Preliminary Findings of an In Vitro Assay Based on Human Complement Activation for the Detection of Chemicals with Phototoxicity Potential

1993 ◽  
Vol 21 (4) ◽  
pp. 509-512
Author(s):  
Dariusz Sladowski ◽  
Sarah Steer ◽  
Lynda Moore ◽  
Ruth Glassborow ◽  
Richard Clothier ◽  
...  
Keyword(s):  
Uv Light ◽  
Alternative Pathway ◽  
Sodium Dodecyl ◽  
Negative Control ◽  
In Vitro Tests ◽  
Classical Pathway ◽  
Vitro Assay ◽  
Alternative Pathways ◽  
The Complement System

Eight known photosensitising chemicals and one non-photoaensitiser were compared for their ability to activate the complement system of human serum in both the presence and the absence of ultraviolet (UV) radiation. Commercially available ELISA systems for the detection of C4d and iC3b fragments were used to investigate the classical and alternative pathways, respectively. The results indicated that, whereas the classical pathway was unaffected, all the photosensitisers tested were capable of activating the alternative pathway in the presence of UV light, and that this activation occurred at different levels, depending upon the chemical itself. The non-photosensitising chemical, sodium dodecyl sulphate, acted as a negative control. It is, therefore, suggested that this type of method could be considered for inclusion in any battery of in vitro tests designed for the detection of potential photosensitising chemicals.

scholarly journals Development of a Novel Cytomedical Treatment that can Protect Entrapped Cells from Host Humoral Immunity

2002 ◽  
Vol 11 (8) ◽  
pp. 787-797 ◽  
Author(s):  
Ryo Suzuki ◽  
Yasuo Yoshioka ◽  
Etsuko Kitano ◽  
Tatsunobu Yoshioka ◽  
Hiroaki Oka ◽  
...  
Keyword(s):  
Humoral Immunity ◽  
Complement System ◽  
Alternative Pathway ◽  
Classical Pathway ◽  
Dependent Manner ◽  
Complement Components ◽  
Alternative Pathways ◽  
Low Cytotoxicity ◽  
The Complement System

Cell therapy is expected to relieve the shortage of donors needed for organ transplantation. When patients are treated with allogeneic or xenogeneic cells, it is necessary to develop a means by which to isolate administered cells from an immune attack by the host. We have developed “cytomedicine, ” which consists of functional cells entrapped in semipermeable polymer, and previously reported that alginate-poly-l-lysine-alginate microcapsules and agarose microbeads could protect the entrapped cells from injury by cellular immunity. However, their ability to isolate from humoral immunity was insufficient. It is well known that the complement system plays an essential role in rejection of transplanted cells by host humoral immunity. Therefore, the goal of the present study was to develop a novel cytomedical device containing a polymer capable of inactivating complement. In the screening of various polymers, polyvinyl sulfate (PVS) exhibited high anticomplement activity and low cytotoxicity. Murine pancreatic β-cell line (MIN6 cell) entrapped in agarose microbeads containing PVS maintained viability and physiological insulin secretion, replying in response to glucose concentration, and resisted rabbit antisera in vitro. PVS inhibited hemolysis of sensitized sheep erythrocytes (EAs) and rabbit erythrocytes by the complement system. This result suggests that PVS inhibits both the classical and alternative complement pathways of the complement system. Next, the manner in which PVS exerts its effects on complement components was examined. PVS was found to inhibit generation of C4a and Ba generation in activation of the classical and alternative pathways, respectively. Moreover, when the EAC1 cells, which were carrying C1 on the EAs, treated with PVS were exposed to C1-deficient serum, hemolysis decreased in a PVS dose-dependent manner. These results suggest that PVS inhibits C1 in the classical pathway and C3 convertase formation in the alternative pathway. Therefore, PVS may be a useful polymer for developing an anticomplement device for cytomedical therapy.

