashi is downregulated, lifting HIFα repression and contributing to trigger HIF-dependent gene expression. Analysis performed in mouse brains revealed that murine Msi1 protein physically interacts with HIF-1α transcript, suggesting that the regulation of HIF by Msi might be conserved in mammalian systems. Thus, Musashi is a novel regulator of HIF that inhibits responses to hypoxia specifically when oxygen is available.

  4. Musashi mediates translational repression of the Drosophila hypoxia inducible factor

    PubMed Central

    Bertolin, Agustina P.; Katz, Maximiliano J.; Yano, Masato; Pozzi, Berta; Acevedo, Julieta M.; Blanco-Obregón, Dalmiro; Gándara, Lautaro; Sorianello, Eleonora; Kanda, Hiroshi; Okano, Hideyuki; Srebrow, Anabella; Wappner, Pablo

    2016-01-01

    Adaptation to hypoxia depends on a conserved α/β heterodimeric transcription factor called Hypoxia Inducible Factor (HIF), whose α-subunit is regulated by oxygen through different concurrent mechanisms. In this study, we have identified the RNA binding protein dMusashi, as a negative regulator of the fly HIF homologue Sima. Genetic interaction assays suggested that dMusashi participates of the HIF pathway, and molecular studies carried out in Drosophila cell cultures showed that dMusashi recognizes a Musashi Binding Element in the 3′ UTR of the HIFα transcript, thereby mediating its translational repression in normoxia. In hypoxic conditions dMusashi is downregulated, lifting HIFα repression and contributing to trigger HIF-dependent gene expression. Analysis performed in mouse brains revealed that murine Msi1 protein physically interacts with HIF-1α transcript, suggesting that the regulation of HIF by Msi might be conserved in mammalian systems. Thus, Musashi is a novel regulator of HIF that inhibits responses to hypoxia specifically when oxygen is available. PMID:27141964

  5. Eosinophil granule cationic proteins regulate the classical pathway of complement.

    PubMed Central

    Weiler, J M; Edens, R E; Bell, C S; Gleich, G J

    1995-01-01

    Major basic protein, the primary constituent of eosinophil granules, regulates the alternative and classical pathways of complement. Major basic protein and other eosinophil granule cationic proteins, which are important in mediating tissue damage in allergic disease, regulate the alternative pathway by interfering with C3b interaction with factor B to assemble an alternative pathway C3 convertase. In the present study, eosinophil peroxidase, eosinophil cationic protein and eosinophil-derived neurotoxin, as well as major basic protein, were examined for capacity to regulate the classical pathway. Eosinophil peroxidase, eosinophil cationic protein and major basic protein inhibited formation of cell-bound classical pathway C3 convertase (EAC1,4b,2a), causing 50% inhibition of complement-mediated lysis at about 0.19, 0.75 and 0.5 micrograms/10(7) cellular intermediates, respectively. Eosinophil-derived neurotoxin had no activity on this pathway of complement. The eosinophil granule proteins were examined for activity on the formation of the membrane attack complex. Major basic protein and eosinophil cationic protein had no activity on terminal lysis. In contrast, eosinophil peroxidase inhibited lysis of EAC1,4b,2a,3b,5b, but had only minimal activity on later events in complement lysis. These polycations were then examined to determine the site(s) at which they regulated the early classical pathway. Eosinophil granule polycationic proteins: (1) reduced the Zmax at all time points but had only minimal effect on the Tmax during the formation of the classical pathway C3 convertase (EAC1,4b,2a); (2) inhibited formation of EAC1,4b,2a proportional to C4 but independent of C2 concentration; (3) inhibited fluid phase formation of C1,4b,2a, as reflected by a decrease in C1-induced consumption of C2 over time; and (4) inhibited C1 activity over time without a direct effect on either C4 or C2. These observations suggest that polycations regulate the early classical pathway by

  6. The complement system: an unexpected role in synaptic pruning during development and disease.

    PubMed

    Stephan, Alexander H; Barres, Ben A; Stevens, Beth

    2012-01-01

    An unexpected role for the classical complement cascade in the elimination of central nervous system (CNS) synapses has recently been discovered. Complement proteins are localized to developing CNS synapses during periods of active synapse elimination and are required for normal brain wiring. The function of complement proteins in the brain appears analogous to their function in the immune system: clearance of cellular material that has been tagged for elimination. Similarly, synapses tagged with complement proteins may be eliminated by microglial cells expressing complement receptors. In addition, developing astrocytes release signals that induce the expression of complement components in the CNS. In the mature brain, early synapse loss is a hallmark of several neurodegenerative diseases. Complement proteins are profoundly upregulated in many CNS diseases prior to signs of neuron loss, suggesting a reactivation of similar developmental mechanisms of complement-mediated synapse elimination potentially driving disease progression.

  7. Bacteria under stress by complement and coagulation.

    PubMed

    Berends, Evelien T M; Kuipers, Annemarie; Ravesloot, Marietta M; Urbanus, Rolf T; Rooijakkers, Suzan H M

    2014-11-01

    The complement and coagulation systems are two related protein cascades in plasma that serve important roles in host defense and hemostasis, respectively. Complement activation on bacteria supports cellular immune responses and leads to direct killing of bacteria via assembly of the Membrane Attack Complex (MAC). Recent studies have indicated that the coagulation system also contributes to mammalian innate defense since coagulation factors can entrap bacteria inside clots and generate small antibacterial peptides. In this review, we will provide detailed insights into the molecular interplay between these protein cascades and bacteria. We take a closer look at how these pathways are activated on bacterial surfaces and discuss the mechanisms by which they directly cause stress to bacterial cells. The poorly understood mechanism for bacterial killing by the MAC will be reevaluated in light of recent structural insights. Finally, we highlight the strategies used by pathogenic bacteria to modulate these protein networks. Overall, these insights will contribute to a better understanding of the host defense roles of complement and coagulation against bacteria.

  8. Complement Activation and Inhibition in Retinal Diseases.

    PubMed

    Kleinman, Mark E; Ambati, Jayakrishna

    2016-01-01

    Within the past several decades, a brigade of dedicated researchers from around the world has provided essential insights into the critical niche of immune-mediated inflammation in the pathogenesis of age-related macular degeneration (AMD). Yet, the question has lingered as to whether disease-initiating events are more or less dependent on isolated immune-related responses, unimpeded inflammation, endogenous pathways of age-related cell senescence and oxidative stress, or any of the other numerous molecular derangements that have been identified in the natural history of AMD. There is now an abundant cache of data signifying immune system activation as an impetus in the pathogenesis of this devastating condition. Furthermore, recent rigorous investigations have revealed multiple inciting factors, including several important complement-activating components, thus creating a new array of disease-modulating targets for the research and development of molecular therapeutic interventions. While the precise in vivo effects of complement activation and inhibition in the progression and treatment of AMD remain to be determined, ongoing clinical trials of the first generation of complement-targeted therapeutics are hoped to yield critical data on the contribution of this pathway to the disease process. PMID:26501209

  9. Tumor necrosis factor alpha-induced angiogenesis depends on in situ platelet-activating factor biosynthesis

    PubMed Central

    1994-01-01

    Tumor necrosis factor (TNF) alpha, a potent inhibitor of endothelial cell growth in vitro, is angiogenic in vivo. Therefore, it was suggested that the angiogenic properties of this agent might be consequent to the production of secondary mediators. Since TNF-alpha stimulates the synthesis of platelet-activating factor (PAF) by monocytes and endothelial cells, we investigated the possible involvement of PAF in the angiogenic effect of TNF-alpha. Angiogenesis was studied in a murine model in which Matrigel was used as a vehicle for the delivery of mediators. In this model the angiogenesis induced by TNF-alpha was shown to be inhibited by WEB 2170, a specific PAF receptor antagonist. Moreover, in mice injected with TNF-alpha, PAF was detected within the Matrigel, 6 and 24 h after TNF-alpha injection. The synthesis of PAF within the Matrigel was concomitant with the early migration of endothelial cells and infiltration of monocytes. No infiltration of lymphocytes or polymorphonuclear leukocytes was observed. Synthetic PAF as well as PAF extracted and purified from mice challenged with TNF-alpha induced a rapid angiogenic response, inhibited by WEB 2170. These results suggest that the angiogenic effect of TNF-alpha is, at least in part, mediated by PAF synthesized from monocytes and/or endothelial cells infiltrating the Matrigel plug. PMID:7516414

  10. D-Xylose as a sugar complement regulates blood glucose levels by suppressing phosphoenolpyruvate carboxylase (PEPCK) in streptozotocin-nicotinamide-induced diabetic rats and by enhancing glucose uptake in vitro

    PubMed Central

    Kim, Eunju; Kim, Yoo-Sun; Kim, Kyung-Mi; Jung, Sangwon; Yoo, Sang-Ho

    2016-01-01

    BACKGROUND/OBJECTIVES Type 2 diabetes (T2D) is more frequently diagnosed and is characterized by hyperglycemia and insulin resistance. D-Xylose, a sucrase inhibitor, may be useful as a functional sugar complement to inhibit increases in blood glucose levels. The objective of this study was to investigate the anti-diabetic effects of D-xylose both in vitro and stretpozotocin (STZ)-nicotinamide (NA)-induced models in vivo. MATERIALS/METHODS Wistar rats were divided into the following groups: (i) normal control; (ii) diabetic control; (iii) diabetic rats supplemented with a diet where 5% of the total sucrose content in the diet was replaced with D-xylose; and (iv) diabetic rats supplemented with a diet where 10% of the total sucrose content in the diet was replaced with D-xylose. These groups were maintained for two weeks. The effects of D-xylose on blood glucose levels were examined using oral glucose tolerance test, insulin secretion assays, histology of liver and pancreas tissues, and analysis of phosphoenolpyruvate carboxylase (PEPCK) expression in liver tissues of a STZ-NA-induced experimental rat model. Levels of glucose uptake and insulin secretion by differentiated C2C12 muscle cells and INS-1 pancreatic β-cells were analyzed. RESULTS In vivo, D-xylose supplementation significantly reduced fasting serum glucose levels (P < 0.05), it slightly reduced the area under the glucose curve, and increased insulin levels compared to the diabetic controls. D-Xylose supplementation enhanced the regeneration of pancreas tissue and improved the arrangement of hepatocytes compared to the diabetic controls. Lower levels of PEPCK were detected in the liver tissues of D-xylose-supplemented rats (P < 0.05). In vitro, both 2-NBDG uptake by C2C12 cells and insulin secretion by INS-1 cells were increased with D-xylose supplementation in a dose-dependent manner compared to treatment with glucose alone. CONCLUSIONS In this study, D-xylose exerted anti-diabetic effects in vivo by

  11. The Role of Hypoxia Inducible Factor-1 Alpha in Bypassing Oncogene-Induced Senescence

    PubMed Central

    Kilic Eren, Mehtap; Tabor, Vedrana

    2014-01-01

    Oncogene induced senescence (OIS) is a sustained anti-proliferative response acutely induced in primary cells via activation of mitogenic oncogenes such as Ras/BRAF. This mechanism acts as an initial barrier preventing normal cells transformation into malignant cell. Besides oncogenic activation and DNA damage response (DDR), senescence is modulated by a plethora of other factors, and one of the most important one is oxygen tension of the tissue. The aim of this study was to determine the impact of hypoxia on RasV12-induced senescence in human diploid fibroblasts (HDFs). We showed here that hypoxia prevents execution of oncogene induced senescence (OIS), through a strong down-regulation of senescence hallmarks, such as SA- β-galactosidase, H3K9me3, HP1γ, p53, p21CIP1 and p16INK4a in association with induction of hypoxia inducible factor-1α (HIF-1α). In addition, hypoxia also decreased marks of H-RasV12-induced DDR in both cell lines through down-regulation of ATM/ATR, Chk1 and Chk2 phosphorylation as well as decreased γ-H2AX positivity. Utilizing shRNA system targeting HIF-1α we show that HIF-1α is directly involved in down regulation of p53 and its target p21CIP1 but not p16INK4a. In line with this finding we found that knock down of HIF-1α leads to a strong induction of apoptotic response, but not restoration of senescence in Ras expressing HDFs in hypoxia. This indicates that HIF-1α is an important player in early steps of tumorigenesis, leading to suppression of senescence through its negative regulation of p53 and p21CIP1. In our work we describe a mechanism through which hypoxia and specifically HIF-1α preclude cells from maintaining senescence-driven anti proliferative response. These findings indicate the possible mechanism through which hypoxic environment helps premalignant cells to evade impingement of cellular failsafe pathways. PMID:24984035

