Antiadhesive properties of a quaternary structure-specific hybridoma antibody against type 1 fimbriae of Escherichia coli.

The relationship between the structure and biological function of type 1 fimbriae of Escherichia coli was investigated using a set of monoclonal antibodies directed against conformation-specific antigenic determinants. Of three monoclonal antibodies tested, only one (clone CD3) prevented adhesion of the vaccine strain to epithelial cells or guinea pig erythrocytes. The antibody produced by CD3, but not that produced by the other two hybridoma clones (AA8 and GG1), precipitated isolated fimbriae by double diffusion in agar gel and was shown to bind in a highly discrete, periodic manner along the length of each of the fimbriae by immunoelectron microscopy. Immunoelectroblots of type 1 fimbrial subunits and polymers electrophoresed in SDS-gels indicated that the antibodies in AA8 and GG1 reacted only with fimbrial monomers (mol wt 17,000), whereas the antibody in CD3 reacted only with polymers of mol wt 102,000 (hexamers) or higher. ELISA inhibition assays demonstrated that dissociated fimbrial subunits lost their reactivity with antibody CD3 but gained reactivity with antibodies AA8 and GG1. Conversely, when allowed to reassemble in vitro in the presence of 5 mM MgCl2, the reassembled fimbriae lost their reactivity with antibodies AA8 and GG1 but regained reactivity with antibody CD3. These results demonstrated that certain antigenic epitopes are dependent on quaternary structural determinants, whereas others are independent of quaternary fimbrial structure and also are inaccessible for antibody binding in fimbriae once they have been assembled. These monoclonal antibodies should prove useful in studies of the structural determinants of the biological function of type 1 fimbriae as well as in studies of fimbrial synthesis, transport, and assembly.

Hybridoma antibodies against protective and nonprotective antigenic determinants of a structurally defined polypeptide fragment of streptococcal M protein.

Hybridoma technology was used to produce a set of monoclonal antibodies against a purified polypeptide fragment of type 24 streptococcal M protein to delineate the protective determinants of M protein exposed on the surface of the virulent streptococci. Several hybridoma antibodies were found to be opsonic against the homologous type streptococci. At least two of these antibodies (IIC3.7 and IIC4.6) protected mice against challenge infections with the homologous, but not a heterologous, serotype of bacteria. One of the hybridoma antibodies that reacted in high dilution (1:204,800) with the isolated M protein failed to react with the M protein on the surface of type 24 streptococci, and thus did not opsonize the homologous organisms or protect mice against challenge infections. Because hybridoma antibodies are directed against a single distinct immunodeterminant, these results indicate that protective immunity may be directed at any one of several distinct antigenic determinants of M protein exposed on the surface of virulent group A streptococci.

Freeze-fracture studies of the developing cell surface. II. Particle-free membrane blisters on glutaraldehyde-fixed corneal fibroblasts are artefacts.

We describe, in sections and by freeze-fracture, four classes of intramembrane particle (IMP)-free membrane blebs or "blisters" associated with glutaraldehyde-fixed embryonic corneal fibroblasts: (a) Single blisters attached to the cell membrane; (b) free (detached) vesicles; (c) myelin figures; (d) multivesicular protrusions which resemble the "mounds" described by others on nerve growth cones. The IMP-free, membrane-bounded blisters contain no ground cytoplasm or organelles, in contrast to blebs on trypsin-isolated fibroblasts, which we show here do contain cytoplasm and IMP-rich membranes. That the IMP-free membrane blisters in embryonic corneas are artefacts of fixation is demonstrated by (a) their absence in replicas of fibroblasts frozen and fractured without prior aldehyde fixation and (b) their absence in sections of fibroblasts fixed in a combination of glutaraldehyde and osmium tetroxide. We suggest that the addition of osmium prevents postfixation movement of membrane lipids, especially the negatively charged "fluid" lipids which others have shown are capable of considerable mobility after aldehyde fixation alone. Recent literature has implicated membrane blistering in secretory processes and in growth of nerves, but before the functional significance of such IMP-free blisters is assessed, membrane mobility of the type shown here should be taken into consideration.

Freeze-fracture studies of the developing cell surface. I. The plasmalemma of the corneal fibroblast.