Schistosoma mansoni: complement activation in human and rodent sera by living parasites of various developmental stages

Parasitology ◽  
1983 ◽  
Vol 87 (1) ◽  
pp. 75-86 ◽  
Author(s):  
A. Ruppel ◽  
U. Rother ◽  
H. Vongerichten ◽  
H. J. Diesfeld
Keyword(s):  
Schistosoma Mansoni ◽  
Complement System ◽  
Developmental Stages ◽  
Alternative Pathway ◽  
Classical Pathway ◽  
Factor B ◽  
The Complement System ◽  
Dose Dependent ◽  
Living Adult

SUMMARYLiving Schistosoma mansoni of various developmental stages were studied with respect to their ability to activate the complement system in sera of humans, mice and rats. Immunofluorescence assays demonstrated that binding of human C3 occurred on fresh schistosomula as well as on schistosomula prepared from mouse lymph-nodes or lungs and on adult schistosomes. However, rodent C3 was deposited only on fresh schistosomula. Deposition of human C3 on the worms' surface required activation of the complement system. The alternative pathway was shown to be involved in deposition of human C3 on schistosomes of all ages, whereas activation of the classical pathway was demonstrable only with fresh schistosomula. Immunoelectrophoretic studies demonstrated a dose-dependent cleavage of human C3 and conversion of factor B by living adult schistosomes. The results demonstrate that the ability of living schistosomes to activate complement in vitro is dependent not only on their developmental stage but also on the species of the serum.

Modulation of CD11b/CD18 on Monocytes and Granulocytes following Hemodialysis Membrane Interaction in vitro

1996 ◽  
Vol 19 (3) ◽  
pp. 156-163 ◽  
Author(s):  
P. Thylén ◽  
E. Fernvik ◽  
J. Lundahl ◽  
J. Hed ◽  
S.H. Jacobson
Keyword(s):  
Complement System ◽  
Blood Donors ◽  
Alternative Pathway ◽  
Classical Pathway ◽  
Dialysis Membrane ◽  
Serum Factors ◽  
Relative Contribution ◽  
Normal Human ◽  
The Complement System

We studied the generation of CD11b/CD18 mobilizing factors in serum after incubation with dialysis membrane fragments of different chemical composition. We also evaluated the relative importance of the alternative and classical pathways of the complement system in the generation of such factors. Monocytes and granulocytes from healthy blood donors were incubated in normal human serum (NHS) and in NHS that had been preincubated with Cuprophan (CU) membrane (NHS-CU), Hemophan (HE) (NHS-HE) or polysulfone (PS) (NHS-PS). NHS-CU caused the highest up-regulation of the CD11b/CD18 receptor on monocytes and granulocytes. The rank in capacity to mobilize CD11b/CD18 on granulocytes was CU>HE>PS (p<0.001), CU>HE (p<0.05) and HE>PS (p<0.001). The rank in capacity to mobilize CD11b/CD18 on monocytes was CU>HE>PS (p<0.001), CU>HE (p<0.05) and HE>PS (p<0.01). NHS-PS induced a lower up-regulation of CD11b/CD18 compared to NHS which indicates that serum factors with the ability to mobilize the CD11b/CD18 receptor on monocytes and granulocytes are deposited on or adsorbed by PS. In order to study the relative contribution of the alternative and classical pathways of the complement system in the generation of CD11b/CD18 mobilizing factors in serum, three different serum preparations (1. both pathways intact. 2. only the alternative intact and 3. only the classical pathway intact) were used. The CU membrane activated the classical pathway to a larger extent than the PS membrane (p<0.01). When only the alternative pathway was intact no difference in the generation of CD11b/CD18 mobilizing factors between the CU and PS membranes was observed. These studies show that CD11b/CD18 mobilizing serum factors are generated after incubation with CU membranes and that such factors are probably adsorbed by PS. The classical pathway of complement activation seems to contribute to the generation of CD11b/CD18 mobilizing factors in serum.

scholarly journals Two New Compounds and Anti-complementary Constituents from Amomum tsao-ko

2013 ◽  
Vol 8 (12) ◽  
pp. 1934578X1300801
Author(s):  
Jiahong Jin ◽  
Zhihong Cheng ◽  
Daofeng Chen
Keyword(s):  
Phenolic Compounds ◽  
High Resolution ◽  
Complement System ◽  
Alternative Pathway ◽  
2D Nmr ◽  
Classical Pathway ◽  
Ms Analysis ◽  
New Compounds ◽  
The Complement System

Two new compounds, (2 R,3 R,4 R)–3′,5′-dimethoxy-3,4,7,4′-tetrahydroxy-flavan (1) and 2-(4-hydroxy-3-methoxybenzoyl)-4-methoxy-benzaldehyde (2), together with 35 known phenolic compounds were obtained from the fruits of Amomum tsao-ko. Structures of the new compounds were elucidated on the basis of spectroscopic means, including 2D NMR, and high-resolution MS analysis. The isolated compounds were tested in vitro for their complement-inhibitory properties against the classical pathway (CP) and alternative pathway (AP). The results showed that 14 compounds exhibited anti-complementary activities against the CP and AP with CH50 values of 0.42 - 4.43 mM and AP50 values of 0.53 −1.51 mM. Preliminary mechanism studies showed that 1,7-bis(4-hydroxyphenyl)–4( E)-hepten-3-one (8) blocked C1q, C2, C3, C4, C5 and C9 components of the complement system, and hydroquinone (15) acted on C1q, C2, C3, C5 and C9 components.

scholarly journals Lysis of oncornaviruses by human serum. Isolation of the viral complement (C1) receptor and identification as p15E.