  12. The diagnostic significance of signal peptide-complement C1r/C1s, Uegf, and Bmp1-epidermal growth factor domain-containing protein-1 levels in pulmonary embolism

    PubMed Central

    Dirican, Nigar; Duman, Ali; Sağlam, Gülcan; Arslan, Akif; Ozturk, Onder; Atalay, Sule; Bircan, Ahmet; Akkaya, Ahmet; Cakir, Munire

    2016-01-01

    BACKGROUND: Pulmonary embolism (PE) is a common and potentially life-threatening disorder. Patients with PE often have nonspecific symptoms, and the diagnosis is often delayed. AIM: The aim of our study was to investigate the role of signal peptide-complement C1r/C1s, Uegf, and Bmp1-epidermal growth factor domain-containing protein 1 (SCUBE1) used in the diagnosis of PE. METHODS: The study was designed prospectively. A total of 57 patients who were admitted to emergency service with clinically suspected PE were included in the study. The patients diagnosed with PE were defined as PE group (n = 32), and the patients with undetectable embolism on computerized tomographic pulmonary angiography were defined as non-PE group (n = 25). Twenty-five age- and sex-matched healthy cases were chosen for the study. Routine biochemical analysis, complete blood count, D-dimer, SCUBE1, and arterial blood gas analysis were performed early after admission. RESULTS: Mean SCUBE1 levels were higher in the PE group (0.90 ng/mL) than in the non-PE (0.38 ng/mL) and control groups (0.47 ng/mL) (P < 0.01). A cutoff point of 0.49 ng/mL for SCUBE1 indicated 100% sensitivity and 64% specificity in patients with PE. Mean D-dimer levels were not different between PE and non-PE groups (P = 0.591). A multivariable logistic regression analysis (with dichotomous PE groups as the response variable; age, gender, chest pain, syncope, diabetes mellitus, chronic obstructive pulmonary disease, hypertension, D-dimer, neutrophil-lymphocytes ratio, and SCUBE1 variables as predictors) showed that the significant and independent predictors of PE diagnosis were SCUBE1 and chest pain. CONCLUSION: This study suggests that serum SCUBE1 measurement might be used as a diagnostic biomarker in PE. PMID:27803754

  13. Haematopoietic growth factor in antithyroid-drug-induced agranulocytosis.

    PubMed

    Andrès, E; Kurtz, J E; Perrin, A E; Dufour, P; Schlienger, J L; Maloisel, F

    2001-08-01

    Drug-induced agranulocytosis (DIA) is often caused by antithyroid drugs. We retrospectively studied the use of granulocyte colony-stimulating factor (G-CSF) therapy in antithyroid-DIA. Data for 20 patients (10 treated with G-CSF) with antithyroid-DIA (neutrophil count <0.5x10(9)/l) were extracted from a cohort study of DIA patients (n=110). G-CSF (300 microg/day subcutaneously) was used where the neutrophil count was <0.1x10(9)/l, or the patient was aged >70 years, or there were severe features of infection or underlying disease. Mean patient age was 62 years (range 34-87); sex ratio (M/F) was 0.05. Carbimazole (n=19) and benzylthiouracile (n=1) were the causative drugs, at mean doses of 30 mg/day (range 20-60) and 100 mg/day (range 50-150), respectively, for a mean of 37 days (range 31-90). Antithyroid drugs were prescribed for Graves' disease (n=8), thyrotoxicosis related to amiodarone intake (n=6) and multinodular goitre (n=6). Clinical features included isolated fever (n=7), pneumonia (n=5), septicaemia or septic shock (n=5) and acute tonsillitis (n=3). Mean neutrophil count was 0.07+/-0.1x10(9)/l. No patient died. Mean durations of haematological recovery, antibiotic therapy and hospitalization were significantly reduced with G-CSF: 6.8+/-4 days vs. 11.6+/-5; 7.5+/-3.8 days vs. 12+/-4.5; and 7.3+/-4.8 days vs. 13+/-6.1, respectively (all p<0.05). G-CSF induced flu-like symptoms in 30% of patients, but reduced overall costs.

  14. Particulate matter phagocytosis induces tissue factor in differentiating macrophages.

    PubMed

    Milano, M; Dongiovanni, P; Artoni, A; Gatti, S; Rosso, L; Colombo, F; Bollati, V; Maggioni, M; Mannucci, P M; Bertazzi, P A; Fargion, S; Valenti, L

    2016-01-01

    Airborne exposure to particulate matter with diameter < 10 mcM (PM10) has been linked to an increased risk of thromboembolic events, but the mechanisms are not completely understood. The aim of this study was to evaluate the effect of PM10 phagocytosis on the release of procoagulant molecules in human differentiating macrophages, and that of PM10 inhalation in an experimental model in rats. Human monocytes were separated from the peripheral blood by the lymphoprep method, differentiated in vitro and treated with standard PM10 or vehicle. Sprague-Dawley rats were instilled intratracheally with PM10 or vehicle alone. The outcome was expression of proinflammatory genes and of tissue factor (TF). In human differentiating macrophages, PM10 exposure upregulated inflammatory genes, but most consistently induced TF mRNA and protein levels, but not TF protein inhibitor, resulting in increased TF membrane expression and a procoagulant phenotype. Differentiation towards the anti-inflammatory M2 phenotype inhibited PM10 -mediated TF expression. TF induction required phagocytosis of PM10 , whereas phagocytosis of inert particles was less effective. PM10 phagocytosis was associated with a gene expression profile consistent with intracellular retention of iron, inducing oxidative stress. Both PM10 and iron activated the stress kinases ERK1/2 pathway, involved in the induction of TF expression. In rats, alveolar exposure to PM10 was associated with pulmonary recruitment of inflammatory cells and resulted in local, but not systemic, induction of TF expression, which was sufficient to increase circulating TF levels. In conclusion, TF induction by differentiating lung macrophages, activated following phagocytosis, contributes to the increased risk of thromboembolic complications associated with PM10 exposure.

  15. Complement Component C3 Binds to Activated Normal Platelets without Preceding Proteolytic Activation and Promotes Binding to Complement Receptor 1

    PubMed Central

    Hamad, Osama A.; Nilsson, Per H.; Wouters, Diana; Lambris, John D.; Ekdahl, Kristina N.; Nilsson, Bo

    2010-01-01

    It has been reported that complement is activated on the surface of activated platelets, despite the presence of multiple regulators of complement activation. To reinvestigate the mechanisms by which activated platelets bind to complement components, the presence of complement proteins on the surfaces of nonactivated and thrombin receptor-activating peptide-activated platelets was analyzed by flow cytometry and Western blot analyses. C1q, C4, C3, and C9 were found to bind to thrombin receptor-activating peptide-activated platelets in lepirudin-anticoagulated platelet-rich plasma (PRP) and whole blood. However, inhibiting complement activation at the C1q or C3 level did not block the binding of C3 to activated platelets. Diluting PRP and chelating divalent cations also had no effect, further indicating that the deposition of complement components was independent of complement activation. Furthermore, washed, activated platelets bound added C1q and C3 to the same extent as platelets in PRP. The use of mAbs against different forms of C3 demonstrated that the bound C3 consisted of C3(H2O). Furthermore, exogenously added soluble complement receptor 1 was shown to bind to this form of platelet-bound C3. These observations indicate that there is no complement activation on the surface of platelets under physiological conditions. This situation is in direct contrast to a number of pathological conditions in which regulators of complement activation are lacking and thrombocytopenia and thrombotic disease are the ultimate result. However, the generation of C3(H2O) represents nonproteolytic activation of C3 and after factor I cleavage may act as a ligand for receptor binding. PMID:20139276

  16. Improvisation: A Complement to Curriculum

    ERIC Educational Resources Information Center

    Ronald, Green A.

    2006-01-01

    With the growth of standardized assessment benchmarks in both the public and private paradigms, testing performance matters to institutions more than ever. In an attempt to take as many hindering variables out of this process, such as test anxiety, socioeconomic influences, and latency in cognition, Improvisation: A Complement to Curriculum seeks…

  17. Pentoxifylline inhibits hypoxia-induced upregulation of tumor cell tissue factor and vascular endothelial growth factor.

    PubMed

    Amirkhosravi, A; Meyer, T; Warnes, G; Amaya, M; Malik, Z; Biggerstaff, J P; Siddiqui, F A; Sherman, P; Francis, J L

    1998-10-01

    Tissue factor (TF), the membrane glycoprotein that initiates blood coagulation, is constitutively expressed by many tumor cells and is implicated in peri-tumor fibrin deposition and hypercoagulability in cancer. Upregulation of tumor TF correlates with enhanced metastatic potential. Furthermore, TF has been colocalized with VEGF in breast cancer, specially at sites of early angiogenesis. There are no data on the effect of hypoxia on tumor cell TF expression. Since hypoxia is known to stimulate VEGF production, we studied whether this also induces tumor cell TF expression. Confluent monolayers of A375 melanoma, MCF-7 breast carcinoma and A549 lung carcinoma were cultured in either 95% air, 5% CO2 (normoxic) or 95% N2, 5% CO2 (hypoxic; 25-30 mmHg) for 24 h. Procoagulant activity (PCA) was measured by amidolytic and clotting assays, surface TF antigen by flow cytometry, early apoptosis by annexin V binding and VEGF levels in culture supernatants by ELISA. Hypoxia significantly increased tumor cell PCA in all three cell lines tested and TF antigen on A375 cells was increased four-fold (P <0.05). Pentoxifylline (PTX), a methylxanthine derivative, significantly inhibited the hypoxia-induced increase in PCA as well as VEGF release in all three cell lines tested. In A375 cells, PTX significantly inhibited TF antigen expression by both normoxic and hypoxic cells. Hypoxia induced a slight (5%) but not significant, increase in early apoptosis. Intravenous injection of hypoxic A375 cells into nude rats produced more pronounced thrombocytopenia (n = 5, P <0.01) and more lung metastases (n = 3, P <0.05) compared to normoxic cells. We conclude that hypoxia increases TF expression by malignant cells which enhances tumor cell-platelet binding and hematogenous metastasis. Hypoxia-induced upregulation of TF appears to parallel that of VEGF, although the mechanism remains unclear.

  18. Pentoxifylline inhibits hypoxia-induced upregulation of tumor cell tissue factor and vascular endothelial growth factor.

    PubMed

    Amirkhosravi, A; Meyer, T; Warnes, G; Amaya, M; Malik, Z; Biggerstaff, J P; Siddiqui, F A; Sherman, P; Francis, J L

    1998-10-01

    Tissue factor (TF), the membrane glycoprotein that initiates blood coagulation, is constitutively expressed by many tumor cells and is implicated in peri-tumor fibrin deposition and hypercoagulability in cancer. Upregulation of tumor TF correlates with enhanced metastatic potential. Furthermore, TF has been colocalized with VEGF in breast cancer, specially at sites of early angiogenesis. There are no data on the effect of hypoxia on tumor cell TF expression. Since hypoxia is known to stimulate VEGF production, we studied whether this also induces tumor cell TF expression. Confluent monolayers of A375 melanoma, MCF-7 breast carcinoma and A549 lung carcinoma were cultured in either 95% air, 5% CO2 (normoxic) or 95% N2, 5% CO2 (hypoxic; 25-30 mmHg) for 24 h. Procoagulant activity (PCA) was measured by amidolytic and clotting assays, surface TF antigen by flow cytometry, early apoptosis by annexin V binding and VEGF levels in culture supernatants by ELISA. Hypoxia significantly increased tumor cell PCA in all three cell lines tested and TF antigen on A375 cells was increased four-fold (P <0.05). Pentoxifylline (PTX), a methylxanthine derivative, significantly inhibited the hypoxia-induced increase in PCA as well as VEGF release in all three cell lines tested. In A375 cells, PTX significantly inhibited TF antigen expression by both normoxic and hypoxic cells. Hypoxia induced a slight (5%) but not significant, increase in early apoptosis. Intravenous injection of hypoxic A375 cells into nude rats produced more pronounced thrombocytopenia (n = 5, P <0.01) and more lung metastases (n = 3, P <0.05) compared to normoxic cells. We conclude that hypoxia increases TF expression by malignant cells which enhances tumor cell-platelet binding and hematogenous metastasis. Hypoxia-induced upregulation of TF appears to parallel that of VEGF, although the mechanism remains unclear. PMID:9798977

  19. Hepatocyte growth factor induction of macrophage chemoattractant protein-1 and osteophyte-inducing factors in osteoarthritis.

