Surface components of Escherichia coli that mediate resistance to the bactericidal activities of serum and phagocytes.
Explore the source record for details and available documents.
Antigens, Bacterial↗
Biomedical subjects
D Bitter-Suermann
Publications and source records attributed to D Bitter-Suermann.
Comparison of the antibacterial effect of uroepithelial cells from healthy donors and children with asymptomatic bacteriuria.
Bacterial attachment to uroepithelial cells (UEC) and the effect of UEC on bacterial growth was investigated in 15 healthy persons and 12 patients suffering from asymptomatic bacteriuria (ABU) with recurrent urinary tract infections (UTI). Desquamated UEC and mannose-resistant Escherichia coli were co-cultivated for up to 90 min. While no difference in bacterial adherence was observed between healthy controls and patients, 33.4% of the bacteria attached to normal UEC were found to be dead under microscopic evaluation (acridine orange staining), whereas no killing effect could be observed in patients' UEC 5 min after the onset of incubation. This phenomenon was confirmed by investigating the E. coli growth rate in the presence of UEC, measured by counting bacterial colony forming units (CFU) on agar plates. While E. coli showed exponential growth in RPMI medium, the addition of normal UEC suppressed bacterial growth (P less than 0.01). UEC from patients with ABU, however, did not show this effect. It has been concluded that bacterial adhesion may initiate an epithelial defence function, present in healthy controls and lacking in ABU patients.
Adolescent↗
Biliary adenocarcinoma. Characterisation of three new human tumor cell lines.
Three human cell lines from adenocarcinomas of the extrahepatic biliary tract were established in permanent tissue culture. Mz-ChA-1 and Mz-ChA-2 were cultured from mechanically dissociated gallbladder adenocarcinoma metastases and SK-ChA-1 was grown from malignant ascites of a patient with primary adenocarcinoma of the extrahepatic biliary tree. Cell doubling times in tissue culture are 3-4 days for Mz-ChA-1 and approximately 2 days for Mz-ChA-2 and SK-ChA-1. All three tumour cell lines were successfully transplanted to nude mice, inducing progressive tumour growth. Histologically, nude mouse tumours resembled the original adenocarcinomas. In vitro formation of gland-like structures were regularly seen in Mz-ChA-1 and Mz-ChA-2 but only occasionally in SK-ChA-1. All three cell lines formed contacts through interdigitating processes with desmosomes and junctional complexes. On scanning electron microscopy, an abundance of microvilli was seen at the cell surfaces. Chromosome analyses of all three tumour cell lines showed a wide range of numerical abnormalities and presence of marker chromosomes. Mz-ChA-1 appears to be highly differentiated with cells producing mucus. Mz-ChA-2 synthesizes components of complement C2, C3 and C5, while Mz-ChA-1 and SK-ChA-1 produce only C3 in detectable quantities. In addition, Mz-ChA-2 supernatants are positive for ferritin and alpha 1-fetoprotein, but not CEA; while Mz-ChA-1 and SK-ChA-1 produce only CEA. Supernatants of all three cell lines are positive for N-acetyl neuraminic acid (NANA), phosphohexoisomerase (PHI) and LDH, and negative for alpha 2-macroglobulin, alpha 1-anti-trypsin, gamma-GT, AP, coeruloplasmin, haptoglobin and albumin. A high cloning efficiency renders these new tumour cell lines suitable for continued studies on clonal heterogeneity in malignant tumours. The establishment of these cell lines in tissue culture facilitates further studies on the biology of upper gastrointestinal tract cancer in man.
Adenocarcinoma↗
Biosynthesis of the third (C3), eighth (C8), and ninth (C9) complement components by guinea pig hepatocyte primary cultures.
In the present report guinea pig hepatocyte primary cultures were established in order to study the synthesis of the eighth (C8) and ninth (C9) complement component. As reference-protein, the third complement component (C3) was measured antigenetically and hemolytically. Synthesis of C8 and C9 was determined by means of the hemolytic activity of the culture supernatant harvested every 24 h during a 6-day incubation period in vitro. The data to be reported demonstrated that the hepatocytes are able to synthesize spontaneously and secrete C8 and C9; in their culture medium a hemolytic activity of about 15-25 X 10(8) em/10(6) cells/24 h for C8 and of 25-90 x 10(8) em/10(6) cells/24 h for C9 were found. The same hepatocyte cultures produced 2500-6000 micrograms/10(6) cells/24 h of C3. Hemolytic C3 activity was also found in the culture media. The synthesis of C8 and C9 could be reversibly inhibited by addition of 30-50 micrograms cycloheximide per ml of culture medium. The kinetics of synthesis show a slight decrease after the first day of culture and a recovery in the following days up to a rate that is two- to threefold higher than that of the first day. The data suggest that hepatocytes could contribute to the production of C8 and C9 present in the plasma.