1978 ◽  
Vol 147 (3) ◽  
pp. 844-853 ◽  
Author(s):  
R M Bartholomew ◽  
A F Esser ◽  
H J Müller-Eberhard
Keyword(s):  
Alternative Pathway ◽  
Leukemia Virus ◽  
Sodium Dodecyl ◽  
Biological Significance ◽  
Surface Protein ◽  
Enzymatic Digestion ◽  
Classical Pathway ◽  
Alternative Pathways ◽  
Moloney Leukemia Virus ◽  
Triton X

Moloney leukemia virus activated both the classical and alternative pathways of human complement. About 500,000 virions were required to detect activation of the classical pathway whereas 5,000 times as many virions were necessary to initiate the alternative pathway, indicating that in this system only the former is of biological significance. Disruption of the virus with Triton X-100 destroyed its ability to initiate the alternative pathway without affecting its ability to activate the classical pathway. After ultracentrifugation of disrupted virus the active component could be recovered in the supernate and was isolated by isoelectric focusing in granulated gels. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic and analysis and cyanogen bromide digestion studies revealed that the activity resided in a methionine-containing protein having a pI of 7.5 and a molecular weight of approximately equal to 15,000 daltons. The purified protein interacts strongly with Clq and efficiently activates Cl. RNase and lipolytic enzymes had no effect on the isolated protein but incubation with trypsin resulted in loss of activity. Enzymatic digestion studies of surface-labeled virus indicate that the active protein is a viral membrane protein. On the basis of these results it is concluded that the complement receptor of Moloney leukemia virus is the surface protein p15E.

scholarly journals GSP1 and GSP2, genetic suppressors of the prp20-1 mutant in Saccharomyces cerevisiae: GTP-binding proteins involved in the maintenance of nuclear organization.

1993 ◽  
Vol 13 (4) ◽  
pp. 2152-2161 ◽  
Author(s):  
P Belhumeur ◽  
A Lee ◽  
R Tam ◽  
T DiPaolo ◽  
N Fortin ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Essential Gene ◽  
Negative Control ◽  
Temperature Sensitive ◽  
Vitro Assay ◽  
Gtp Binding ◽  
Genetic Suppressors ◽  
Multicopy Suppressors

The temperature-sensitive mutation prp20-1 of Saccharomyces cerevisiae exhibits a pleiotropic phenotype associated with a general failure to maintain a proper organization of the nucleus. Its mammalian homolog, RCC1, is not only reported to be involved in the negative control of chromosome condensation but is also believed to assist in the coupling of DNA replication to the entry into mitosis. Recent studies on Xenopus RCC1 have strongly suggested a further role for this protein in the formation or maintenance of the DNA replication machinery. To elucidate the nature of the various components required for this PRP20 control pathway in S. cerevisiae, we undertook a search for multicopy suppressors of a prp20 thermosensitive mutant. Two genes, GSP1 and GSP2, were identified that encode almost identical polypeptides of 219 and 220 amino acids. Sequence analyses of these proteins show them to contain the ras consensus domains involved in GTP binding and metabolism. The levels of the GSP1 transcript are about 10-fold those of GSP2. As for S. cerevisiae RAS2, GSP2 expression exhibits carbon source dependency, while GSP1 expression does not. GSP1 is an essential gene, and GSP2 is not required for cell viability. We show that GSP1p is nuclear, that it can bind GTP in an in vitro assay, and finally, that a mutation in GSP1p which activates small ras-like proteins by increasing the stability of the GTP-bound form causes a dominant lethal phenotype. We believe that these two gene products may serve in regulating the activities of the multicomponent PRP20 complex.