    PubMed

    Dankbar, Berno; Neugebauer, Katja; Wunrau, Christina; Tibesku, Carsten O; Skwara, Adrian; Pap, Thomas; Fuchs-Winkelmann, Susanne

    2007-05-01

    In osteoarthritis (OA), hepatocyte growth factor (HGF) is supposed to play a role in cartilage repair. Because the development of osteophytes is a major characteristic of OA and thought to be part of an attempted repair process, the purpose of this study was to determine whether HGF may be involved in osteophyte formation. HGF levels in synovial fluids from 41 patients assessed by enzyme immunosorbant assay were correlated with disease severity and osteophyte formation, evaluated by anteroposterior weight-bearing radiographs. Detection of HGF, c-Met, and CD68 in cartilage and synovial tissues was assessed by immunohistochemistry. Effects of HGF on the secretion of TGF-beta1 and BMP-2 by chondrocytes, fibroblast-like synovial cells (FLS), and macrophages as well as HGF-induced secretion of MCP-1 by FLS and chondrocytes were determined by ELISA. HGF was detected in all synovial fluids and concentrations correlated highly with disease severity and osteophyte formation (p < 0.001). Immunohistochemistry revealed weak synovial staining for HGF, whereas increasing numbers of HGF expressing chondrocytes were detected depending on disease severity. In addition, an increased number of macrophages in synovial specimens was observed, which was likewise severity dependent. In a series of subsequent in vitro studies, HGF remarkable induced MCP-1 secretion by FLS in a dose-dependent manner. No effect on TGF-beta1 and BMP-2 secretion by FLS and chondrocytes was evident upon HGF stimulation, whereas secretion of these growth factors by PMA-differentiated THP-1 cells was significantly increased by HGF. The results indicate that HGF may facilitate osteophyte development by promoting MCP-1-mediated entry of monocytes/macrophages into the OA-affected joint and/or by stimulating macrophage-derived growth factors.

  20. Complement plays a minimal role in Sm-p80-mediated protection against Schistosoma mansoni.

    PubMed

    Karmakar, Souvik; Zhang, Weidong; Ahmad, Gul; Alam, Mayeen U; Winn, Richard; Torben, Workineh; Le, Loc; Tillery, Kory A; Siddiqui, Afzal A

    2014-01-01

    Sm-p80, the large subunit of Schistosoma masoni calpain, is a leading antigen candidate for a schistosome vaccine. Prophylactic and antifecundity efficacy of Sm-p80 has been tested using a variety of vaccine approaches. However, the mechanism of Sm-p80-mediated killing is still unknown. In this study, potential role of complement in Sm-p80-mediated protection was studied using both in vitro (cobra venom factor inhibition) and in vivo using mice deficient in C3 (C3 -/-; B6.129S4-C3tm1Crr/J). In the absence of C3, Sm-p80-based vaccine was able to provide significant reduction in adult worm burden following challenge with schistosome cercariae in mice suggesting the effector functions of complement may be limited in this vaccine-induced protection.

  1. Hypoxia inducible factor in hepatocellular carcinoma: A therapeutic target

    PubMed Central

    Lin, Daniel; Wu, Jennifer

    2015-01-01

    Hepatocellular carcinoma (HCC) is one of the most commonly diagnosed and deadly cancers worldwide; its incidence has been rising in the United States due to the increase in hepatitis C associated cirrhosis and the growing epidemic of obesity. There have been no effective therapeutic options in the advanced disease setting beyond sorafenib, a multi-targeted tyrosine kinase inhibitor that showed significant survival benefit. Because of this, there is an urgent need to search for novel pathways in sorafenib experienced patients. This review will focus on the role of hypoxia and hypoxia-inducible factor alpha (HIF-1α) in cancer development, specifically in HCC. We will discuss the biology of HIF-1α, the pathways with which it interacts, and the function of HIF-1α in HCC. Furthermore, we will review studies highlighting the relevance of HIF-1α in the clinical setting, as well as the pre-clinical data supporting its further investigation. Finally, we will conclude with a discussion of the potential role of a HIF-1α mRNA antagonist for the treatment of HCC, and hypothesize the ways in which such an inhibitor may be best utilized in the management of advanced HCC. Hypoxia plays a significant role in the development of HCC. HIF-1α is a key transcription factor involved in the hypoxic response of cancer cells. It activates transcription of genes responsible for angiogenesis, glucose metabolism, proliferation, invasion and metastasis in HCC. Its involvement in multiple, essential tumor pathways makes it an attractive potential therapeutic target in HCC. PMID:26576101

  2. Protective responses to sublytic complement in the retinal pigment epithelium.

    PubMed

    Tan, Li Xuan; Toops, Kimberly A; Lakkaraju, Aparna

    2016-08-01

    The retinal pigment epithelium (RPE) is a key site of injury in inherited and age-related macular degenerations. Abnormal activation of the complement system is a feature of these blinding diseases, yet how the RPE combats complement attack is poorly understood. The complement cascade terminates in the cell-surface assembly of membrane attack complexes (MACs), which promote inflammation by causing aberrant signal transduction. Here, we investigated mechanisms crucial for limiting MAC assembly and preserving cellular integrity in the RPE and asked how these are compromised in models of macular degeneration. Using polarized primary RPE and the pigmented Abca4(-/-) Stargardt disease mouse model, we provide evidence for two protective responses occurring within minutes of complement attack, which are essential for maintaining mitochondrial health in the RPE. First, accelerated recycling of the membrane-bound complement regulator CD59 to the RPE cell surface inhibits MAC formation. Second, fusion of lysosomes with the RPE plasma membrane immediately after complement attack limits sustained elevations in intracellular calcium and prevents mitochondrial injury. Cholesterol accumulation in the RPE, induced by vitamin A dimers or oxidized LDL, inhibits these defense mechanisms by activating acid sphingomyelinase (ASMase), which increases tubulin acetylation and derails organelle traffic. Defective CD59 recycling and lysosome exocytosis after complement attack lead to mitochondrial fragmentation and oxidative stress in the RPE. Drugs that stimulate cholesterol efflux or inhibit ASMase restore both these critical safeguards in the RPE and avert complement-induced mitochondrial injury in vitro and in Abca4(-/-) mice, indicating that they could be effective therapeutic approaches for macular degenerations. PMID:27432952

  3. Complement in disease: a defence system turning offensive.

    PubMed

    Ricklin, Daniel; Reis, Edimara S; Lambris, John D

    2016-07-01

    Although the complement system is primarily perceived as a host defence system, a more versatile, yet potentially more harmful side of this innate immune pathway as an inflammatory mediator also exists. The activities that define the ability of the complement system to control microbial threats and eliminate cellular debris - such as sensing molecular danger patterns, generating immediate effectors, and extensively coordinating with other defence pathways - can quickly turn complement from a defence system to an aggressor that drives immune and inflammatory diseases. These host-offensive actions become more pronounced with age and are exacerbated by a variety of genetic factors and autoimmune responses. Complement can also be activated inappropriately, for example in response to biomaterials or transplants. A wealth of research over the past two decades has led to an increasingly finely tuned understanding of complement activation, identified tipping points between physiological and pathological behaviour, and revealed avenues for therapeutic intervention. This Review summarizes our current view of the key activating, regulatory, and effector mechanisms of the complement system, highlighting important crosstalk connections, and, with an emphasis on kidney disease and transplantation, discusses the involvement of complement in clinical conditions and promising therapeutic approaches.

  4. LPS-inducible factor(s) from activated macrophages mediates cytolysis of Naegleria fowleri amoebae

    SciTech Connect

    Cleary, S.F.; Marciano-Cabral, F.

    1986-03-01

    Soluble cytolytic factors of macrophage origin have previously been described with respect to their tumoricidal activity. The purpose of this study was to investigate the mechanism and possible factor(s) responsible for cytolysis of the amoeba Naegleria fowleri by activated peritoneal macrophages from B6C3F1 mice. Macrophages or conditioned medium (CM) from macrophage cultures were incubated with /sup 3/H-Uridine labeled amoebae. Percent specific release of label served as an index of cytolysis. Bacille Calmette-Guerin (BCG) and Corynebacterium parvum macrophages demonstrated significant cytolysis of amoebae at 24 h with an effector to target ratio of 10:1. Treatment of macrophages with inhibitors of RNA or protein synthesis blocked amoebicidal activity. Interposition of a 1 ..mu..m pore membrane between macrophages and amoebae inhibited killing. Inhibition in the presence of the membrane was overcome by stimulating the macrophages with LPS. CM from SPS-stimulated, but not unstimulated, cultures of activated macrophages was cytotoxic for amoebae. The activity was heat sensitive and was recovered from ammonium sulfate precipitation of the CM. Results indicate that amoebicidal activity is mediated by a protein(s) of macrophage origin induced by target cell contact or stimulation with LPS.

  5. Comparing brain-derived neurotrophic factor and ciliary neurotrophic factor secretion of induced neurotrophic factor secreting cells from human adipose and bone marrow-derived stem cells.

    PubMed

    Razavi, Shahnaz; Razavi, Mohamad Reza; Zarkesh Esfahani, Hamid; Kazemi, Mohammad; Mostafavi, Fatemeh Sadat

    2013-08-01

    Adipose derived stem cells (ADSCs) and bone marrow stem cells (BMSCs) may be equally beneficial in treating neurodegenerative diseases. However, ADSCs have practical advantages. In this study, we aimed to induce neurotrophic factors secreting cells in human ADSCs. Then, we compared the level of brain-derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF) secretion in neurotrophic factors secreting cells from human adipose and bone marrow-derived stem cells. Isolated human ADSCs and BMSCs were induced to neurotrophic factor (NTF)-secreting cells. The levels of expression and secretion of BDNF and CTNF of induced cells were assessed using immunocytochemical, Real-Time polymerase chain reaction, and enzyme linked immunosorbent assay (ELISA). The level of BDNF significantly increased in both the induced mesenchymal stem cells (MSCs) relative to ADSCs and the BMSCs (P < 0.01). Moreover, ELISA analysis showed that the release of BDNF in the induced BMSCs was almost twofold more than the induced ADSCs. Overall, NTF-secreting factor cells derived BMSCs and ADSCs could secret a range of different growth factors. Therefore, the variation in neurotrophic factors of different induced MSC populations suggest the possible beneficial effect of each specific kind of neurotrophic factor secreting cells for the treatment of a particular neurodegenerative disease. PMID:23944834

  6. Polysomnographic correlates of inflammatory complement components in young healthy males.

    PubMed

    Hussain, M Ejaz; Golam Sarwar, Abu Hasnath M; Alam, Mohd Shoeb; Noohu, Majumi M; Zannat, Wassilatul; Pandi-Perumal, Seithikurippu R; Bahammam, Ahmed S; Manzar, Md Dilshad

    2016-01-01

    A growing body of evidence has delineated the predominant role of humoral mediators of inflammation in linking sleep with immunity. Nonetheless, characterization of the relationship between complement components with inflammatory functions and objective sleep measures has not been performed. In this study we investigated the relationships between objective measures of sleep and complement components with inflammatory functions. Thirty-six healthy male university students (age, 23.94±4.23 years; BMI, 23.44±2.67 kg/m(2)) completed the study. An RMS Quest 32 polysomnograph (PSG) was used for sleep recording. Non-fasting blood was collected before subjects went to bed on the second night in the sleep laboratory to estimate complement component 3 (C-3), complement component 4 (C-4), complement factor-H (Factor-H), C1-inhibitor (C1INH), complement factor I (CFI) and other inflammatory mediators, such as IL-6 and sICAM-1. Multiple linear regression analysis was used to assess the association between PSG sleep measures and inflammatory mediators. Higher values of C-3 and lower values of sICAM-1, C1INH, and CFI (adjusted model, R2=0.211, p<0.041) predicted longer sleep duration. Lower C-3 (adjusted model, R2=0.078, p<0.055) predicted higher N1 (%). Higher levels of C1INH and CFI and lower values of C-4 (model adjusted R2=0.269, p<0.008) predicted higher N3 (%). Higher C-3, higher C-4, lower IL-6, lower C1INH and lower CFI (model adjusted R2=0.296, p<0.007) predicted higher REM (%). Poor sleep measures were associated with increased levels of pro-inflammatory complement components and decreased anti-inflammatory complement components. PMID:27656278

  7. A Molecular Insight into Complement Evasion by the Staphylococcal Complement Inhibitor Protein Family1

    PubMed Central

    Ricklin, Daniel; Tzekou, Apostolia; Garcia, Brandon L.; Hammel, Michal; McWhorter, William J.; Sfyroera, Georgia; Wu, You-Qiang; Holers, V. Michael; Herbert, Andrew P.; Barlow, Paul N.; Geisbrecht, Brian V.; Lambris, John D.