The freeze-fracture technique was used to study changes in the corneal fibroblast cell membrane during morphogenesis in chick embryos. Fibroblasts migrate into the acellular primary corneal stroma on day 6 of embryogenesis, moving between the orthogonal layers of collagen fibrils which serve as their substratum. Morphometric analysis of the intramembrane particles (IMP) reveals their concentration on the P face to decrease from 756 to 534/mum2 from day 6 to day 14. After day 14, fibroblast migration and cell division cease and the stroma condenses due to dehydration, so that by day 18 all of the layers of fibroblasts are extremely flattened and the cornea has taken on its mature, transparent form. The cell membranes of the terminally differentiated, highly compacted fibroblasts are rich in IMP (1,300/MUM2, P face). In seeking to relate the particle increase to cell differentiation, we analyzed synthetic events taking place at this time, but no correlation, we analyzed synthetic events taking place at this time, but no correlation with 25SO4 or proline-3H incorporation was found. The event which seems best correlated with the doubling of P face particles between days 15 and 18 is the dehydration and condensation of the stroma, an event which is associated with cessation of both cell division and migration. Thyroxine stimulates premature condensation of the stroma, whereas thiouracil delays condensation, but neither of these treatments affects IMP concentration. Interestingly, IMP concentration on the filopodia of migrating fibroblasts is similar to that on the cell bodies, suggesting that the new membrane has the same composition as the pre-existing membrane. Observations are also presented on tight and gap junctions between fibroblasts and on the relation of extracellular matrix to the outer etched surface of the fibroblast plasmalemma.

Location of a fibronectin domain involved in newt epidermal cell migration.

The interaction of migrating newt epidermal cells with the extracellular matrix protein, fibronectin, was studied. Pieces of nitrocellulose coated with intact human plasma fibronectin or proteolytically derived fragments were implanted into wounded limbs so that the coated nitrocellulose served as wound bed for migrating epidermal cells as they attempted to form a wound epithelium. Epidermal cells migrated very poorly on nitrocellulose pieces coated with (a) a 27-kD amino-terminal heparin-binding fragment, (b) a 46-kD gelatin-binding fragment, (c) a combined 33- and 66-kD carboxy-terminal heparin-binding preparation representing peptide sequences in the A and B chains, respectively, or (d) a 31-kD carboxy-terminal fragment from the A chain, containing a free sulfhydryl group. In contrast, epidermal cells readily migrated onto nitrocellulose coated with a mixture of fragments from the middle of the molecule (80-125kD) that bind neither heparin nor gelatin. Attempts to block migration on fibronectin-coated nitrocellulose using IB10, a monoclonal antibody that blocks Chinese hamster ovary cell attachment to fibronectin, were unsuccessful despite saturation of the epitope against which IB10 is directed. In contrast, a polyclonal anti-fibronectin antibody did inhibit migration. These results show that the ability of fibronectin to support newt epidermal cell migration is not shared equally by all regions of the molecule, but is restricted to a domain in the middle third. They also suggest that the site supporting migration is separate and distinct from the site mediating Chinese hamster ovary cell attachment.

New protective antigen of group A streptococci.

It is widely believed that the surface M protein of group A streptococci is the predominant surface protein of these organisms containing opsonic epitopes. In the present study, we identified a new surface protein, distinct from M protein, that evokes protective antibodies. A type 18 M-negative mutant was found to be both resistant to phagocytosis in human blood and virulent in mice. The wild-type strain, but not the M-negative mutant, was opsonized by antisera against purified recombinant M18 protein or a synthetic peptide copying the NH2-terminus of M18. However, antisera raised against a crude pepsin extract of the M-negative mutant opsonized both strains, indicating the presence of a protective antigen in addition to type 18 M protein. This antiserum was used to identify and purify a 24-kDa protein fragment (Spa, streptococcal protective antigen) that evoked antibodies that opsonized the M18 parent and the M-negative mutant. The results of passive mouse protection tests confirmed the presence of protective epitopes within Spa. The deduced amino acid sequence of a 636-bp 5' fragment of the spa18 gene showed no homology with sequences in GenBank. These studies reveal the presence of a new protective antigen of certain strains of group A streptococci that may prove to be an important component of vaccines to prevent streptococcal infections.

Pathoadaptive mutations: gene loss and variation in bacterial pathogens.

Pathogenicity-adaptive, or pathoadaptive, mutations represent a genetic mechanism for enhancing bacterial virulence without horizontal transfer of specific virulence factors. Pathoadaptive evolution can be important within single infections and for defining the population structure of a pathogenic species.

Production and preliminary characterization of monoclonal antibodies to rat plasma fibronectin.