Animals↗
NZB mouse system for production of monoclonal antibodies to weak bacterial antigens: isolation of an IgG antibody to the polysaccharide capsules of Escherichia coli K1 and group B meningococci.
A system for the production of monoclonal antibodies, particularly of the IgG type, against weakly immunogenic bacterial polysaccharide antigens is described. This system, which is based on the autoimmune NZB mouse strain, has been used to produce a monoclonal IgG2a antibody against the meningococcus group B and Escherichia coli K1 polysaccharides, identical homopolymers of alpha (2----8)-linked units of N-acetylneuraminic acid that are extremely poor immunogens. Comparison of the humoral immune responses of normal BALB/c mice and autoimmune NZB mice to hyperimmunization with group A, B, and C meningococci showed that, although both strains mounted a weak meningococcal B polysaccharide-specific IgM response, only the NZB strain mounted an IgG response. Similarly, NZB mice mounted a stronger IgG response to the more immunogenic group C meningococcal polysaccharide than did BALB/c mice, although this difference was less pronounced than that observed with meningococcal B polysaccharide. No difference between the two strains of mice was demonstrable with the strongly antigenic group A meningococcal polysaccharide. These results indicate that the NZB system may be generally useful for the production of monoclonal antibodies against weakly antigenic bacterial determinants.
Animals↗
Interaction of Ross River virus with the complement system.
In the absence of virus-specific antibody, Ross River virus failed to activate either the classical or alternative complement pathways. Instead, it inhibited the cleavage of C3 via both pathways. The virus did not appear to act by disrupting C3bBb complexes or by preventing cleavage of factor B by factor D. Instead Ross River virus was found to interfere with the actual cleavage of C3 by activated factor B (C3bBb) of the alternative pathway and C4b2a of the classical pathway.
Alphavirus↗
traT gene sequences, serum resistance and pathogenicity-related factors in clinical isolates of Escherichia coli and other gram-negative bacteria.
The R6-5 plasmid-specified outer membrane protein, TraT protein, has previously been shown to mediate resistance to bacterial killing by serum. Colony hybridization with a 700 bp DNA fragment carrying most of the traT gene was used to examine the prevalence of traT in Gram-negative bacteria, particularly strains of Escherichia coli, isolated from clinical specimens. traT was found in isolates of E. coli, Salmonella, Shigella and Klebsiella, but not in Pseudomonas, Aeromonas or Plesiomonas, nor in the few isolates of Enterobacter, Proteus, Acinetobacter, Citrobacter, Serratia or Yersinia that were examined. It was detected in a significantly higher proportion of the E. coli strains isolated from the blood of patients with bacteraemia/septicaemia or from faeces of patients with enteric infections (50-70%) than in that of strains isolated from normal faeces (20-40%). The incidence of traT in strains isolated from cases of urinary tract infections was variable. traT was found to be frequently associated with production of the K1 capsule and with the carriage of ColV plasmids, but not with the carriage of R plasmids, nor with serum resistance or the production of haemolysin.
Autoradiography↗
An in vitro system to study listericidal capacity of macrophages from separate mice: resident macrophages exhibit different activation patterns.
An in vitro system with macrophages from individual mice was established to study their listericidal capacity. Because no antibiotics were used, bacterial killing was really due to macrophages in short-term culture. To restrict the extracellular growth of bacteria, cell culture medium was changed at 1-h intervals. We demonstrated that intracellular growth of listeria in macrophage pools from untreated animals varies considerably. Obviously, preactivated macrophages are constantly present, so that the common procedure of using macrophage pools from several animals is no longer acceptable. In addition, we demonstrated that in vitro mixtures of listeria-immune macrophages of one animal with cells from untreated animals at different ratios exhibit enhanced bacterial killing above a mere additive effect. Consequently, by using macrophages from individual untreated mice, we found that cells of different animals exhibited various activation stages, although unstimulated, inbred specific-pathogen-free mice of the same age, weight, and sex were used. When equal numbers of macrophages from untreated separate animals were mixed in vitro, intracellular growth of listeria was only moderate; that is, the number of preactivated macrophages of the individual animals determined listerial growth in the pooled preparation. Furthermore, we showed that identical doses of phorbol myristate acetate exerted different effects on the listericidal activities of macrophages as a function of their preactivation states. These experiments clearly demonstrate the advantage of using macrophages from individual mice for in vitro studies of macrophage activation.