    2010-01-01

    Staphylococcus aureus possesses an impressive arsenal of complement evasion proteins that help the bacterium escape attack of the immune system. The staphylococcal complement inhibitor (SCIN) protein exhibits a particularly high potency and was previously shown to block complement by acting at the level of the C3 convertases. However, many details about the exact binding and inhibitory mechanism remained unclear. In this study, we demonstrate that SCIN directly binds with nanomolar affinity to a functionally important area of C3b that lies near the C terminus of its β-chain. Direct competition of SCIN with factor B for C3b slightly decreased the formation of surface-bound convertase. However, the main inhibitory effect can be attributed to an entrapment of the assembled convertase in an inactive state. Whereas native C3 is still able to bind to the blocked convertase, no generation and deposition of C3b could be detected in the presence of SCIN. Furthermore, SCIN strongly competes with the binding of factor H to C3b and influences its regulatory activities: the SCIN-stabilized convertase was essentially insensitive to decay acceleration by factor H and the factor I- and H-mediated conversion of surface-bound C3b to iC3b was significantly reduced. By targeting a key area on C3b, SCIN is able to block several essential functions within the alternative pathway, which explains the high potency of the inhibitor. Our findings provide an important insight into complement evasion strategies by S. aureus and may act as a base for further functional studies. PMID:19625656

  8. Mechanics of Coriolis stimulus and inducing factors of motion sickness.

    PubMed

    Isu, N; Shimizu, T; Sugata, K

    2001-12-01

    To specify inducing factors of motion sickness comprised in Coriolis stimulus, or cross-coupled rotation, the sensation of rotation derived from the semicircular canal system during and after Coriolis stimulus under a variety of stimulus conditions, was estimated by an approach from mechanics with giving minimal hypotheses and simplifications on the semicircular canal system and the sensory nervous system. By solving an equation of motion of the endolymph during Coriolis stimulus, rotating angle of the endolymph was obtained, and the sensation of rotation derived from each semicircular canal was estimated. Then the sensation derived from the whole semicircular canal system was particularly considered in two cases of a single Coriolis stimulus and cyclic Coriolis stimuli. The magnitude and the direction of sensation of rotation were shown to depend on an angular velocity of body rotation and a rotating angle of head movement (amplitude of head oscillation when cyclic Coriolis stimuli) irrespective of initial angle (center angle) of the head relative to the vertical axis. The present mechanical analysis of Coriolis stimulus led a suggestion that the severity of nausea evoked by Coriolis stimulus is proportional to the effective value of the sensation of rotation caused by the Coriolis stimulus.

  9. Bead arrays for antibody and complement profiling reveal joint contribution of antibody isotypes to C3 deposition.

    PubMed

    Ayoglu, Burcu; Szarka, Eszter; Huber, Krisztina; Orosz, Anita; Babos, Fruzsina; Magyar, Anna; Hudecz, Ferenc; Rojkovich, Bernadette; Gáti, Tamás; Nagy, György; Schwenk, Jochen M; Sármay, Gabriella; Prechl, József; Nilsson, Peter; Papp, Krisztián

    2014-01-01

    The development of antigen arrays has provided researchers with great tools to identify reactivities against self or foreign antigens from body fluids. Yet, these approaches mostly do not address antibody isotypes and their effector functions even though these are key points for a more detailed understanding of disease processes. Here, we present a bead array-based assay for a multiplexed determination of antigen-specific antibody levels in parallel with their properties for complement activation. We measured the deposition of C3 fragments from serum samples to reflect the degree of complement activation via all three complement activation pathways. We utilized the assay on a bead array containing native and citrullinated peptide antigens to investigate the levels of IgG, IgM and IgA autoantibodies along with their complement activating properties in serum samples of 41 rheumatoid arthritis patients and 40 controls. Our analysis revealed significantly higher IgG reactivity against the citrullinated fibrinogen β and filaggrin peptides as well as an IgA reactivity that was exclusive for citrullinated fibrinogen β peptide and C3 deposition in rheumatoid arthritis patients. In addition, we characterized the humoral immune response against the viral EBNA-1 antigen to demonstrate the applicability of this assay beyond autoimmune conditions. We observed that particular buffer compositions were demanded for separate measurement of antibody reactivity and complement activation, as detection of antigen-antibody complexes appeared to be masked due to C3 deposition. We also found that rheumatoid factors of IgM isotype altered C3 deposition and introduced false-positive reactivities against EBNA-1 antigen. In conclusion, the presented bead-based assay setup can be utilized to profile antibody reactivities and immune-complex induced complement activation in a high-throughput manner and could facilitate the understanding and diagnosis of several diseases where complement

  10. Bead Arrays for Antibody and Complement Profiling Reveal Joint Contribution of Antibody Isotypes to C3 Deposition

    PubMed Central

    Ayoglu, Burcu; Szarka, Eszter; Huber, Krisztina; Orosz, Anita; Babos, Fruzsina; Magyar, Anna; Hudecz, Ferenc; Rojkovich, Bernadette; Gáti, Tamás; Nagy, György; Schwenk, Jochen M.; Sármay, Gabriella; Prechl, József

    2014-01-01

    The development of antigen arrays has provided researchers with great tools to identify reactivities against self or foreign antigens from body fluids. Yet, these approaches mostly do not address antibody isotypes and their effector functions even though these are key points for a more detailed understanding of disease processes. Here, we present a bead array-based assay for a multiplexed determination of antigen-specific antibody levels in parallel with their properties for complement activation. We measured the deposition of C3 fragments from serum samples to reflect the degree of complement activation via all three complement activation pathways. We utilized the assay on a bead array containing native and citrullinated peptide antigens to investigate the levels of IgG, IgM and IgA autoantibodies along with their complement activating properties in serum samples of 41 rheumatoid arthritis patients and 40 controls. Our analysis revealed significantly higher IgG reactivity against the citrullinated fibrinogen β and filaggrin peptides as well as an IgA reactivity that was exclusive for citrullinated fibrinogen β peptide and C3 deposition in rheumatoid arthritis patients. In addition, we characterized the humoral immune response against the viral EBNA-1 antigen to demonstrate the applicability of this assay beyond autoimmune conditions. We observed that particular buffer compositions were demanded for separate measurement of antibody reactivity and complement activation, as detection of antigen-antibody complexes appeared to be masked due to C3 deposition. We also found that rheumatoid factors of IgM isotype altered C3 deposition and introduced false-positive reactivities against EBNA-1 antigen. In conclusion, the presented bead-based assay setup can be utilized to profile antibody reactivities and immune-complex induced complement activation in a high-throughput manner and could facilitate the understanding and diagnosis of several diseases where complement

  11. Exploitation of complement regulatory proteins by Borrelia and Francisella.

    PubMed

    Madar, Marian; Bencurova, Elena; Mlynarcik, Patrik; Almeida, André M; Soares, Renata; Bhide, Katarina; Pulzova, Lucia; Kovac, Andrej; Coelho, Ana V; Bhide, Mangesh

    2015-06-01

    Pathogens have developed sophisticated mechanisms of complement evasion such as binding to the host complement regulatory proteins (CRPs) on their surface or expression of CRP mimicking molecules. The ability of pathogens to evade the complement system has been correlated with pathogenesis and host selectivity. Hitherto, little work has been undertaken to determine whether Borrelia and Francisella exploit various CRPs to block complement attack. Seventeen Borrelia (twelve species) and six Francisella (three subspecies) strains were used to assess their ability to bind human, sheep and cattle CRPs or mimic membrane associated complement regulators. A series of experiments including affinity ligand binding experiments, pull-down assays and mass spectrometry based protein identification, revealed an array of CRP binding proteins of Borrelia and Francisella. Unlike Francisella, Borrelia strains were able to bind multiple human CRPs. Three strains of Borrelia (SKT-4, SKT-2 and HO14) showed the presence of a human CD46-homologous motif, indicating their ability to possess putative human CD46 mimicking molecules. Similarly, five strains of Borrelia and two strains of Francisella may have surface proteins with human CD59-homologous motifs. Among ovine and bovine CRPs, the only CRP bound by Francisella (LVS, Tul4 strain) was vitronectin, while ovine C4BP, ovine factor H and bovine factor H were bound to Borrelia strains SKT-2, DN127 and Co53. This study presents an array of proteins of Borrelia and Francisella that bind CRPs or may mimic membrane-CRPs, thus enabling multiphasic complement evasion strategies of these pathogens.

  12. Exogenous tumour necrosis factor α induces suppression of autoimmune arthritis

    PubMed Central

    2008-01-01

    Introduction Our previous studies showed that arthritic Lewis (LEW) rats produced the highest levels of tumour necrosis factor (TNF)α in the recovery phase of adjuvant arthritis (AA), suggesting a correlation between high TNFα levels and reduced severity of arthritis. To further explore this correlation, we compared the TNFα secretion profile of the AA-resistant Wistar Kyoto (WKY) rats with that of LEW rats, determined the effect of exogenous TNFα on the course of AA in LEW rats, and examined various mechanisms involved in TNFα-induced disease modulation. Methods A cohort each of LEW and WKY rats was immunised subcutaneously with heat-killed Mycobacterium tuberculosis H37Ra (Mtb). At different time points thereafter, subgroups of rats were killed and their draining lymph node cells were tested for cytokine production. Another group of LEW rats was injected with TNFα intraperitoneally daily for a total of 10 injections, 3 before and 6 after Mtb challenge, and then observed for signs of AA. In parallel, TNFα-treated rats were examined for changes in other cytokines, in CD4+CD25+ T cell frequency, and in indoleamine 2,3-dioxygenase (IDO) mRNA expression levels. Results LEW rats displayed a TNFα secretion profile that was opposite to that of the WKY rats. Furthermore, TNFα treatment significantly downmodulated the severity of AA in LEW rats, and decreased the interferon (IFN)-γ secretion in response to the pathogenic determinant of the disease-related antigen. No significant alterations were observed in other parameters tested. Conclusion The role of endogenous TNFα in the induction and propagation of arthritis is well established. However, exogenous TNFα can downmodulate the course of AA, displaying an immunoregulatory functional attribute of this cytokine. PMID:18380898

  13. Activated human platelets induce factor XIIa-mediated contact activation.

    PubMed

    Bäck, Jennie; Sanchez, Javier; Elgue, Graciela; Ekdahl, Kristina Nilsson; Nilsson, Bo

    2010-01-01

    Earlier studies have shown that isolated platelets in buffer systems can promote activation of FXII or amplify contact activation, in the presence of a negatively charge substance or material. Still proof is lacking that FXII is activated by platelets in a more physiological environment. In this study we investigate if activated platelets can induce FXII-mediated contact activation and whether this activation affects clot formation in human blood. Human platelets were activated with a thrombin receptor-activating peptide, SFLLRN-amide, in platelet-rich plasma or in whole blood. FXIIa and FXIa in complex with preferentially antithrombin (AT) and to some extent C1-inhibitor (C1INH) were generated in response to TRAP stimulation. This contact activation was independent of surface-mediated contact activation, tissue factor pathway or thrombin. In clotting whole blood FXIIa-AT and FXIa-AT complexes were specifically formed, demonstrating that AT is a potent inhibitor of FXIIa and FXIa generated by platelet activation. Contact activation proteins were analyzed by flow cytometry and FXII, FXI, high-molecular weight kininogen, and prekallikrein were detected on activated platelets. Using chromogenic assays, enzymatic activity of platelet-associated FXIIa, FXIa, and kallikrein were demonstrated. Inhibition of FXIIa in non-anticoagulated blood also prolonged the clotting time. We conclude that platelet activation triggers FXII-mediated contact activation on the surface and in the vicinity of activated platelets. This leads specifically to generation of FXIIa-AT and FXIa-AT complexes, and contributes to clot formation. Activated platelets may thereby constitute an intravascular locus for contact activation, which may explain the recently reported importance of FXII in thrombus formation. PMID:19878657

