The sensitivity of myeloid progenitor cells from normal subjects (N-CFU-GM) and from leukemic patients in complete remission (LR-CFU-GM) to 4-hydroperoxycyclophosphamide (4-HC) were compared to the sensitivity of leukemic progenitor cells (L-CFU) to this drug. The results were expressed as the dose of 4-HC needed to kill 90% (TD 90) of the progenitor cells. The mean TD 90 were respectively for N-CFU-GM : 59 (+/- 11 S.E.M.) nM ml-1 and for L-CFU 79 (+/- 6 S.E.M.) nM ml-1. Thus, L-CFU were equally sensitive to 4-HC as N-CFU-GM. Moreover, the mean TD 90 for LR-CFU-GM was 87 (+/- 5 S.E.M.) nM ml-1. Thus, the sensitivity of N-CFU-GM and LR-CFU-GM did not differ significantly from that of L-CFU. These results are not encouraging for the use of 4-HC in vitro to eliminate the residual leukemic cells from autologous bone marrow of AML patients in complete remission. The sensitivity of L-CFU was modified neither by previous cytoreductive therapy (different from cyclophosphamide) nor by the time elapsed since diagnosis of AML.

Acute Disease↗

PubMed1985

The modulation of immune complex aggregation by classical pathway-mediated reactions.

Classical pathway (CP)-triggered reactions of complement-modulated immune complex (IC) aggregation (tetanus toxoid/human anti-tetanus toxoid-IgG; ICs of equivalence) were investigated turbidimetrically during the early stages of reaction. Monospecific Fab'- or Fab-fragments (rabbit) directed against certain complement components were used to block the complement function in normal human serum (NHS). Additionally, parts of the reactions were studied using purified complement components. C1q in serum generated by the addition of EDTA as well as purified C1q were found to increase the IC aggregation. In contrast to C1q, macromolecular C1 is able to inhibit IC aggregation, whereas additional participation of C-1 INH reversed this process. The cooperation of the remaining CP proteins (C4, C2, C4bp, and I) reconstituted the inhibition capacity of the complement. Whereas C3 supported significantly inhibition, a significant influence of other effector pathway (EP) components (C5-C9) was not detectable turbidimetrically.

Antigen-Antibody Complex↗

PubMed1985

Effect of EDTA and citrate on the functional activity of the first component of complement, C1, and the C1q subcomponent.

The first component of complement, C1, is a calcium-dependent complex of the three distinct subcomponents, C1q, C1r, and C1s. Earlier observations revealed that treatment of C1 with EDTA led to a loss of hemolytic C1 activity even after recalcification. Therefore, it was of interest to study whether EDTA has an additional effect on C1 and its subcomponents, beside its chelating capacity. The chelating effect of EDTA was compared to that of citrate. It was found that treatment of C1 or C1 with EDTA followed by addition of Ca++ led to a loss of hemolytic activity up to 90%, depending on EDTA concentration. Even pretreatment of EDTA with varying amounts of Ca++ did not prevent the inactivation of C1 or C1. In contrast, after dissociation of C1 or C1 by citrate, 100% of the original C1q activity is recoverable on addition of C1q deficient serum as source of C1r and C1s. EDTA-treated serum, however, showed a concentration-dependent loss of hemolytic C1q activity, indicating an inhibitory effect of EDTA on C1q. EDTA-treated C1q, fluid phase or bound to EA, was no longer able to form an hemolytically active C1 complex by interaction with C1r and C1s.

Calcium↗

PubMed1985

Comparison of the exoproducts of gram-negative bacteria by SDS-Page.

The protein exoproducts released during exponential growth of Gram-negative bacteria were analysed and compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-Page). The following bacterial strains were tested: Escherichia coli, Klebsiella pneumoniae, Citrobacter freundii, Enterobacter cloacae, Serratia liquefaciens, Serratia rubidaea, Proteus mirabilis, Proteus vulgaris, Salmonella minnesota, Pseudomonas aeruginosa and Pseudomonas fluorescens. It is demonstrated by SDS-Page that members of one species show identical protein pattern, whereas different species show besides comparable protein bands a species characteristic pattern. All members of Enterobacteriaceae were shown to release proteins whose molecular weights fell into the following size regions: Each strain was shown to synthesize a polypeptide of molecular weight 34,000 and one or more polypeptides within the molecular weight range 25,000-29,000. This profile was shown to be clearly different from that of Pseudomonas strains where 20 or more distinct polypeptides ranging from 12,500 to 160,000 Mr were detectable.