We have produced several monoclonal antibodies which appear to be directed against different antigenic determinants of rat plasma fibronectin. Fibronectin was purified from rat plasma by affinity chromatography on gelatin-Sepharose and arginine-Sepharose columns. Mice were immunized and hybridomas were prepared by fusing spleen cells with Sp2/0-Ag14 myeloma cells using poly(ethylene glycol). Three hybridomas (RFN1, RFN2 and RFN3) were selected for characterization. All are IgG molecules, one is IgG2a, one IgG2b and one IgG1. Titers of ascites fluids produced using these hybridomas range from 102 400 to greater than 409 600. The antibodies cross-reacted to different degrees with human fibronectin. Rat fibronectin was radioactively labeled and cleaved using human polymorphonuclear leukocyte elastase. Four major peptides, Mr approx. 160 000, 140 000, 60 000 and 30 000 were produced. Each of the hybridoma antibodies immunoprecipitated different elastase peptides. RFN1 precipitated the Mr 160 000 peptide, RFN2 precipitated the Mr 160 000 and the Mr 140 000 peptide and RFN3 precipitated the Mr 60 000 peptide as well as low molecular weight material migrating at the buffer front. These antibodies will be useful in studies of structure/function relationships of rat fibronectin.

Immunocytochemical localization of fibronectin in human fibroblast cultures using a cell surface replica technique.

The pericellular fibronectin-containing matrices of human foreskin fibroblasts cultured in ascorbate-supplemented medium were examined using surface replicas. An extensive filamentous network is present over and between adjacent cells, with a considerable amount at points of cell-to-cell contact. Indirect immunocytochemical localization of the distribution of fibronectin and procollagen type III within the matrix was done using the peroxidase-antiperoxidase (PAP) sandwich technique. The PAP molecule with the surrounding diaminobenzidine reaction product appears as a globular particle of approximately 39 nm in surface replicas. The apparent size of the marker was larger (60-80 nm) when bound to pericellular fibronectin, due presumably to the binding of more than one PAP complex to each fibronectin molecule. The immunocytochemical data suggest that fibronectin is a component of most, if not all, matrix fibrils. Some of the smallest filaments of the matrix (5-10 nm) exhibit a periodic, beaded appearance, with a repeat distance of approximately 70-100 nm. After either anti-fibronectin or anti-procollagen type III labeling, the filaments were decorated at regular 70-100 nm intervals with the globular marker. We suggest that the periodicity may be due to fibronectin molecules bound to collagen microfibrils at regular intervals. Our results demonstrate the usefulness of combined surface replica and immunocytochemical techniques for analysis of matrix components of cultured cells.

Collagen-induced arthritis in mice: synergistic effect of E. coli lipopolysaccharide bypasses epitope specificity in the induction of arthritis with monoclonal antibodies to type II collagen.

DBA/1 mice develop a chronic peripheral arthritis after immunization with type II collagen termed collagen-induced arthritis. We have localized the main arthritogenic determinants of CB11, a CNBr-generated arthritogenic fragment of chick type II collagen (CII), using 3 smaller peptide fragments of CB11 generated by endoproteinase LysC, LysC1 (CII 124-290), LysC2 (CII 291-374) and LysC3 (CII 375-402) and a panel of monoclonal antibodies (mAb) specific to CB11. MAb specific to the arthritogenic region of CB11 were also used to study the synergistic effect of E. coli lipopolysaccharide (LPS) on antibody-mediated arthritis in naive DBA/1 mice. LysC2 contained a minimum essential arthritogenic fragment of type II collagen: LysC2 induced arthritis by active immunization, also, a combination of four mAb specific to LysC2 passively transferred arthritis to naive mice. A single i.p. injection of LPS (50 micrograms/mouse) reduced the threshold values of the arthritogenic dose of mAb from 1 mg to 50 micrograms/clone per mouse, and decreased the number of mAb required for inducing arthritis from 4 to 2 clones. These observations suggest that LysC2, an 84 amino acid residue fragment, contains the main arthritogenic determinants within chick CB11. Importantly, LPS, a strong inducer of pro-inflammatory cytokines, negates the required multiple epitope specificity of autoantibodies in the passive transfer model and acts synergistically in the induction of arthritis by autoantibody.

Cytokine induction in murine bladder tissue by type 1 fimbriated Escherichia coli.

The local cytokine response to uropathogenic phenotype Escherichia coli KBC211 infection exhibits characteristics of both TH1 and TH2 profiles. Interleukin (IL)-6, MIP-2, IL-12, IL-18, and tumor necrosis factor-alpha (TNF-alpha) are expressed, but IL-4, IL-5, and IL-10 are also present at low levels. This is clearly a complex response that should be explored more fully. The relative contributions of the bladder epithelium and other cells of the bladder wall should also be determined. Epithelial cytokine responses may be considerable, and because these cells are the first to encounter the pathogen, they will be of great importance in the immune response to pathogenic E. coli.

p53 and anti-p53 antibodies as possible markers of a switch towards a neoplastic phenotype in patients infected by Helicobacter pylori.