  14. Hypoxia and Hypoxia-Inducible Factors in Leukemias

    PubMed Central

    Deynoux, Margaux; Sunter, Nicola; Hérault, Olivier; Mazurier, Frédéric

    2016-01-01

    Despite huge improvements in the treatment of leukemia, the percentage of patients suffering relapse still remains significant. Relapse most often results from a small number of leukemic stem cells (LSCs) within the bone marrow, which are able to self-renew, and therefore reestablish the full tumor. The marrow microenvironment contributes considerably in supporting the protection and development of leukemic cells. LSCs share specific niches with normal hematopoietic stem cells with the niche itself being composed of a variety of cell types, including mesenchymal stem/stromal cells, bone cells, immune cells, neuronal cells, and vascular cells. A hallmark of the hematopoietic niche is low oxygen partial pressure, indeed this hypoxia is necessary for the long-term maintenance of hematopoietic stem/progenitor cells. Hypoxia is a strong signal, principally maintained by members of the hypoxia-inducible factor (HIF) family. In solid tumors, it has been well established that hypoxia triggers intrinsic metabolic changes and microenvironmental modifications, such as the stimulation of angiogenesis, through activation of HIFs. As leukemia is not considered a “solid” tumor, the role of oxygen in the disease was presumed to be inconsequential and remained long overlooked. This view has now been revised since hypoxia has been shown to influence leukemic cell proliferation, differentiation, and resistance to chemotherapy. However, the role of HIF proteins remains controversial with HIFs being considered as either oncogenes or tumor suppressor genes, depending on the study and model. The purpose of this review is to highlight our knowledge of hypoxia and HIFs in leukemic development and therapeutic resistance and to discuss the recent hypoxia-based strategies proposed to eradicate leukemias. PMID:26955619

  15. Chimeric mouse human IgG3 antibodies with an IgG4-like hinge region induce complement-mediated lysis more efficiently than IgG3 with normal hinge.

    PubMed

    Norderhaug, L; Brekke, O H; Bremnes, B; Sandin, R; Aase, A; Michaelsen, T E; Sandlie, I

    1991-10-01

    We have altered the amino acid sequence of the hinge and the first constant domain (CH1) of mouse/human chimeric IgG3 antibodies by site-directed mutagenesis, so as to make the sequences identical to those of IgG4. All the mutant antibodies with altered hinge region were more active in complement activation and complement-mediated lysis than native IgG3. The mutations in CH1, however, did not alter the activity. This demonstrates the importance of the hinge region in modulating this effector function. The results show that the primary structure of neither CH1 nor the hinge of IgG4 is responsible for the lack of complement activation shown by this subclass.

  16. Role of hypoxia-inducible factor 1{alpha} in modulating cobalt-induced lung inflammation.

    PubMed

    Saini, Yogesh; Kim, Kyung Y; Lewandowski, Ryan; Bramble, Lori A; Harkema, Jack R; Lapres, John J

    2010-02-01

    Hypoxia plays an important role in development, cellular homeostasis, and pathological conditions, such as cancer and stroke. There is also growing evidence that hypoxia is an important modulator of the inflammatory process. Hypoxia-inducible factors (HIFs) are a family of proteins that regulate the cellular response to oxygen deficit, and loss of HIFs impairs inflammatory cell function. There is little known, however, about the role of epithelial-derived HIF signaling in modulating inflammation. Cobalt is capable of eliciting an allergic response and promoting HIF signaling. To characterize the inflammatory function of epithelial-derived HIF in response to inhaled cobalt, a conditional lung-specific HIF1alpha, the most ubiquitously expressed HIF, deletion mouse, was created. Control mice showed classic signs of metal-induced injury following cobalt exposure, including fibrosis and neutrophil infiltration. In contrast, HIF1alpha-deficient mice displayed a Th2 response that resembled asthma, including increased eosinophilic infiltration, mucus cell metaplasia, and chitinase-like protein expression. The results suggest that epithelial-derived HIF signaling has a critical role in establishing a tissue's inflammatory response, and compromised HIF1alpha signaling biases the tissue towards a Th2-mediated reaction. PMID:19915160

  17. The Complement System and Adverse Pregnancy Outcomes

    PubMed Central

    Regal, Jean F.; Gilbert, Jeffrey S.; Burwick, Richard M.

    2015-01-01

    Adverse pregnancy outcomes significantly contribute to morbidity and mortality for mother and child, with lifelong health consequences for both. The innate and adaptive immune system must be regulated to insure survival of the feta allograft, and the complement system is no exception. An intact complement system optimizes placental development and function and is essential to maintain host defense and fetal survival. Complement regulation is apparent at the placental interface from early pregnancy with some degree of complement activation occurring normally throughout gestation. However, a number of pregnancy complications including early pregnancy loss, fetal growth restriction, hypertensive disorders of pregnancy and preterm birth are associated with excessive or misdirected complement activation, and are more frequent in women with inherited or acquired complement system disorders or complement gene mutations. Clinical studies employing complement biomarkers in plasma and urine implicate dysregulated complement activation in components of each of the adverse pregnancy outcomes. In addition, mechanistic studies in rat and mouse models of adverse pregnancy outcomes address the complement pathways or activation products of importance and allow critical analysis of the pathophysiology. Targeted complement therapeutics are already in use to control adverse pregnancy outcomes in select situations. A clearer understanding of the role of the complement system in both normal pregnancy and complicated or failed pregnancy will allow a rational approach to future therapeutic strategies for manipulating complement with the goal of mitigating adverse pregnancy outcomes, preserving host defense, and improving long term outcomes for both mother and child. PMID:25802092

  18. The complement system and adverse pregnancy outcomes.

    PubMed

    Regal, Jean F; Gilbert, Jeffrey S; Burwick, Richard M

    2015-09-01

    Adverse pregnancy outcomes significantly contribute to morbidity and mortality for mother and child, with lifelong health consequences for both. The innate and adaptive immune system must be regulated to insure survival of the fetal allograft, and the complement system is no exception. An intact complement system optimizes placental development and function and is essential to maintain host defense and fetal survival. Complement regulation is apparent at the placental interface from early pregnancy with some degree of complement activation occurring normally throughout gestation. However, a number of pregnancy complications including early pregnancy loss, fetal growth restriction, hypertensive disorders of pregnancy and preterm birth are associated with excessive or misdirected complement activation, and are more frequent in women with inherited or acquired complement system disorders or complement gene mutations. Clinical studies employing complement biomarkers in plasma and urine implicate dysregulated complement activation in components of each of the adverse pregnancy outcomes. In addition, mechanistic studies in rat and mouse models of adverse pregnancy outcomes address the complement pathways or activation products of importance and allow critical analysis of the pathophysiology. Targeted complement therapeutics are already in use to control adverse pregnancy outcomes in select situations. A clearer understanding of the role of the complement system in both normal pregnancy and complicated or failed pregnancy will allow a rational approach to future therapeutic strategies for manipulating complement with the goal of mitigating adverse pregnancy outcomes, preserving host defense, and improving long term outcomes for both mother and child.

  19. The Emerging Role of Complement Lectin Pathway in Trypanosomatids: Molecular Bases in Activation, Genetic Deficiencies, Susceptibility to Infection, and Complement System-Based Therapeutics

    PubMed Central

    Evans-Osses, Ingrid; de Messias-Reason, Iara; Ramirez, Marcel I.

    2013-01-01

    The innate immune system is evolutionary and ancient and is the pivotal line of the host defense system to protect against invading pathogens and abnormal self-derived components. Cellular and molecular components are involved in recognition and effector mechanisms for a successful innate immune response. The complement lectin pathway (CLP) was discovered in 1990. These new components at the complement world are very efficient. Mannan-binding lectin (MBL) and ficolin not only recognize many molecular patterns of pathogens rapidly to activate complement but also display several strategies to evade innate immunity. Many studies have shown a relation between the deficit of complement factors and susceptibility to infection. The recently discovered CLP was shown to be important in host defense against protozoan microbes. Although the recognition of pathogen-associated molecular patterns by MBL and Ficolins reveal efficient complement activations, an increase in deficiency of complement factors and diversity of parasite strategies of immune evasion demonstrate the unsuccessful effort to control the infection. In the present paper, we will discuss basic aspects of complement activation, the structure of the lectin pathway components, genetic deficiency of complement factors, and new therapeutic opportunities to target the complement system to control infection. PMID:23533355

  20. CHEMOSENSITIZATION BY A NON-APOPTOGENIC HEAT SHOCK PROTEIN 70-BINDING APOPTOSIS INDUCING FACTOR MUTANT

    EPA Science Inventory

    Chemosensitization by a non-apoptogenic heat shock protein 70-binding apoptosis inducing factor mutant

    Abstract
    HSP70 inhibits apoptosis by neutralizing the caspase activator Apaf-1 and by interacting with apoptosis inducing factor (AIF), a mitochondrial flavoprotein wh...

  1. Fluorescence bimolecular complementation enables facile detection of ribosome assembly defects in Escherichia coli.

    PubMed

    Sharma, Himanshu; Anand, Baskaran

    2016-09-01

    Assembly factors promote the otherwise non-spontaneous maturation of ribosome under physiological conditions inside the cell. Systematic identification and characterization of candidate assembly factors are fraught with bottlenecks due to lack of facile assay system to capture assembly defects. Here, we show that bimolecular fluorescence complementation (BiFC) allows detection of assembly defects that are induced by the loss of assembly factors. The fusion of N and C-terminal fragments of Venus fluorescent protein to the ribosomal proteins uS13 and uL5, respectively, in Escherichia coli facilitated the incorporation of the tagged uS13 and uL5 onto the respective ribosomal subunits. When the ribosomal subunits associated to form the 70S particle, the complementary fragments of Venus were brought into proximity and rendered the Venus fluorescent. Assembly defects that inhibit the subunits association were provoked by either the loss of the known assembly factors such as RsgA and SrmB or the presence of small molecule inhibitors of ribosome maturation such as Lamotrigine and several ribosome-targeting antibiotics and these showed abrogation of the fluorescence complementation. This suggests that BiFC can be employed as a surrogate measure to detect ribosome assembly defects proficiently by circumventing the otherwise cumbersome procedures. BiFC thus offers a facile platform not only for systematic screening to validate potential assembly factors but also to discover novel small molecule inhibitors of ribosome assembly toward mapping the complex assembly landscape of ribosome.

  2. Featured Article: Hypoxia-inducible factor-1α dependent nuclear entry of factor inhibiting HIF-1

    PubMed Central

    Liang, Ke; Ding, Xue-qin; Lin, Chen

    2015-01-01

    The regulation of hypoxia-inducible factor-1 (HIF-1) transcriptional activity in the nucleus is related to factor inhibiting HIF-1 (FIH-1). FIH-1 hydrolyzes asparagine at the C-terminal of HIF-1α, preventing the interaction between HIF-1α and its associated cofactors, and leading to suppressed activation of HIF-1. FIH-1 is a cytosolic protein and its entry to the nucleus has to be coordinated with HIF-1α. The present study was undertaken to examine the correlation between HIF-1α and FIH-1 in their nuclear entry. Human umbilical vein endothelial cells were treated with dimethyloxalylglycine at a final concentration of 100 µM for 4 h, resulting in an accumulation of HIF-1α and an increase of FIH-1 in the nucleus as determined by Western blot analysis. Pretreatment of the cells with copper (Cu) chelator tetraethylenepentamine at 50 µM in cultures for 24 h reduced both HIF-1α protein levels and the HIF-1α entry to the nucleus, along with decreased FIH-1 protein levels in the nucleus but no changes in the total FIH-1 protein levels in the cells. These effects were prevented by simultaneous addition of 50 µM CuSO4 with tetraethylenepentamine. Gene-silencing of HIF-1α significantly inhibited FIH-1 entry to the nucleus, but did not affect the total protein levels of FIH-1 in the cells. This work demonstrates that the nuclear entry of FIH-1 depends on HIF-1α. Cu deficiency caused a decrease of HIF-1α, leading to suppression of FIH-1 entry to the nucleus. PMID:25687434

  3. Complement deposition in glomerular diseases.

    PubMed

    di Belgiojoso, G B; Tarantino, A; Durante, A; Guerra, L

    1975-01-01

    Biopsies from 400 patients affected by glomerular diseases, both "primary" and secondary to systemic diseases, have been studied by immunofluorescence. Staining was performed for immunoglobulins fibrogen and C1q, C4, C3 and C3A. C1q, C4 and C3 were positive in a high percentage of cases in focal glomerulosclerosis, membranoproliferative glomerulonephritis, lupus nephritis and essential cryoglobulinaemia glomerulonephritis. C1q and C4 were very rarely present in focal proliferative glomerulonephritis and rheumatoid purpura glomerulonephritis. C3A was found frequently only in acute glomerulonephritis. Results are discussed with reference to their diagnostic value and to information about mechanisms of complement activation.