Animals↗
Monoclonal antibodies to enterobacterial common antigen and to Escherichia coli lipopolysaccharide outer core: demonstration of an antigenic determinant shared by enterobacterial common antigen and E. coli K5 capsular polysaccharide.
We established hybridoma cell lines producing monoclonal antibodies against enterobacterial common antigen (ECA) and a substructure of the outer core of different Escherichia coli lipopolysaccharides (LPSs). Anti-ECA antibodies 865 and 898 reacted with ECA in extracts of heated E. coli and with ECA-bound R1 and R4 core-containing LPS preparations, as well as with a purified sample of ECA from Salmonella montevideo. Antibody 865, but not antibody 898, cross-reacted with K5 capsular polysaccharide, suggesting that 4-linked alpha-N-acetylglucosamine is part of an antigenic determinant shared by both K5 polysaccharide and ECA. Anti-LPS antibody 786 recognized an outer core structure common to E. coli K-12, B, R2, and R4 core type LPS, but not to R1 and R3 core type LPS. Its most probable target is the trisaccharide sequence Hexp(1----2)-alpha-D -Glcp(1----3) alpha-D-Glcp----(Hepp) (where Hex is hexose, p is phosphate, Glc is glucose, and Hep is heptose), the first glucose being the immunodominant moiety. These monoclonal antibodies may be used not only for the detection of ECA, K5, and LPS core structures but also for analysis of the molecular forms resolved on polyacrylamide gels (banding patterns) of both ECA and LPS, independently of one another.
Antibodies, Monoclonal↗
Biosynthesis of enterobacterial common antigen.
Cultures of Salmonella typhimurium pulse-labeled with N-acetyl-D-[3H]glucosamine ([3H]GlcNAc) incorporated isotope into a GlcNAc-linked lipid that was tentatively identified as GlcNAc-pyrophosphorylundecaprenol. The incorporation of [3H]GlcNAc into this compound was abolished when cells were pulse-labeled in the presence of the antibiotic tunicamycin. Tunicamycin also abolished the in vivo synthesis of the haptenic form of enterobacterial common antigen (ECA) in S. typhimurium as determined by the passive hemagglutination test. These data indicated that the synthesis of the GlcNAc-linked lipid is related to ECA synthesis. Support for this conclusion was provided by the following observations. Cultures of Escherichia coli and S. typhimurium incorporated [3H]GlcNAc into cell envelope components that migrated as a homologous series of polymers when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The [3H]GlcNAc-labeled polymers were not detected in mutants of E. coli and S. typhimurium defective in ECA synthesis due to lesions in either the rfe or rff gene clusters. These polymers were identified as ECA based on Western blot analyses employing anti-ECA monoclonal antibody. The incorporation of [3H]GlcNAc into ECA polymers was abolished by tunicamycin when the drug was added to cultures to give a minimum concentration of 3 micrograms/ml. In addition, pulse-chase experiments provided evidence for a precursor-product relationship between the GlcNAc-linked lipid and ECA. These results strongly suggest that the GlcNAc-linked lipid is involved in the biosynthesis of ECA in a manner analogous to the role of carrier lipid in the biosynthesis of O-antigen and peptidoglycan.
Acetylglucosamine↗
Monoclonal antibodies against complement 3 neoantigens for detection of immune complexes and complement activation. Relationship between immune complex levels, state of C3, and numbers of receptors for C3b.
