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W Bredt

Publications and source records attributed to W Bredt.

At least 37 records · Page 2Linked to original sources
PubMed1987

Use of adherence protein of Mycoplasma pneumoniae as antigen for enzyme-linked immunosorbent assay (ELISA).

Antibodies against the adherence protein of Mycoplasma pneumoniae are regularly found in patients with M. pneumoniae infection. Therefore, this 168-kilodalton (kDa) protein was used as an antigen in a dot-ELISA for serological diagnosis of M. pneumoniae disease. M. pneumoniae proteins were separated by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), gels were stained with Coomassie Blue, and the 168-kDa protein band was cut out and eluted using a special electroelution device. Isolated proteins or sonicated whole-cell antigens, respectively, were immobilized on a 96-well filtration plate with a nitrocellulose bottom (dot-ELISA). The test procedure was performed as in conventional ELISA tests, using alkaline phosphatase-labeled antihuman IgM or IgG antibodies, respectively, to detect antigen-antibody complexes. All results were confirmed by immunoblotting. The dot-ELISA using the 168-kDa antigen proved to be sensitive and specific. The specificity was tested on 53 sera of M. pneumoniae infections and on 490 serum specimens of patients with other respiratory diseases due to other pathogens, or with clinical conditions such as pancreatitis, meningitis or endocarditis. With regard to IgM antibodies, no false-positive reactions were found in non-M. pneumoniae diseases against the 168-kDa antigen, but there were such reactions against other M. pneumoniae proteins in immunoblots.

Adhesins, Bacterial↗
PubMed1986

A 168-kilodalton protein of Mycoplasma pneumoniae used as antigen in a dot enzyme-linked immunosorbent assay.

The attachment protein of Mycoplasma pneumoniae (molecular weight 168 kd) was used as antigen in a special enzyme-linked immunosorbent assay (dot ELISA) and compared with a sonicate of the whole organism. In control sera the intensity of the 168-kd band on immunoblots correlated well with the ELISA IgG values derived from isolated protein. The diagnostic significance of the 168-kd antigen was tested on paired sera from 33 patients with Mycoplasma pneumoniae infection (24 children, 9 adults). The ELISA values with the isolated protein were slightly lower than with cell antigen, but the protein also showed a lower basic activity in controls. In first sera of specimens of children collected within the first week of infection the 168-kd IgM response was more distinct than that to the whole cell antigen. Similarly the IgG response to the purified protein antigen differed significantly from the controls already in the first serum. In sera of adult patients the increased levels of IgG antibody were more evident with the 168-kd protein antigen. Use of the protein 168 kd as antigen increased the sensitivity of the ELISA for detecting early stages of disease, especially in children.

Adolescent↗
PubMed1986

Reaction pattern of human anti-Mycoplasma pneumoniae antibodies in enzyme-linked immunosorbent assays and immunoblotting.

In serological diagnosis of Mycoplasma pneumoniae disease the frequently used complement fixation test is based on a cross-reacting glycolipid. Recently enzyme-linked immunoassays have been developed to overcome this lack of specificity. To study the involvement of the various proteins and the influence of age on the level of antiprotein antibodies present, we investigated by enzyme-linked immunoassays (immunoglobulin M [IgM] and IgG) and immunoblotting the sera of healthy persons of different age groups as well as sera of patients (including paired sera) with M. pneumoniae infection. In sera of children with nonrespiratory diseases and in healthy blood donors the IgM antibodies rose during the first 2 years of life to a relatively constant background level (optical density at 405 nm of 0.15 to 0.21). In contrast IgG remained low up to the seventh year and then increased to moderate levels (optical density of 0.15). The blotting patterns showed few IgM bands in the age group of 20 to 30 years. IgG blots revealed, up to 7 years, only very few reactions with a 168-kilodalton protein, but in higher age groups a considerable number of reactions with proteins of 193, 168, 84, 69, and 56 kilodaltons were detected. In the sera of patients, positive IgM blots were most numerous in the third week, whereas the number of IgG blots increased up to the fourth and the fifth week. At this time all sera contained antibodies against the 168-kilodalton protein, which is identical with the adhesin of M. pneumoniae. In a patient with acute infection, who had a high preinfection IgG level, no IgM response developed. The data indicate a relatively high background of antibodies against M. pneumoniae proteins in older age groups, suggesting a requirement for paired sera. Furthermore, reinfections of adults may occur without a concomitant IgM response.