  4. Methoxychlor induces atresia by altering Bcl2 factors and inducing caspase activity in mouse ovarian antral follicles in vitro.

    PubMed

    Basavarajappa, Mallikarjuna S; Karman, Bethany N; Wang, Wei; Gupta, Rupesh K; Flaws, Jodi A

    2012-12-01

    Methoxychlor (MXC) is an organochlorine pesticide widely used in many countries against various species of insects that attack crops and domestic animals. MXC reduces fertility by increasing atresia (death) of antral follicles in vivo. MXC also induces atresia of antral follicles after 96 h in vitro. The current work tested the hypothesis that MXC induces morphological atresia at early time points (24 and 48 h) by altering pro-apoptotic (Bax, Bok, Casp3, and caspase activity) and anti-apoptotic (Bcl2 and Bcl-xL) factors in the follicles. The results indicate that at 24 h, MXC increased Bcl-xL and Bax mRNA levels and increased the ratio of Bax/Bcl2. At 48-96 h, MXC induced morphological atresia. At 24-96 h, MXC increased caspase activities. These data suggest that MXC may induce atresia by altering Bcl2 factors and inducing caspase activities in antral follicles.

  5. Complement in the Homeostatic and Ischemic Brain

    PubMed Central

    Alawieh, Ali; Elvington, Andrew; Tomlinson, Stephen

    2015-01-01

    The complement system is a component of the immune system involved in both recognition and response to pathogens, and it is implicated in an increasing number of homeostatic and disease processes. It is well documented that reperfusion of ischemic tissue results in complement activation and an inflammatory response that causes post-reperfusion injury. This occurs following cerebral ischemia and reperfusion and triggers secondary damage that extends beyond the initial infarcted area, an outcome that has rationalized the use of complement inhibitors as candidate therapeutics after stroke. In the central nervous system, however, recent studies have revealed that complement also has essential roles in synaptic pruning, neurogenesis, and neuronal migration. In the context of recovery after stroke, these apparent divergent functions of complement may account for findings that the protective effect of complement inhibition in the acute phase after stroke is not always maintained in the subacute and chronic phases. The development of effective stroke therapies based on modulation of the complement system will require a detailed understanding of complement-dependent processes in both early neurodegenerative events and delayed neuro-reparatory processes. Here, we review the role of complement in normal brain physiology, the events initiating complement activation after cerebral ischemia-reperfusion injury, and the contribution of complement to both injury and recovery. We also discuss how the design of future experiments may better characterize the dual role of complement in recovery after ischemic stroke. PMID:26322048

  6. Nitric oxide mediates angiogenesis induced in vivo by platelet-activating factor and tumor necrosis factor-alpha.

    PubMed Central

    Montrucchio, G.; Lupia, E.; de Martino, A.; Battaglia, E.; Arese, M.; Tizzani, A.; Bussolino, F.; Camussi, G.

    1997-01-01

    We evaluated the role of an endogenous production of nitric oxide (NO) in the in vitro migration of endothelial cells and in the in vivo angiogenic response elicited by platelet-activating factor (PAF), tumor necrosis factor-alpha (TNF), and basic fibroblast growth factor (bFGF). The NO synthase inhibitor, N omega-nitro-L-arginine-methyl ester (L-NAME), but not its enantiomer D-NAME, prevented chemotaxis of endothelial cells induced in vitro by PAF and by TNF. The motogenic activity of TNF was also inhibited by WEB 2170, a specific PAF-receptor antagonist. In contrast, chemotaxis induced by bFGF was not prevented by L-NAME or by WEB 2170. Angiogenesis was studied in vivo in a murine model in which Matrigel was used as a vehicle for the delivery of mediators. In this model, the angiogenesis induced by PAF and TNF was inhibited by WEB 2170 and L-NAME but not by D-NAME. In contrast, angiogenesis induced by bFGF was not affected by L-NAME or by WEB 2170. TNF, but not bFGF, induced PAF synthesis within Matrigel. These results suggest that NO mediates the angiogenesis induced by PAF as well as that induced by TNF, which is dependent on the production of PAF. In contrast, the angiogenic effect of bFGF appears to be both PAF and NO independent. Images Figure 3 Figure 4 PMID:9250168

  7. Characterization and expression analysis of a complement component gene in sea cucumber ( Apostichopus japonicus)

    NASA Astrophysics Data System (ADS)

    Chen, Zhong; Zhou, Zunchun; Yang, Aifu; Dong, Ying; Guan, Xiaoyan; Jiang, Bei; Wang, Bai

    2015-12-01

    The complement system plays a crucial role in the innate immune system of animals. It can be activated by distinct yet overlapping classical, alternative and lectin pathways. In the alternative pathway, complement factor B (Bf) serves as the catalytic subunit of complement component 3 (C3) convertase, which plays the central role among three activation pathways. In this study, the Bf gene in sea cucumber ( Apostichopus japonicus), termed AjBf, was obtained by rapid amplification of cDNA ends (RACE). The full-length cDNA of AjBf was 3231 bp in length barring the poly (A) tail. It contained an open reading frame (ORF) of 2742 bp encoding 913 amino acids, a 105 bp 5'-UTR (5'-terminal untranslated region) and a 384 bp 3'-UTR. AjBf was a mosaic protein with six CCP (complement control protein) domains, a VWA (von Willebrand factor A) domain, and a serine protease domain. The deduced molecular weight of AjBf protein was 101 kDa. Quantitative real time PCR (qRT-PCR) analysis indicated that the expression level of AjBf in A. japonicus was obviously higher at larval stage than that at embryonic stage. Expression detection in different tissues showed that AjBf expressed higher in coelomocytes than in other four tissues. In addation, AjBf expression in different tissues was induced significantly after LPS or PolyI:C challenge. These results indicated that AjBf plays an important role in immune responses to pathogen infection.

  8. Elongation factor-1 alpha is a selective regulator of growth factor withdrawal and ER stress-induced apoptosis.

    PubMed

    Talapatra, S; Wagner, J D O; Thompson, C B

    2002-08-01

    To identify genes that contribute to apoptotic resistance, IL-3 dependent hematopoietic cells were transfected with a cDNA expression library and subjected to growth factor withdrawal. Transfected cells were enriched for survivors over two successive rounds of IL-3 withdrawal and reconstitution, resulting in the identification of a full-length elongation factor 1 alpha (EF-1alpha) cDNA. Ectopic EF-1alpha expression conferred protection from growth factor withdrawal and agents that induce endoplasmic reticulum stress, but not from nuclear damage or death receptor signaling. Overexpression of EF-1alpha did not lead to growth factor independent cell proliferation or global alterations in protein levels or rates of synthesis. These findings suggest that overexpression of EF-1alpha results in selective resistance to apoptosis induced by growth factor withdrawal and ER stress. PMID:12107828

  9. Hepatocyte growth factor induces tubulogenesis of primary renal proximal tubular epithelial cells.

    PubMed

    Bowes, R C; Lightfoot, R T; Van De Water, B; Stevens, J L

    1999-07-01

    Hepatocyte growth factor (HGF)-induced tubulogenesis has been demonstrated with renal epithelial cell lines grown in collagen gels but not with primary cultured renal proximal tubular epithelial cells (RPTEs). We show that HGF selectively induces proliferation and branching morphogenesis of primary cultured rat RPTEs. Additional growth factors including fibroblast growth factor (FGF)-1, epidermal growth factor (EGF), FGF-7, or insulin-like growth factor-1 (IGF-1) did not selectively induce tubulogenesis. However, when administered in combination, these factors initiated branching morphogenesis comparable to HGF alone and greatly augmented HGF-induced proliferation and branching. Microscopic analysis revealed that branching RPTEs were undergoing tubulogenesis and formed a polarized epithelium. TGF-beta1 blocked HGF- or growth factor cocktail (GFC; HGF, FGF-1, EGF, IGF-1)-induced proliferation and branching morphogenesis. Adding TGF-beta1 after GFC-induced tubulogenesis had occurred caused a progressive regression of the tubular structures, a response associated with an increase in apoptosis of the RPTEs. Primary cultured RPTEs are capable of undergoing HGF-induced tubulogenesis. Unlike cell lines, combinations of growth factors differentially augment the response. PMID:10362020

  10. Complement activation in the context of stem cells and tissue repair

    PubMed Central

    Schraufstatter, Ingrid U; Khaldoyanidi, Sophia K; DiScipio, Richard G

    2015-01-01

    The complement pathway is best known for its role in immune surveillance and inflammation. However, its ability of opsonizing and removing not only pathogens, but also necrotic and apoptotic cells, is a phylogenetically ancient means of initiating tissue repair. The means and mechanisms of complement-mediated tissue repair are discussed in this review. There is increasing evidence that complement activation contributes to tissue repair at several levels. These range from the chemo-attraction of stem and progenitor cells to areas of complement activation, to increased survival of various cell types in the presence of split products of complement, and to the production of trophic factors by cells activated by the anaphylatoxins C3a and C5a. This repair aspect of complement biology has not found sufficient appreciation until recently. The following will examine this aspect of complement biology with an emphasis on the anaphylatoxins C3a and C5a. PMID:26435769

  11. Factor XIa induced activation of the intrinsic cascade in vivo.

    PubMed

    ten Cate, H; Biemond, B J; Levi, M; Wuillemin, W A; Bauer, K A; Barzegar, S; Buller, H R; Hack, C E; ten Cate, J W; Rosenberg, R D

    1996-03-01

    Coagulation factor XI is a glycoprotein of the contact factor system. Its deficiency is associated with a highly variable bleeding tendency, thus a role in relation to hemostasis appears to exist. However, the importance of factor XI for stimulating intrinsic coagulation in vivo has not yet been determined. To study the procoagulant effects of human factor XIa in vivo, we infused the purified enzyme into normal chimpanzees (100 micrograms) in the absence or presence of the thrombin inhibitor rec-hirudin (1.0 mg/kg loading dose plus 0.3 mg/kg body wt continuous infusion). Factor XIa elicited an immediate activation of factors IX, X, and prothrombin, as measured by their respective activation fragments. However, whereas the activation of factors IX and X was immediate and shortlasting, (peak increments of 6- and 1.4-fold of baseline at 5 minutes after injection), the conversion of prothrombin gradually increased, reaching a summit of 6-fold baseline values after 60 min, and remaining elevated during the course of the experiments. Thrombin-antithrombin complexes also remained elevated during the study period. In the presence of hirudin, the initial activation of factors IX, X, and prothrombin was unchanged, however the further increment in prothrombin fragment F1 + 2 was markedly inhibited. These results demonstrate that factor XIa is a potential agonist of the intrinsic cascade in vivo, which activity is enhanced in the presence of thrombin.

  12. Quartz crystal microbalance-with dissipation monitoring (QCM-D) for real time measurements of blood coagulation density and immune complement activation on artificial surfaces.