Characterization of an in vitro assay for import of 3-phosphoglycerate kinase into the glycosomes of Trypanosoma brucei

1990 ◽  
Vol 10 (9) ◽  
pp. 4545-4554
Author(s):  
J M Sommer ◽  
J A Thissen ◽  
M Parsons ◽  
C C Wang
Keyword(s):  
Trypanosoma Brucei ◽  
Protein Import ◽  
Sodium Dodecyl ◽  
Phosphoglycerate Kinase ◽  
Proteinase K ◽  
Vitro Assay ◽  
Protein Core ◽  
Triton X ◽  
Gamma S

Glycosomes are microbody organelles found in kinetoplastida, where they serve to compartmentalize the enzymes of the glycolytic pathway. In order to identify the mechanism by which these enzymes are targeted to the glycosome, we have modified the in vitro import assay developed by Dovey et al. (Proc. Natl. Acad. Sci. USA 85:2598-2602, 1988). This assay measures the uptake of in vitro-translated Trypanosoma brucei glycosomal 3-phosphoglycerate kinase (gPGK) by purified glycosomes. Up to 50% of the total 35S-gPGK in the glycosomal fraction was resistant to extraction by 3 M urea or treatment with proteinase K (500 micrograms/ml). The glycosome-associated 35S-gPGK could be chemically cross-linked to the endogenous glycosomal proteins to form a sodium dodecyl sulfate-resistant complex, suggesting that it is close to the intraglycosomal protein matrix. Deoxycholate solubilized the glycosome and thereby rendered the glycosome-associated 35S-gPGK fully susceptible to proteinase K. However, the glycosome-associated 35S-gPGK was not digested by proteinase K in the presence of Triton X-100, which cannot dissolve the glycosomal protein core. The 35S-gPGK synthesized in vitro was able to bind directly to protein cores, where it became resistant to urea extraction and proteinase K digestion. However, the 35S-gPGK-protein core complex exhibited a much higher density than the 35S-gPGK-glycosome complex and was readily separable in sucrose gradients. Thus, in our in vitro import assay, the 35S-gPGK appeared to associate with intact glycosomes, possibly reflecting import of protein into the organelle. Complete denaturation of the 35S-gPGK in 8 M urea prior to the assay enhanced the efficiency of its association with glycosomes. Native gPGK did not compete with the association of in vitro-translated gPGK unless it was denatured. The assay exhibited time and temperature dependence, but it did not require externally added ATP and was not inhibited by the nonhydrolyzable analogs adenosine-5'-(beta,gamma-imido)-triphosphate and gamma-S-ATP. However, the presence of 20 to 30 microM ATP inside the glycosome may fulfill the requirement for protein import.

scholarly journals Antibacterial Effect of Silver Nanoparticles on Klebsiella spp

2020 ◽  
Vol 1 (2) ◽  
pp. 8-15
Author(s):  
Gislanne Stéphanne Estevam da Silva ◽  
Rivaldo Leon Bezerra Cabral ◽  
Nathalie de Sena Pereira ◽  
José Heriberto Oliveira do Nascimento ◽  
Dany G kramer
Keyword(s):  
Silver Nanoparticles ◽  
Bacterial Resistance ◽  
Brain Heart Infusion ◽  
Trisodium Citrate ◽  
Antimicrobial Activities ◽  
Positive Control ◽  
Negative Control ◽  
In Vitro Tests ◽  
Klebsiella Spp

Silver nanoparticles (AgNP) can be incorporated into medical devices, such as tissues, to circumvent bacterial resistance such as Klebsiella spp, which can lead to skin and mucosal infections. Thus, the aim of the present study was to synthesize silver nanoparticles for later incorporation into cotton fabrics and in vitro tests against Klebsiella spp. The AgNP colloidal solution was synthesized (AgNO3 - 0.1 mM, 100 mM trisodium citrate, polyvinylpyrrolidone - 0.24 g, H2OH2) and then impregnated into the cotton fabric pretreated with poly diallyl dimethylammonium chloride (PDDA) of 100/500 tissue, shaken for 30 minutes). The material produced was analyzed by the FTIR; DLS and reflectance spectroscopy. The tests of the antimicrobial activities were by the microdilution technique against Klebsiella spp, in tubes containing Brain Heart Infusion (BHI), with the solution of silver (1); Tissue containing AgNP - 4 mm (2); Negative control (3) and positive control - ceftriaxone (4). Regarding MIC, the inhibitory activity occurred of the dilutions between 1/2 and 1/16. The AgNP particles had an average size of 24.75 nm. As synthesized AgNPs demonstrate the excellent antimicrobial activity against Klebsiella spp, with special emphasis on applications in nanotechnology and nanomedicine, targeting multiresistant antibiotic bacteria.