Adult↗
PubMed1985

Adherence inhibition assay: a specific serological test for detection of antibodies to Mycoplasma pneumoniae.

Antibodies directed against the adherence-mediating protein of Mycoplasma pneumoniae were measured by an adherence inhibition assay. Pretreatment with antibody-containing sera reduced the attachment of sheep erythrocytes to Mycoplasma pneumoniae layers grown in flat-bottom microtiter plates. The degree of attachment of erythrocytes was estimated by lysis with distilled water and measurement of absorbance in a microtiter reader. Sera tested included 126 sera from patients with suspected Mycoplasma pneumoniae respiratory infection and 60 sera from patients with serologically confirmed respiratory infection of other origin. Examination of human sera by both complement fixation using glycolipid as antigen, and the adherence inhibition assay indicated a high degree of specificity of the latter. Furthermore, titer increases parallel to complement fixation titers were found in six paired sera, and testing of separated IgM showed high reactivity in this immunoglobulin fraction. The adherence inhibition assay is a reproducible method which is relatively easy to perform. It may be of importance especially when a non-specific complement fixation reaction due to cross-reaction is suspected.

Animals↗
PubMed1985

Mycoplasma pneumoniae induces cytotoxic activity in guinea pig bronchoalveolar cells.

Precultured guinea pig alveolar macrophages (AM) and freshly harvested alveolar cells (FHAC) activated by interaction with Mycoplasma pneumoniae were cytotoxic for xenogeneic 75selenomethionine-labeled tumor target cells. Phagocytosis of whole opsonized or nonopsonized M. pneumoniae cells was more effective in eliciting cytotoxicity than uptake of sonicated microorganisms. The addition of living mycoplasma cells to the assay system enhanced the cytotoxic effect considerably. Target cells were significantly more susceptible to the cytotoxic action of phagocytes if they were coated with mycoplasma antigen or cocultured together with M. pneumoniae. The activation of the phagocytes could be inhibited by 2-deoxy-D-glucose but not by antimicrobial substances suppressing mycoplasma protein synthesis. It was accompanied by 51Cr release without detectable signs of cell damage. The supernatants of activated cells were cytotoxic for approximately 24 h. Inhibition, release, and cytotoxic activity indicate the necessity of an intact metabolism of the effector cells and suggest a secretion of cytotoxic substances.

Animals↗
PubMed1984

Effect of monoclonal antibodies to the attachment-tip on experimental Mycoplasma pneumoniae infection of hamsters. A preliminary report.

Mycoplasma pneumoniae, an important human pathogen, is an extra-cellular parasite, colonizing mucosal surfaces. Attachment to epithelial cells of the host is therefore an important mechanism of pathogenicity, and inhibition of adhesion might protect the host. M. pneumoniae predominantly adheres with a special organelle, the attachment-tip, to host cells. In vitro studies confirmed the observation that monoclonal antibodies (MOAB) to the tip inhibited adherence to erythrocytes. In animal experiments, high numbers of virulent M. pneumoniae exposed for 4 h at 37 C to MOAB and kept in suspension with MOAB 1:100 were inoculated intranasally into hamsters. A significant reduction in the lung lesion score, but not in the numbers of organisms in lung tissue or wash fluid of the upper respiratory tract, was seen in hamsters 14 days after inoculation of MOAB-treated organisms, as compared with controls. These observations, although preliminary, may have implications for the understanding of pathogenesis and for vaccine development.

Adhesins, Bacterial↗
PubMed1983

Analysis of polypeptides of mutants of Mycoplasma pneumoniae that lack the ability to haemadsorb.