Helicobacter pylori is a definite carcinogen whose mechanism of action is still unknown. The aim of this work was (1) to determine the presence of p53 protein and related antibodies in patients affected by various gastric pathologies and chronically infected with H. pylori, and (2) to try to discover a test to be used as a marker of a possible switch towards a neoplastic phenotype.

M protein mediated adhesion of M type 24 Streptococcus pyogenes stimulates release of interleukin-6 by HEp-2 tissue culture cells.

We investigated the contributions of lipoteichoic acid and M protein to reversible and irreversible adhesion of group A streptococci and the effects of such adhesion on release of interleukin-6. Streptococci in which lipoteichoic acid was masked by the hyaluronate capsule were readily washed from HEp-2 cells, indicating no attachment. Unencapsulated, M-negative streptococci in which lipoteichoic acid was exposed were removed more slowly, indicating loose attachment. Only unencapsulated streptococci that expressed both lipoteichoic acid and M protein remained stably adherent to HEp-2 cells throughout multiple washes. Streptococci expressing both M protein and lipoteichoic acid induced release of interleukin-6 from HEp-2 cells, whereas an isogenic, M-negative mutant failed to induce release of interleukin-6. These data suggest that lipoteichoic acid mediates reversible adhesion and that M protein is required for irreversible adhesion and for inducing release of interleukin-6 from HEp-2 cells.

Linker insertion analysis of the FimH adhesin of type 1 fimbriae in an Escherichia coli fimH-null background.

The gene encoding the Escherichia coli FimH adhesin of type 1 fimbriae has been subjected to linker insertion mutagenesis. Amino acid changes were introduced at a number of positions spanning the entire sequence in order to probe the structure-function relationship of the FimH protein. The effect of these mutations on the ability of bacteria to express a D-mannose binding phenotype was assessed in a fimH null mutant (MS4) constructed by allelic exchange in the E. coli K-12 strain PC31. Mutations mapping at amino acid residues 36, 58 and 279 of the mature FimH protein were shown to completely abolish binding to D-mannose receptors. Differences in the level of fimbriation were also observed as a result of some of the mutations in the fimH gene. These mutants may prove useful in dissecting receptor-ligand interactions by defining regions of the FimH protein that are important in erythrocyte binding.

Chlamydia pneumoniae infections prevent the programmed cell death on THP-1 cell line.

Chlamydia pneumoniae is an obligate intracellular bacterium which frequently causes airway infection in humans and has been implicated in chronic inflammatory disease and atherosclerosis. Here we show that infection with C. pneumoniae protects THP-1 cells against the apoptosis which spontaneously occurs in macrophages in the absence of an activation signal. Analysis by flow cytometry at different post-infection times revealed that 50+/-7% of THP-1 cells were apoptotic at 48 h after onset of the experiments, whereas C. pneumoniae-infected cultures (multiplicity of infection, MOI=30) displayed only 18+/-4% of cells in apoptosis. At MOI=20 and MOI=10 the cells susceptible to apoptosis at 48 h were 28+/-5% and 35+/-6% respectively. Moreover, the results show that heat-inactivated bacteria do not give significant protection against apoptosis, even at higher MOI (MOI=30), while UV-treated Chlamydia did provide a degree of protection against apoptosis. These data suggest that the anti-apoptotic effect of C. pneumoniae requires a heat-labile component released during infection, and that the effect is not lipopolysaccharide-dependent.

Commonly used antibiotics induce expression of Hsp 27 and Hsp 60 and protect human lymphocytes from apoptosis.

Heat shock proteins (Hsps) are abundant molecular chaperones participating in the cytoprotection. The kinetics of synthesis of Hsps closely correlates with the kinetics of development of resistance to cell death. In this study, we analysed the probable involvement of Hsp 27 and Hsp 60 in the protection of cells undergoing apoptosis. Human lymphocytes cultured in the presence of ampicillin or ceftriaxone produced Hsp 60 and Hsp 27, estimated by immunoblotting in a time-dependent manner and the increased levels of Hsp 60 and Hsp 27 correlated with enhanced resistance of the lymphocytes to apoptosis, as determined by flow cytometry. Cultures treated with ampicillin or ceftriaxone also exhibited smaller numbers of apoptotic cells than untreated cultures when exposed to the apoptosis-inducing agent staurosporine (1 mM). In contrast, cloramphenicol induced the production of only small amounts of Hsp 60, and no resistance apoptosis. Further studies are needed to clarify the potential role of Hsp 27 and Hsp 60 in the resistance of human cells to apoptosis and the effects of antibiotics on this phenomenon.