C3-bearing immune complexes and C3 activation products were detected by using two monoclonal antibodies, one specific for a neoantigenic determinant on C3c and the other for C3d. To quantitate immune complexes, the anti-C3c or anti-C3d antibodies were fixed to microtiter plates and reacted with test plasma. The binding of C3-bearing immune complexes in this plasma was then measured with radioisotope- or enzyme-labeled anti-human IgG. To test for C3 breakdown products, solid-phase monoclonal antibody to the C3d neoantigen was reacted with EDTA-plasma samples, and fixed iC3b or C3d was measured with a polyclonal anti-C3 antibody. Patients with autoimmune diseases, such as systemic lupus erythematosus, rheumatoid arthritis, and Sjogren's syndrome, and paracoccidioidomycosis were found to contain immune complexes bearing C3b/iC3b or C3d. In most conditions, there were more C3d-containing immune complexes than C3b/iC3b. Although CR1 (C3b receptors) rapidly converted immune complex-bound iC3b to C3dg/C3d and lupus patients had reduced CR1, no correlation between the state of C3 on circulating immune complexes or levels of immune complexes and CR1 numbers was seen. However, levels of C3-fixing ICs correlated with levels of C3 activation products. This assay system with monoclonal antibodies to neoantigens expressed on activated, but not native, C3 provides sensitive and specific means for detecting and classifying C3-fixing immune complexes and for assessing C3 activation.
Antibodies, Monoclonal↗
Influence of genetically inherited complement deficiencies on humoral immune response in guinea pigs.
To assess the role of complement in the induction of the humoral immune response, we studied the antibody response of guinea pigs genetically deficient in the second component of the classical complement pathway (C2D-GP) to bacteriophage phi X 174--a T cell-dependent antigen--in comparison with normal guinea pigs and C4D-GP, for which a disturbance in induction of antibody response has been described. We were able to establish a clear dose-response relationship: with low doses of antigen (1 X 10(9) PFU/kg), the antibody response of both complement-deficient strains was grossly impaired as compared with normal guinea pigs. After primary immunization, the peak antibody titer was diminished (1 log10) and declined rapidly; after secondary immunization, the diminution became even more distinct. Both complement-deficient strains had unusual secondary antibody responses almost identical to their primary ones, and amplification of antibody titer, as well as regular isotype switch from IgM to IgG, was absent. By increasing the antigen dose (2 X 10(9) PFU/kg), the antibody responses of the complement-deficient guinea pigs tend to normalize, and when high doses of antigen (1 X 10(10) PFU/kg) were used, the behavior of the complement-deficient animals was nearly indistinguishable from that of normal animals. Partial restoration of the immune response was seen when substituting the genetic complement deficiency by giving serum as source of the missing complement component. The important contribution of the C2 deficiency is given by the now compelling evidence that it is not the missing individual component itself, but rather the common block in sequential activation of C3 via the classical pathway in both complement deficiencies, that is responsible for the impaired humoral immune response, especially at low antigen doses. We therefore postulate that an intact classical pathway contributes to reaching a normal humoral immune response.
Animals↗
Cell surface antigens on the guinea-pig macrophage: identification by monoclonal antibodies and association with the activation state.
Ten monoclonal antibodies (moAb) directed against cell surface antigens of guinea-pig monocytes and macrophages (mph) were produced and characterized. The corresponding antigens are not present on granulocytes, T lymphocytes, an Ia-positive B-cell line or other haematopoietic cells. In binding or cytotoxicity assays, the moAb demonstrated characteristics patterns of reactivity, with mph being in different stages of differentiation or activation. Three moAb (342, 322, 249) recognized 'lineage antigens' (i.e. antigens continuously expressed during maturation of monocytes to mph and after stimulation or activation of the cells). MoAb 342 possibly defines a major cell surface determinant, being present on 90% of mph. The antigens detected by moAb 305, 320, 321 and 344 characterize mature mph. They were not expressed on monocytes, but were expressed on the majority of resident, elicited or activated peritoneal mph. MoAb 253, 310 or 257 defined discrete subpopulations of elicited and--with the exception of moAb 257--activated mph. The corresponding antigens were not present on monocytes or resident mph, but appeared on the cell surface during in vivo or in vitro stimulation of the cells. There was no indication of a contribution of the moAb-defined antigens to the presentation of antigen, mitogen or alloantigen by the mph to T cells. The functional significance of the antigens thus remains to be elucidated. Our studies indicate that cells committed to the monocyte/mph lineage share a family of differentiation antigens, distinguishing them from other cell lines. The moAb provide useful tools for further investigation of the activation of mph and allow the rapid detection of mph in different tissues.
Animals↗
Monoclonal antibody detection of IncF group plasmid-encoded TraT protein in clinical isolates of Escherichia coli.