Haemadsorption-negative mutants of Mycoplasma pneumoniae were isolated which varied in their capacity to adsorb erythrocytes of various animal species suggesting adherence to erythrocytes is mediated by different binding mechanisms. Trypsin treatment of the wild-type strain resulted in loss of haemadsorbing activity; several polypeptides, some of which regenerated with haemadsorbing activity following further incubation, were also trypsin sensitive. The haemadsorption-negative mutants could be divided into two groups according to their polypeptide pattern. In the first group (11 mutants) the PAGE pattern was identical to that of the wild-type strain. The second group comprised 7 mutants which differed from the wild-type by lack of one or more polypeptides with molecular weights of 190 000, 90 000 or 40 000. During growth attachment to glass was weak or absent in the mutants. Surface hydrophobicity as measured by hydrophobic-interaction chromatography was nearly comparable in mutants and parent strain.

Adhesiveness↗
PubMed1983

Influence of cell shape and surface charge on attachment of Mycoplasma pneumoniae to glass surfaces.

Attachment of Mycoplasma pneumoniae to glass is reduced in the presence of protein, and fatty acid-free bovine serum albumin is more effective than Cohn fraction V. Cultures in the early log phase (pH 7.45 to 7.25) and cultures in the stationary or decline phase (pH 6.9 to 6.4) were more sensitive to this inhibiting effect of protein-containing buffer. Treatment of the glass surface with bovine serum albumin, concanavalin A, or polylysine reduced attachment of the mycoplasma cells. The inhibiting effects of both proteins in buffer or on the glass surface could be overcome by the addition of glucose. Modification of the mycoplasma surface charge by blocking of carboxyl groups or neutralization of ionic lipids by tetracaine altered the attachment level, whereas fibronectin and its corresponding antiserum were without effect. The results suggest that the mycoplasma interaction with glass is a complex multifactorial process. In protein-free buffer both hydrophobic and electrostatic forces are involved; in protein-containing fluid, other factors seem to be involved. The energy required for this type of attachment could be necessary for maintenance of cell shape or synthesis of polypeptides.

Adhesiveness↗
PubMed1982

Adherence of mycoplasmas: phenomena and possible role in the pathogenesis of disease.

Mycoplasma pneumoniae attaches to a variety of surfaces. Adherence to inert surfaces such as glass requires an intact energy metabolism. Interaction with sheep erythrocytes occurs via a binding protein on the mycoplasma surface. The protein reacts with a receptor containing sialic acid. Adherence to other erythrocytes may involve different mechanisms. Different results have been reported on interaction with tissue cells. The various mechanisms probably cooperate and thereby facilitate the colonization of the human respiratory tract.

Adhesiveness↗
PubMed1982

Release of Mycoplasma pneumoniae substances after phagocytosis by guinea pig alveolar macrophages.

Antibody-opsonized Mycoplasma pneumoniae cells with various radioactive markers were sedimented onto monolayers of guinea pig alveolar macrophages (AM). After 2 h of incubation, about 50% of the activity of [3H]palmitate-labeled mycoplasmas was associated with AM. Nonspecific attachment of the opsonized mycoplasmas to AM-free plastic surface areas was negligible. The occurrence of phagocytosis was proven by electron microscopy and monitoring of AM surface-bound antigen by 125I-labeled F(ab)2 fragments. The activity of [3H]palmitic acid-labeled mycoplasmas was only slowly released into the supernatant. About 55% of the activity remained AM-associated up to 70 h after phagocytosis. After phagocytosis of [3H]thymidine-labeled cells, about 70% of the radioactivity found non-precipitable by trichloracetic acid. 3H-amino acid-labeled protein was released to 50% within 8 h. Supernatants and AM were tested for M. pneumoniae antigen with enzyme-linked immunosorbent assay. Considerable amounts of antigenically active material could be found in the supernatant within 8 h. This antigen was totally inactivated by heat (80 degrees C). Trypsin treatment (1 mg/ml, 10 min) reduced the antigenicity by 80%. The results suggest a selective release of microbial material after phagocytosis.