Inherited complement deficiencies

1984 ◽  
Vol 306 (1129) ◽  
pp. 419-430 ◽  
Keyword(s):  
Immune Complex ◽  
Complement System ◽  
Lupus Erythematosus ◽  
Alternative Pathway ◽  
Strong Association ◽  
Factor H ◽  
Complement Deficiency ◽  
Classical Pathway ◽  
The Complement System

Isolated genetic deficiencies of individual components of the complement system have been described in man for all the components of the classical pathway and the membrane attack complex as well as for Factor I, Factor H and properdin. It is only for Factor B and Factor D of the alternative pathway that homozygous deficiency states are not so far known. Complement deficiency states provide the most direct way of looking at the role of the complement system in vivo and emphasize the importance of complement in resistance to bacterial infection and in particular to infection with Neisseria . This association is not unexpected since in vitro studies have shown complement to be an efficient enhancer of phagocytosis and inflammation. The particularly frequent occurrence of neisserial infection may be ascribed to the ability of these organisms to survive in phagocytic cells so that the plasma cytolytic activity provided by complement is needed to kill them. On the other hand the strong association between complement deficiencies and immune-complex diseases - especially systemic lupus erythematosus — was unexpected and seems paradoxical in view of the large part played by complement in the pathogenesis of immune complex mediated tissue damage. The paradox can be explained in part by the necessity for an intact complement system in the solubilization and the proper handling of immune complexes. It is also likely that complement deficiency can allow the persistence of low virulence organisms that produce disease solely by an immune complex mechanism. Recently described deficiencies of complement receptors and their effects in vivo are described.

The Studies in Various Murine Strains with Defects in Activation of Complement Cascade (CC) Reveal Both Pivotal and Pleiotropic Role of CC in Mobilization of Hematopoietic Stem/Progenitor Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 774-774
Author(s):  
Marcin Wysoczynski ◽  
Ryan Reca ◽  
Wu Wan ◽  
Magda Kucia ◽  
Marina Botto ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Molecular Level ◽  
Alternative Pathway ◽  
Classical Pathway ◽  
Complement Cascade ◽  
Hematopoietic Stem ◽  
Alternative Pathways ◽  
Dependent Pathway ◽  
Poor Mobilizers

Abstract We reported that complement cascade (CC) becomes activated in bone marrow (BM) during mobilization of hematopoietic stem/progenitor cells (HSPC) by i) immunoglobulin (Ig)-dependent pathway and/or by ii) alternative Ig-independent pathway and, as result of this, iii) several potent bioactive CC anaphylatoxins (C3a, desArgC3a, C5a and desArgC5a) are released (Blood2003;101,3784; Blood2004;103,2071; Blood2005;105,40). To learn more on the role of CC and innate immunity in this process, we compared mobilization in mice that possess defects in CC activation by i) classical pathway (C1q−/−, Ig-deficient), ii) both classical and alternative pathway (C2fB−/−) and in animals iii) that do not generate CC-derived anaphylatoxins (C3−/−, C5−/−). For mobilization, we employed G-CSF and zymosan that activate classical and alternative pathways of CC, respectively. First, we found by ELISA that CC activation in fact correlates with the level of HSPC mobilization. Next, studies in mice deficient in CC activation revealed that CC plays both pivotal and pleiotropic roles in this process. Accordingly, while C1q−/− and C3−/− mice turned out to be easy mobilizers, mobilization was very poor in Ig-deficient, C2fB−/− and C5−/− mice that demonstrate that C3 and C5 cleavage fragments differently control the mobilization of HSPC. To explain this at molecular level, we found that C3 cleavage fragments (C3a, desArgC3a) directly interact with HSPC and increase their responsiveness to SDF-1 gradient and thus prevent uncontrolled egress of HSPC from BM. It explains why C1q−/− and C3−/− mice that do not generate C3 cleavage fragments in BM release easily HSPC into circulation. In contrast, C5 cleavage fragments (C5a, desArgC5a) increase permeability of BM-endothelium and thus are crucial for the egress of HSPC from BM to occur. This explains why mice that do not activate efficient CC such as Ig-deficient, C2fB−/− and C5−/− animals are poor mobilizers. We conclude that the mobilization of HSPC is i) dependent on C activation by the classical or alternative pathway and balanced differently by C3 and C5 cleavage fragments that enhance retention or promote egress of HSPC respectively. Thus, modulation of C activation in BM may help to develop new more efficient strategies for both HSPC mobilization and their homing/engraftment.

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