The TraT protein specified by IncF group plasmids mediates surface exclusion and bacterial resistance to the lethal activities of serum. In this study, an anti-TraT protein monoclonal antibody was generated which failed to react with TraT+ bacteria but which efficiently detected solubilized TraT protein in Western blots and in an enzyme-linked immunosorbent assay. Use of this antibody to screen clinical and nonclinical isolates of Escherichia coli for the production of TraT protein revealed its presence in a modest proportion (38%) of normal fecal strains, a significantly higher proportion of clinical strains (51 to 73%), and an even higher proportion (78 to 88%) of clinical strains concomitantly producing the K1 capsule, an important virulence factor of E. coli.
Antibodies, Monoclonal↗
Release of prostaglandin E and thromboxane from macrophages by stimulation with factor H.
Recently novel actions of factor H of complement other than regulation of alternative pathway activation have been described. We examined the influence of H on the arachidonic acid (AA) metabolism of macrophages. Guinea-pig peritoneal macrophages cultured for up to 18 h under serum free conditions were supplied with homologous factor H. H, tested over a concentration range of 12.5-100 micrograms/ml, promoted an indomethacin sensitive release of prostaglandin E and thromboxane B2 in a dose-dependent manner. Stimulation of AA conversion to prostanoids in response to H was shown to be specific as evidenced by immunoabsorption experiments. This novel effect attests to the potential of H to act not only as regulatory protein of the complement pathway but also as an inducer of cellular release reactions. Moreover, these findings emphasize the close functional links that exist between the three main constituents of the inflammatory process: macrophages, the complement system and the AA cascade.
Animals↗
Expression of Ia antigens on macrophages is reduced after stimulation with homologous C3b.
The complement cleavage product C3b was examined for its ability to influence the expression of Ia antigens on macrophages. Monoclonal anti-Ia antibodies were applied for quantitation of Ia antigens by using FACS analysis. Albumin-elicited guinea pig peritoneal macrophages were cultured for 48 hr in the presence of C3b. C3b at final concentrations varying from 10 to 80 micrograms/ml reduced the proportion of Ia-positive macrophages from 87 to 25%. The C3b-stimulated loss of Ia antigens from the macrophage surface was mediated by metabolites of the cyclooxygenase pathway as shown by the inhibitory effect of indomethacin. Exposure of macrophages to C3b for 5 min was sufficient to induce Ia reduction, seen after subsequent 48-hr incubation. C3b generated in vivo in the course of inflammatory reactions might therefore influence the interaction of immunocompetent cells by modulating macrophage Ia expression.
Animals↗
Quantitative determination of complement components produced by purified hepatocytes.
In this report we describe, on a quantitative basis, the secretion of complement components by hepatocytes. Primary cultures were established after isolation of the cells from guinea-pig liver and the synthesis of C3, C5, C4 and C2 was measured. The cells were isolated by collagenase perfusion of the liver followed by differential centrifugation. The contamination of the hepatocyte suspension with non-parenchymal cells was less than 1%. At 24 h after plating the cells the kinetics of complement production were measured. C3 and C5 content in the culture medium harvested at different time intervals was determined by a sensitive ELISA. Secretion of C2 and C4 was measured haemolytically using C2 or C4 deficient guinea-pig serum. Under the conditions used hepatocytes secreted C3 at a rate of about 100 ng/10(6) cells/h with a plateau of secretion after 24 h of culture corresponding to about 350,000 molecules/cell/h. C5 secretion was detectable after 3-6 h of culture. The C5 secretion rate was about 15 ng/10(6) cells/24 h. The functional activity of C4 and C2 in the supernatants amounted to about 80 SFU/cell/h if the culture medium was changed every 3 h but dropped significantly if the medium was changed every 12 h. The decrease of the haemolytic activity became stronger if the medium was changed every 24 h. Cycloheximide reversibly inhibited the complement production. Our results show that guinea-pig hepatocytes synthesize considerably more C3 and C5 compared to peritoneal macrophages supporting the hypothesis that hepatocytes provide the major source of plasma complement.
Animals↗
Stimulation of prostaglandin E and thromboxane synthesis in macrophages by purified C3b.
Complement cleavage product C3b was examined for its effect on macrophages. Albumin-elicited guinea pig peritoneal macrophages kept in short-term culture were challenged with purified homologous C3b, and release of oxygen and arachidonic acid metabolites was studied. C3b at concentrations ranging from 20 to 80 micrograms/ml induced synthesis of large quantities of the arachidonic acid cyclooxygenation products thromboxane B2 (TXB2) and prostaglandin E (PGE) but failed to trigger an oxidative burst. Our findings link together macrophages, complement, and arachidonate metabolites as major participants in inflammatory responses.
Animals↗