    PubMed

    Andersson, Marcus; Andersson, Jonas; Sellborn, Anders; Berglin, Mattias; Nilsson, Bo; Elwing, Hans

    2005-07-15

    A recently developed variant of quartz crystal microbalance (QCM) called QCM-with dissipation monitoring (QCM-D) allows simultaneous and simple measurements of changes in adsorbed mass as well as the viscoelastic property (D-factor) of deposited protein layers on the sensor surface. We have taken the QCM-D technology a step further and demonstrated its advantages in the study of protein assembly as a consequence of surface induced immune complement activation, or contact activated blood coagulation. In the present study we have continued our QCM-D investigations of surface assembly of fibrin clot formation and complement activation and incubated differently modified quartz sensor surfaces in blood plasma and sera. Polymer surfaces used were spin-coated polyethylene, poly(ethylene terephtalate), poly(methylmetacrylate) and poly(dimethylsiloxane). Also used were sputtered titanium and heparin grafted surfaces. In this investigation we found that we could describe the surface induced coagulation with four independent parameters: (1) Time of onset of coagulation, (2) fibrin deposition rate, (3) total frequency shift at stable plateau, and (4) fibrin clot density. The most important finding was that the blood plasma clot density can be assessed with the use of D determinations and that the clot density varied significantly with the chemical composition of the surface. However, the D-factor did not give any new analytical information about the possible complement activation mechanisms. Nevertheless, the QCM-D was found to be a reliable tool for the analysis of surface induced complement activation. We also compared the QCM-D technique with traditional enzyme immuno assay (EIA) measurements of soluble products from the surface activation of the complement and coagulation systems. We found that the results from EIA and QCM-D measurements corresponded well for the complement activation but not for the coagulation, probably due to the biological complexity of the coagulation

  13. Expression of hypoxia-inducible factor 3α in hepatocellular carcinoma and its association with other hypoxia-inducible factors

    PubMed Central

    LIU, PING; FANG, XIEFAN; SONG, YANG; JIANG, JIAN-XIN; HE, QIAN-JIN; LIU, XIANG-JIE

    2016-01-01

    The functional role of hypoxia-inducible factor (HIF)-3α in the development of hepatocellular carcinoma (HCC) is not yet fully understood. The aim of the present study was to elucidate the association between HIF-3α expression and the clinicopathological features as well as prognosis of HCC patients. In addition, we investigated the association between HIF-3α expression and the expression of HIF-1α and HIF-2α in tumor tissues. The protein levels of HIF-3α were determined using immunohistochemical analysis of paraffin sections of 126 paired HCC and peritumoral tissues. PLC/PRF/5 cells, a human HCC cell line, were transfected with HIF-1α and HIF-2α vectors and HIF-3α mRNA and protein expression was detected using quantitative polymerase chain reaction and western blot analysis, respectively. The expression of HIF-3α was upregulated in 46.0% (58/126) and downregulated in 42.9% (54/126) of tumor tissues, respectively, when compared to peritumoral tissues. HIF-3α protein expression was not associated with peripheral blood vessel invasion, overall survival, or disease-free survival in HCC patients (P>0.05). In HCC tissues, the levels of HIF-3α protein were positively correlated with HIF-2α, but not with HIF-1α expression in HCC tissues. HIF-3α was upregulated in PLC/PRF/5 and Hep3B cells overexpressed with HIF-1α or HIF-2α. The hypoxic microenvironment of liver cancer did not lead to elevated HIF-3α protein expression, indicating that HIF-3α is regulated differently from HIF-1α in vivo. The correlation between HIF-3α and HIF-2α expression at the cellular and tissue levels indicated that HIF-3α may be a target gene of HIF-2α. The hypoxic microenvironment did not lead to elevation of HIF-3α protein expression in liver cancer; thus, HIF-3α may be a target gene of HIF-2α. PMID:27284334

  14. Interaction of toxic venoms with the complement system

    PubMed Central

    Birdsey, Vanessa; Lindorfer, Jean; Gewurz, H.

    1971-01-01

    Thirty-nine venoms from various vertebrate and invertebrate species were tested for their ability to consume haemolytic complement (C) activity upon incubation in fresh guinea-pig serum. Nineteen had `anti-complementary' activity, and these were provisionally sorted into the following groups: Pattern I—exemplified by the Naja haje (Egyptian cobra) and six other Elapidae species (all cobras), which induced selective consumption of C3—C9, and led to formation of a stable C3—C9-consuming intermediate; Pattern II—exemplified by the Agkistrodon rhodostoma (Malayan pit viper), Bitis arietans (puff adder), Bothrops jararaca (South American pit viper), Bothrops atrox (Fer de Lance) and three other species, which induced marked consumption of C4 and C2, as well as C3—C9, but did not form a stable C3—C9-consuming intermediate; and individual animals, e.g. the Lachesis muta (bushmaster), which induced other patterns (III—VI) of complement component consumption. Active fractions of representative venoms were partially purified by ion exchange and gel filtration chromatography and their interactions with the complement system characterized further. It is anticipated that these enzymes, with a capacity to activate the complement system in unique ways, will prove to be of further experimental usefulness. PMID:4398349

  15. Complement the hemostatic system: an intimate relationship.

    PubMed

    Weitz, Ilene Ceil

    2014-05-01

    The complement system is important part of our innate immune system and interacts directly with the hemostatic system. Disorders of complement activation or dysregulation resulting in excess complement generation, such as Paroxysmal Nocturnal Hemoglobinuria (PNH), atypical Hemolytic uremic Syndrome (aHUS) and antiphospholipid syndrome (APLS) have been associated with significant thrombophilia. Terminal Complement (C5b-9) deposition on endothelial and tumor cell membranes has also been reported in a variety of cancer. Recent developments in complement inhibition have given us new insights into the mechanism of thrombosis in these disorders.

  16. Malaria inhibits surface expression of complement receptor-1 in monocyte/macrophages causing decreased immunecomplex internalization

    PubMed Central

    Fernandez-Arias, Cristina; Lopez, Jean Pierre; Hernandez-Perez, Jean Nikolae; Bautista-Ojeda, Maria Dolores; Branch, OraLee; Rodriguez, Ana

    2013-01-01

    Complement receptor 1 (CR1) expressed on the surface of phagocytic cells binds complement-bound IC playing an important role in the clearance of circulating immunecomplexes (IC). This receptor is critical to prevent accumulation of IC, which can contribute to inflammatory pathology. Accumulation of circulating IC is frequently observed during malaria, although the factors contributing to this accumulation are not clearly understood. We have observed that the surface expression of CR1 on monocyte/macrophages and B cells is strongly reduced in mice infected with Plasmodium yoelii, a rodent malaria model. Monocyte/macrophages from these infected mice present a specific inhibition of complement-mediated internalization of IC caused by the decreased CR1 expression. Accordingly, mice show accumulation of circulating IC and deposition of IC in the kidneys that inversely correlates with the decrease in CR1 surface expression. Our results indicate that malaria induces a significant decrease on surface CR1 expression in the monocyte/macrophage population that results in deficient internalization of IC by monocyte/macrophages. To determine whether this phenomenon is found in human malaria patients, we have analyzed 92 patients infected with either P. falciparum (22) or P. vivax (70), the most prevalent human malaria parasites. The levels of surface CR1 on peripheral monocyte/macrophages and B cells of these patients show a significant decrease compared to uninfected control individuals in the same area. We propose that this decrease in CR1 plays an essential role in impaired IC clearance during malaria. PMID:23440418

  17. Progress in Parallel Schur Complement Preconditioning for Computational Fluid Dynamics

    NASA Technical Reports Server (NTRS)

    Barth, Timothy J.; Chan, Tony F.; Tang, Wei-Pai; Chancellor, Marisa K. (Technical Monitor)

    1997-01-01

    We consider preconditioning methods for nonself-adjoint advective-diffusive systems based on a non-overlapping Schur complement procedure for arbitrary triangulated domains. The ultimate goal of this research is to develop scalable preconditioning algorithms for fluid flow discretizations on parallel computing architectures. In our implementation of the Schur complement preconditioning technique, the triangulation is first partitioned into a number of subdomains using the METIS multi-level k-way partitioning code. This partitioning induces a natural 2X2 partitioning of the p.d.e. discretization matrix. By considering various inverse approximations of the 2X2 system, we have developed a family of robust preconditioning techniques. A computer code based on these ideas has been developed and tested on the IBM SP2 and the SGI Power Challenge array using MPI message passing protocol. A number of example CFD calculations will be presented to illustrate and assess various Schur complement approximations.

  18. Intracellular sensing of complement C3 activates cell autonomous immunity

    PubMed Central

    Tam, Jerry C.H.; Bidgood, Susanna R.; McEwan, William A.; James, Leo C.

    2014-01-01

    Pathogens traverse multiple barriers during infection including cell membranes. Here we show that during this transition pathogens carry covalently attached complement C3 into the cell, triggering immediate signalling and effector responses. Sensing of C3 in the cytosol activates MAVS-dependent signalling cascades and induces proinflammatory cytokine secretion. C3 also flags viruses for rapid proteasomal degradation, thereby preventing their replication. This system can detect both viral and bacterial pathogens but is antagonized by enteroviruses, such as rhinovirus and poliovirus, which cleave C3 using their 3C protease. The antiviral Rupintrivir inhibits 3C protease and prevents C3 cleavage, rendering enteroviruses susceptible to intracellular complement sensing. Thus, complement C3 allows cells to detect and disable pathogens that have invaded the cytosol. PMID:25190799

  19. The Semantics of Complementation in English: A Cognitive Semantic Account of Two English Complement Constructions

    ERIC Educational Resources Information Center

    Smith, Michael B.

    2009-01-01

    Studies on complementation in English and other languages have traditionally focused on syntactic issues, most notably on the constituent structures of different complement types. As a result, they have neglected the role of meaning in the choice of different complements. This paper investigates the semantics of complementation within the…

  20. Atorvastatin neutralises the thrombin-induced tissue factor expresion in endothelial cells via geranylgeranyl pyrophosphate.

    PubMed

    Martínez-Sales, Vicenta; Vila, Virtudes; Ferrando, Marcos; Reganon, Edelmiro

    2011-01-01

    Statins may have beneficial effects in atherogenesis given their antithrombotic properties involving non-lipid mechanisms that modify endothelial function of tissue factor induction by thrombin. In this study, we investigate the effect of atorvastatin on tissue factor (TF) activity in thrombin-stimulated endothelial cells and its regulation through mevalonate or its derivatives. First subculture of human umbilical endothelial cells was used for this study. Cells were treated with thrombin and atorvastatin for different time intervals and dosage. Tissue factor activity was measured as Factor Xa generation induced by Tissue Factor-Factor VIIa complex on confluent cells. Our results show that atorvastatin prevents the thrombin-induced up-regulation of tissue factor activity in a concentration-dependent manner. Mevalonate and geranylgeranyl pyrophosphate reversed this inhibitory effect of atorvastatin on tissue factor activity, while the presence of farnesyl pyrophosphate did not prevent the atorvastatin effect on thrombin-induced tissue factor activity. Rho-kinase inhibitor did not affect the thrombin stimulation of tissue factor activity. High amount of hydrophobic isoprenoid groups decreases the thrombin-induced TF activity and may promote endothelial cell anti-thrombotic action. Rho kinase pathways do not have a major role in the thrombin-mediated TF activity. The inhibitory effect of atorvastatin on thrombin-induced TF activity was partially reversed by MVA and GGPP but not FPP.

  1. The complement system contributes to the pathology of experimental autoimmune encephalomyelitis by triggering demyelination and modifying the antigen-specific T and B cell response.

    PubMed

    Hundgeburth, Lorenz C; Wunsch, Marie; Rovituso, Damiano; Recks, Mascha S; Addicks, Klaus; Lehmann, Paul V; Kuerten, Stefanie

    2013-03-01

    So far, studies of the human autoimmune disease multiple sclerosis (MS) have largely been hampered by the absence of a pathogenic B cell component in its animal model, experimental autoimmune encephalomyelitis (EAE). To overcome this shortcoming, we have previously introduced the myelin basic protein (MBP)-proteolipid protein (PLP) MP4-induced EAE, which is B cell and autoantibody-dependent. Here we show that MP4-immunized wild-type C57BL/6 mice displayed a significantly lower disease incidence when their complement system was transiently depleted by a single injection of cobra venom factor (CVF) prior to immunization. Considering the underlying pathomechanism, our data suggest that the complement system is crucial for MP4-specific antibodies to trigger CNS pathology. Demyelinated lesions in the CNS were colocalized with complement depositions. In addition, B cell deficient JHT mice reconstituted with MP4-reactive serum showed significantly attenuated clinical and histological EAE after depletion of complement by CVF. The complement system was also critically involved in the generation of the MP4-specific T and B cell response: in MP4-immunized wild-type mice treated with CVF the MP4-specific cytokine and antibody response was significantly attenuated compared to untreated wild-type mice. Taken together, we propose two independent mechanisms by which the complement system can contribute to the pathology of autoimmune encephalomyelitis. Our data corroborate the role of complement in triggering antibody-dependent demyelination and antigen-specific T cell immunity and also provide first evidence that the complement system can modify the antigen-specific B cell response in EAE and possibly MS.