Bacterial Proteins↗

PubMed1985

Antimicrobial activity of clavines.

The antimicrobial activity of two clavine-type ergot alkaloids (agroclavine, festuclavine), and 16 derivatives against four human pathogenic bacteria and Candida albicans was determined. It is shown that all ergolines tested with one exception exhibit antibacterial properties against one to four bacteria species. The most active compounds are 6-allyl-6-norfestuclavine (minimal inhibitory concentration (MIC) 30 micrograms/ml against Staphylococcus aureus), 1-propyl-6-norfestuclavine (MIC 60 micrograms/ml against Escherichia coli), 6-cyano-6-norfestuclavine (MIC 250 micrograms/ml against Pseudomonas aeruginosa), and 1-methyl-agroclavine (MIC 200 micrograms/ml against Proteus vulgaris). 1-Allyl-6-norfestuclavine and 1-propyl-6-norfestuclavine showed a broad action spectrum: the growth of all four bacteria species and of Candida albicans was inhibited. The most effective antifungal compounds are 1-propyl-6-norfestuclavine and 6-cyano-6-norfestuclavine (MIC 250 micrograms/ml). Three alkaloids of different structure (codeine, emetine, quinine) are inactive up to 500 micrograms/ml against the bacteria species and C. albicans. The acute toxicity (mouse) is remarkably diminished by the modifications of the natural clavines.

Animals↗

PubMed1984

Enzyme-linked immunosorbent assay for C1q in human serum by use of monoclonal antibodies.

A sandwich ELISA system has been developed for the detection of C1q in human serum. It is specific, uses monoclonal antibodies, is sensitive into the nanogram range and is rapidly performed. Therefore, it may be a helpful tool for clinical routine diagnosis, e.g., detecting abnormal C1q levels in patients with rheumatic disorders. Various combinations of poly- and monoclonal antibodies were tested in a sandwich assay. One of these combinations, in particular, resulted in a highly reproducible standard curve: C1q bound to solid-phase polyclonal anti-C1q was detected by the monoclonal antibody 242 G3. In this assay, the C1q concentration in sera of normal individuals was found to be 160 micrograms/ml (mean value of 70 normal human sera). This ELISA detected nanogram levels of C1q and gave results comparable to those obtained by haemolytic C1q titration. One nanogram of C1q corresponded to ca. 2.6 X 10(10) effective C1q molecules. With this technique, selective C1q deficient sera as well as sera from patients with rheumatoid diseases were analysed.

Antibodies↗

PubMed1984

Purification and characterization of human, guinea pig and mouse C1q by fast protein liquid chromatography (FPLC).

A simple and rapid procedure for the purification of C1q from human, guinea pig and mouse serum is described. This procedure allows the purification of C1q within one and a half days using euglobulin precipitation, chromatography on Superose 6B, followed by chromatography on Mono S by Fast Protein Liquid Chromatography (FPLC). The highly purified, hemolytically active C1q is free of any immunoglobulins. Since the purification of C1q of three different species was performed by the same purification procedure, a comparison of the subunit compositions was made under reducing and non-reducing conditions on SDS-PAGE. The yield was found to be more than 50%.

Animals↗

PubMed1984

Immunohistological differential diagnosis of inflammatory colonic diseases.

Immunohistological investigations were carried out on human colonic tissue from, I healthy mucosa, 2 slightly inflamed mucosa, 3 mucosa with ulcerative colitis, 4 mucosa with Crohn's colitis, using antibodies against immunoglobulins and complement components. All our antibodies, including F(ab')2 fragments, demonstrated a progressive increase of labelled cells from healthy mucosa through slightly inflamed mucosa to mucosa with ulcerative colitis, in contrast to a complete absence of labelled cells in cases of Crohn's disease. The results are discussed with regard to their pathogenesis and their clinical significance for the differentiation of ulcerative colitis and Crohn's colitis.

Colitis↗

PubMed1984

Electron microscopic study showing antibody-independent binding of C1q, a subcomponent of the first component of complement, to serum-sensitive salmonellae.