Animals↗
PubMed1982

Effects of ionophores and dicyclohexylcarbodiimide on Mycoplasma gallisepticum adherence to erythrocytes.

To test the influence of the electrochemical ion gradient across mycoplasma membranes on the capacity of organisms to adhere to host cells, Mycoplasma gallisepticum cells were treated with valinomycin, carbonylcyanide m-chlorophenylhydrazone, and N,N'-dicyclohexylcarbodiimide (DCCD) singly or in combination. Uptake of [3H]tetraphenylphosphonium by the treated cells was employed as a measure of the effects of the ionophores on membrane potential. In the absence of K+, valinomycin increased, whereas carbonylcyanide m-chlorophenylhydrazone, and DCCD decreased [3H]tetraphenylphosphonium uptake. However, with a high level of K+ or with DCCD, uptake of [3H]tetraphenylphosphonium in the presence of valinomycin decreased below control levels, indicating that, generally, the ionophores affected membrane potential in the expected manner. The treated organisms were tested for their capacity to attach to glutaraldehyde-fixed human erythrocytes. DCCD was the best inhibitor of mycoplasma attachment, and in combination with valinomycin attachment, capacity decreased by about 40%. The combination of valinomycin plus carbonylcyanide m-chlorophenylhydrazone was less effective; it decreased attachment by about 15 to 25%. It was concluded that the dissipation of ion gradients across cell membranes decreases only partially mycoplasma adherence, in line with previous findings that isolated mycoplasma membranes retain the major part of the attachment capacity of intact cells.

Adhesiveness↗
PubMed1981

Adhesion of mycoplasmas to eukaryotic cells.

Many pathogenic mycoplasmas are surface parasites, adhering to the epithelial linings of the respiratory and urogenital tracts. Since mycoplasmas lack cell walls their plasma membrane comes in close contact with that of their host, allowing exchange of components between the two membranes and possibly fusion. The tight association of the parasite with its host is illustrated in scanning electron micrographs of Mycoplasma pneumoniae and M. gallisepticum adhering to human red blood cells. Specialized structure at the tips of the mycoplasma cells appear to function as attachment organelles. Our main aim has been to chemically define the receptors on the host cell and the binding sites on the mycoplasma cells responsible for adhesion. Glycophorin (the major sialoglycoprotein of human red blood cells) serves as the main or sole receptor for M. gallisepticum whereas M. pneumoniae binds to additional receptors on human red blood cells. Trypsin treatment of M. pneumoniae cells abolishes their ability to attach to human red cells, suggesting the protein nature of the binding sites. M. pneumoniae membranes solubilized by detergents were subjected to affinity chromatography on glycophorin-Sepharose so that membrane components with high affinity for glycophorin could be isolated. The fraction isolated consisted of several proteins (relative molecular mass 25 000 and 45 000). The binding of this fraction to red cells was relatively low but appeared to be specific, as it was inhibited by glycophorin but not by its hydrophobic moiety. The possibility is discussed that the exposure of the binding sites on the mycoplasma cell surface is influenced by the electrochemical ion gradient across the membrane.

Adhesiveness↗
PubMed1981

[Chlamydial disease of the eye: a report on 27 cases. Clinical picture, differential diagnosis, treatment (author's transl)].

During the past five years a presumptive diagnosis of ocular chlamydial disease has been made in 90 patients consulting the outpatient department of Freiburg University Eye Clinic. The diagnosis was confirmed by different methods (giemsa stains, chlamydia cultures and serology, electron microscopy and histology) in 27 cases. The clinical spectrum included inclusion blennorrhea in newborns as well as inclusion conjunctivitis, TRIC keratoconjunctivitis and sporadic trachoma in adults. Presumably the incidence of the disease is much higher than indicated by our figures. We suspect a very high number of undiagnosed cases. These patients usually do not receive proper therapy (tetracyclin or erythromycin for at least three weeks) and their disease may run a protracted course with the risk of permanent eye damage.

Adolescent↗