  2. Biological effects of short-term, high-concentration exposure to methyl isocyanate. VI. In vitro and in vivo complement activation studies

    SciTech Connect

    Kolb, W.P.; Savary, J.R.; Troup, C.M.; Dodd, D.E.; Tamerius, J.D.

    1987-06-01

    The ability of MIC to induce complement activation in vitro and in vivo was investigated. For the in vitro studies, both human and guinea pig serum or EDTA-plasma samples were exposed to 1167 to 1260 ppm MIC vapor for 15 min at room temperature. The human serum samples exposed to MIC showed significant reduction in Factor B, C2, C4, C3, C5, and total hemolytic complement CH/sub 50/ activity levels. The C3, C5, and CH/sub 50/ functional activities in guinea pig serum were more sensitive to MIC-mediated reduction than the corresponding activity reductions observed in the human serum samples. The human and single guinea pig EDTA-plasma samples exposed to MIC vapor showed no evidence of C3 consumption but did show significant reductions in CH/sub 50/ levels. Thus, MIC vapor was able to active, and thereby reduce serum complement C3 activity in vitro by a complement-dependent process. For the in vivo studies, five pairs of guinea pigs were exposed to 644 to 702 ppm MIC vapor until one of the pair died (11-15 min). MIC exposure was then discontinued, the surviving guinea pig was sacrificed, and EDTA-plasma was obtained from both animals and analyzed for complement consumption. Clear evidence was obtained to indicate that complement activation had occurred in these animals exposed to MIC for 11 to 15 min. In addition, the complement activation profile observed in these guinea pigs was qualitatively similar to that seen in the guinea pig serum samples exposed to MIC vapor in vitro. The total protein concentration present in plasma samples obtained from guinea pigs that had died from MIC exposure was elevated significantly. The possible contribution of complement activation to the fatal reaction(s) observed in these MIC-treated animals is discussed.

  3. Meningococcal disease and the complement system

    PubMed Central

    Lewis, Lisa A; Ram, Sanjay

    2014-01-01

    Despite considerable advances in the understanding of the pathogenesis of meningococcal disease, this infection remains a major cause of morbidity and mortality globally. The role of the complement system in innate immune defenses against invasive meningococcal disease is well established. Individuals deficient in components of the alternative and terminal complement pathways are highly predisposed to invasive, often recurrent meningococcal infections. Genome-wide analysis studies also point to a central role for complement in disease pathogenesis. Here we review the pathophysiologic events pertinent to the complement system that accompany meningococcal sepsis in humans. Meningococci use several often redundant mechanisms to evade killing by human complement. Capsular polysaccharide and lipooligosaccharide glycan composition play critical roles in complement evasion. Some of the newly described protein vaccine antigens interact with complement components and have sparked considerable research interest. PMID:24104403

  4. The complement system in inflammatory bowel disease.

    PubMed

    Jain, Umang; Otley, Anthony R; Van Limbergen, Johan; Stadnyk, Andrew W

    2014-09-01

    Complement is well appreciated to be a potent innate immune defense against microbes and is important in the housekeeping act of removal of apoptotic and effete cells. It is also understood that hyperactivation of complement, or the lack of regulators, may underlie chronic inflammatory diseases. A pipeline of products to intervene in complement activation, some already in clinical use, is being studied in various chronic inflammatory diseases. To date, the role of complement in inflammatory bowel disease has not received a lot of research interest. Novel genetically modified laboratory animals and experiments using antagonists to complement effector molecules have kindled important research observations implicating the complement system in inflammatory bowel disease pathogenesis. We review the evidence base for the role and potential therapeutic manipulation of the complement cascade in inflammatory bowel disease.

  5. Overview of Complement Activation and Regulation

    PubMed Central

    Noris, Marina; Remuzzi, Giuseppe

    2013-01-01

    Summary Complement is an important component of the innate immune system that is crucial for defense from microbial infections and for clearance of immune complexes and injured cells. In normal conditions complement is tightly controlled by a number of fluid-phase and cell surface proteins to avoid injury to autologous tissues. When complement is hyperactivated, as occurs in autoimmune diseases or in subjects with dysfunctional regulatory proteins, it drives a severe inflammatory response in numerous organs. The kidney appears to be particularly vulnerable to complement-mediated inflammatory injury. Injury may derive from deposition of circulating active complement fragments in glomeruli, but complement locally produced and activated in the kidney also may have a role. Many kidney disorders have been linked to abnormal complement activation, including immune-complex–mediated glomerulonephritis and rare genetic kidney diseases, but also tubulointerstitial injury associated with progressive proteinuric diseases or ischemia-reperfusion. PMID:24161035

  6. Membrane-bound complement regulatory activity is decreased on vaccinia virus-infected cells.

    PubMed Central

    Baranyi, L; Okada, N; Baranji, K; Takizawa, H; Okada, H

    1994-01-01

    Decay accelerating factor (DAF), membrane cofactor protein (MCP), complement receptor 1 and mouse Crry are cell surface-bound complement regulatory proteins capable of inhibiting C3 convertase activity on cell membranes, and therefore provide a substantial protection from attack by homologous complement activated either by the classical or by the alternative pathway. Decrease in complement regulatory activity might lead to spontaneous complement deposition and subsequent cell injury. MoAb 5I2 can inhibit the complement regulatory activity of molecules on rat cells, resulting in deposition of homologous complement. The antigen recognized by 5I2 MoAb in rats is homologous to mouse Crry. Fifteen to 20 h after infection with vaccinia virus, in vitro cultured KDH-8 rat hepatoma cells show a strong decrease in expression of Crry-like antigen, and proved to be sensitive to complement deposition when 1:5 diluted normal rat serum was added to the culture medium as a source of complement. Addition of complement to the cultured KDH-8 cells infected with a very low dose of vaccinia virus (1 plaque-forming unit (PFU)/1000 cells) substantially reduced spreading of virus infection in the cell culture, while inactivation of complement by heat or zymosan treatment abrogated the protective effect. PMID:7923872

  7. Growth factor involvement in tension-induced skeletal muscle growth

    NASA Technical Reports Server (NTRS)

    Vandenburgh, Herman W.

    1987-01-01

    New muscle tissue culture techniques were developed to grow embryonic skeletal myofibers which are able to differentiate into more adultlike myofibers. Studies on mechanical simulation of cultured muscle cell growth will now be more directly applicable to mechanically-induced growth in adult muscle, and lead to better models for understanding muscle tissue atrophy caused by disuse in the microgravity of space.

  8. Alternative Complement Pathway Deregulation Is Correlated with Dengue Severity

    PubMed Central

    Nascimento, Eduardo J. M.; Silva, Ana M.; Cordeiro, Marli T.; Brito, Carlos A.; Gil, Laura H. V. G.; Braga-Neto, Ulisses; Marques, Ernesto T. A.

    2009-01-01

    Background The complement system, a key component that links the innate and adaptive immune responses, has three pathways: the classical, lectin, and alternative pathways. In the present study, we have analyzed the levels of various complement components in blood samples from dengue fever (DF) and dengue hemorrhagic fever (DHF) patients and found that the level of complement activation is associated with disease severity. Methods and Results Patients with DHF had lower levels of complement factor 3 (C3; p = 0.002) and increased levels of C3a, C4a and C5a (p<0.0001) when compared to those with the less severe form, DF. There were no significant differences between DF and DHF patients in the levels of C1q, immunocomplexes (CIC-CIq) and CRP. However, small but statistically significant differences were detected in the levels of MBL. In contrast, the levels of two regulatory proteins of the alternative pathway varied widely between DF and DHF patients: DHF patients had higher levels of factor D (p = 0.01), which cleaves factor B to yield the active (C3bBb) C3 convertase, and lower levels of factor H (p = 0.03), which inactivates the (C3bBb) C3 convertase, than did DF patients. When we considered the levels of factors D and H together as an indicator of (C3bBb) C3 convertase regulation, we found that the plasma levels of these regulatory proteins in DHF patients favored the formation of the (C3bBb) C3 convertase, whereas its formation was inhibited in DF patients (p<0.0001). Conclusion The data suggest that an imbalance in the levels of regulatory factors D and H is associated with an abnormal regulation of complement activity in DHF patients. PMID:19707565

  9. Complement and contact activation in term neonates after fetal acidosis

    PubMed Central

    Sonntag, J.; Wagner, M.; Strauss, E.; Obladen, M.

    1998-01-01

    AIMS—To evaluate complement and contact activation after fetal acidosis.
METHODS—Fifteen term neonates with hypoxic-ischaemic encephalopathy after umbilical arterial pH < 7.10 were compared with 15 healthy neonates with umbilical arterial pH > 7.20. Determinations of the complement function and C1-inhibitor activity were performed as kinetic tests 22-28 hours after birth. C1q, C1-inhibitor, and factor B concentrations were determined by radial immunodiffusion and those of C3a, C5a, and factor XIIa by enzyme immunoabsorbent assay.
RESULTS—Median complement function (46 vs 73 %), C1q (4.3 vs 9.1 mg/dl), and factor B (5.2 vs 7.7 mg/dl) decreased after fetal acidosis. The activated split products C3a (260 vs 185 µg/l), C5a (5.0 vs 0.6 µg/l), and factor XIIa (3.2 vs 1.3 µg/l) increased in the neonates after fetal acidosis. No differences were found in the concentration and activity of C1-inhibitor.
CONCLUSIONS—Complement and contact activation occurred in the newborns with hypoxic-ischaemic encephalopathy. Activation of these systems generates mediators which can trigger inflammation and tissue injury.

 PMID:9577283

  10. Fine-structure mapping and complementation analysis of nif (nitrogen fixation) genes in Klebsiella pneumoniae.

    PubMed Central

    MacNeil, T; MacNeil, D; Roberts, G P; Supiano, M A; Brill, W J

    1978-01-01

    Four hundred and eighty-nine independent Nif- strains containing 260 point, 130 millimicron-induced, and 99 deletion mutations in nif in the Klebsiella pneumoniae chromosome were isolated. Three hundred and ninety insertion and point mutations were mapped with millimicron-induced deletions carried on 44 plasmids derived from pTM4010, a recombinant R factor containing the his-nif region of K. pneumoniae. The 99 chromosomal deletions in the nif region were mapped with 69 derivatives of pTM4010 carrying insertion and point mutations in nif. Complementation analysis between 84 derivatives of pTM4010 carrying nif mutations and Rec- derivatives of the 390 Nif- mutants identified 14 genes. The nif mutations were ordered into 49 deletion groups with a gene order of his...nifQBALFMVSNEKDHJ. Complementation analysis of millimicron-induced, amber, frameshift, and deletion mutations indicates there are five polycistronic and two monocistronic operons: nifQ nifB, nifA nifL, nifF, nifM nifV nifS, nifN nifE, nifK nifD nifH, and nifJ. Transcription is from right to left in all polycistronic operons. PMID:361693

  11. TCDD Induces the Hypoxia-Inducible Factor (HIF)-1α Regulatory Pathway in Human Trophoblastic JAR Cells

    PubMed Central

    Liao, Tien-Ling; Chen, Su-Chee; Tzeng, Chii-Reuy; Kao, Shu-Huei

    2014-01-01

    The exposure to dioxin can compromise pregnancy outcomes and increase the risk of preterm births. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) has been demonstrated to induce placental hypoxia at the end of pregnancy in a rat model, and hypoxia has been suggested to be the cause of abnormal trophoblast differentiation and placental insufficiency syndromes. In this study, we demonstrate that the non-hypoxic stimulation of human trophoblastic cells by TCDD strongly increased hypoxia inducible factor-1 alpha (HIF-1α) stabilization. TCDD exposure induced the generation of reactive oxygen species (ROS) and nitric oxide. TCDD-induced HIF-1α stabilization and Akt phosphorylation was inhibited by pretreatment with wortmannin (a phosphatidylinositol 3-kinase (PI3K) inhibitor) or N-acetylcysteine (a ROS scavenger). The augmented HIF-1α stabilization by TCDD occurred via the ROS-dependent activation of the PI3K/Akt pathway. Additionally, a significant increase in invasion and metallomatrix protease-9 activity was found in TCDD-treated cells. The gene expression of vascular endothelial growth <