Effective serum-mediated killing of sensitive gram-negative bacteria requires all the complement components. In the preimmune phase the antibody-independent interaction of the first component of complement, C1, with the bacteria might be especially important. Electron microscopic studies showed that the C1 subcomponent C1q binds only to the serum-sensitive R form of Salmonella minnesota and not to the serum-resistant S form.

Cell Wall↗

PubMed1984

Characterization of C1q by monoclonal antibodies.

The effect of a purified monoclonal anti-C1q antibody (Ab 242 G3) on the function of C1q, a subcomponent of the first component of complement C1, was studied. No inhibition of purified activated C1 was observed, whereas binding of the Ab to fluid phase C1q, to C1q bound to immune complexes (EAC1q), or to serum C1 in fluid phase resulted in a dose-dependent inhibition of the hemolytic activity of C1. In contrast, when the effect of the Ab on serum C1 bound to immune complexes (EAC1) was measured, no inhibition but a dose-dependent enhancement of the hemolytic activity was obtained. The dose-response curve of the Ab-treated cell bound serum C1 was indistinguishable from that of activated C1. Isolated Fab fragments of this Ab did not cause an increase in C1 activity. After separation of the A, B, and C chains of C1q by SDS-PAGE, Ab 242 G3 reacted in immunoblotting selectively within the C chain. These data indicate that cross-linking of C1q via the C chain of C1q might lead to an internal activation of C1. One out of seven monoclonal antibodies generated against mouse macrophages (M phi was found) to recognize isolated heterologous C1q. This antibody was shown to be cytotoxic and to react in a strain independent way with mouse M phi derived from bone marrow cells as well as with M phi from the peritoneal activity. However, it did not react with mouse granulocytes, thymocytes, T- and B-lymphocytes. The hemolytic activity of fluid phase C1q was inhibited to 50% at a 2 X 10(-4) dilution of hybridoma supernatant, whereas a 100-fold higher concentration was required to inhibit C1q bound to immune complexes (EAC1q) to the same extent. It was demonstrated that this antibody recognizes the isolated globular, Fc-binding portions of the C1q molecule and react with the A and B chains. Since M phi have been shown to synthesize C1q, the Fc-recognizing subcomponent of the first component of complement, evidence was provided that endogenous C1q can serve as an Fc receptor on M phi during secretion. This was demonstrated by a dose-dependent inhibition of Fc receptor activity for EIgG by the F (ab')2 fragment of this monoclonal antibody. In a fluorescence activated cell sorter (FACS) analysis Ab 146 F (ab')2 recognizes up to 75% of unstimulated NMRI peritoneal exudate cells (PEC), 60% and 53% of cells stimulated by thioglycollate and ConA, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1984

Generation of the classical pathway C3 convertase (EAC4b2a) by proteolytic enzymes.

The formation of EAC 4b2a is a two step reaction: first, the temperature- and time-independent binding of C2 to EAC4b2a resulting in EAC4b2 , secondly, the enzymatically triggered conversion of EAC4b2 to EAC4b2a . In the classical cascade of complement activation, the generation of C3 convertase activity is triggered by the C1 esterase, C1-s, which is part of C-1. Evidence is presented that the enzymes trypsin, chymotrypsin, plasmin, and pronase are also able to activate EAC4b2 to EAC4b2a . Kinetic studies showed that the formation of C3 convertase by these enzymes was dependent on concentration, temperature, and time. The optimal conditions were found as follows: trypsin, 2 micrograms/ml (final conc.) for 8 min at 23 degrees C; chymotrypsin 165 micrograms/ml for 18 min at 23 degrees C; plasmin 0.8 units/ml for 15 min at 23 degrees C; pronase 1.25 microgram/ml for 15 min at 23 degrees C. Even under optimal (tmax) conditions the number of generated EAC4b2a differed from enzyme to enzyme: trypsin (= 100%), pronase (58.3%), chymotrypsin (47.9%), and plasmin (12.9%). The enzymes were also able to generate C3 convertase activity from C2 which was adsorbed to EAC1i4b , a C1 inactivator treated and therefore hemolytically inactive intermediate ( EAC1i4b2 ). These findings underline the biological importance of C1 esterase replacing enzymes.

Animals↗