[Bartonella (Rochalimaea) infections: cat-scratch disease and bacillary angiomatosis].
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AIDS-Related Opportunistic Infections↗
Biomedical subjects
W Bredt
Publications and source records attributed to W Bredt.
Comparison of serological tests for the detection of antibodies against Chlamydia trachomatis and Chlamydia pneumoniae in rheumatological patients.
In cases of reactive arthritis, a suspected Chlamydia trachomatis infection is often detected by serological methods. However, mostly tests with genus-specific antigens are used, neglecting the fact that antibodies against Chlamydia pneumoniae are highly prevalent in the adult population. Therefore we tested sera of 129 patients with various rheumatological disorders and of 18 healthy persons in parallel with a genus-specific test (IPAZYME) and with the species-specific microimmunofluorescence test for C. trachomatis and C. pneumoniae antibodies. The data showed that 55% of the 64 IPA-positive results were caused by antibodies (IgG) against Chlamydia pneumoniae, only 6% by anti-Chlamydia trachomatis IgG and 20% by both specificities. For IgA antibodies, the percentages were 44%, 12.5% and 12.5% respectively. In 12 IPA-positive cases, the MIF showed no reaction. 58% of all 147 sera tested with MIF had IgG antibodies against C. pneumoniae, 5% had anti-C. trachomatis IgG and 8% IgG against both species. The percentages for IgA were 29%, 2% and 2%, respectively. IgM positivity in MIF disappeared after absorption with rheumatoid factor absorbent. No significant differences were found between the various groups of patients. The data suggest that due to the high prevalence of anti-C. pneumoniae antibody, genus-species tests cannot be used as screening tests for the serological diagnosis of C. trachomatis infections.
Adolescent↗
Comparison of host responses after intranasal infection of guinea-pigs with Mycoplasma genitalium or with Mycoplasma pneumoniae.
Guinea-pigs were infected intranasally with Mycoplasma genitalium or Mycoplasma pneumoniae. The lung lesions produced by the two mycoplasmas were comparable in extent and histological pattern. Sera of both animal groups taken 2 weeks after infection reacted strongly in the complement fixation test with the M. pneumoniae glycolipid extract. In an ELISA using the respective adherence proteins (P1-protein of M. pneumoniae and MgPa of M. genitalium), strong specific activity, but also considerable cross-reactions were found. Epitope analysis by using overlapping octapeptides of a P1-region immunologically active in human M. pneumoniae infections and of the corresponding MgPa-region revealed six common epitopes but also one M. genitalium and two M. pneumoniae specific determinants. For analysis of a possible pathogenicity of M. genitalium in the human respiratory tract species-specific tests have to be developed.
Administration, Intranasal↗
Effects of growth phase and antibiotics on quantitative DNA/RNA hybridization.
Synthetic probes complementary to ribosomal RNA are increasingly used in the detection of bacteria. Many applications, however, require the quantitation of bacteria. We therefore tested the influence of growth phase and representative antibiotics (ampicillin, chloramphenicol and gentamycin) on the outcome of DNA/RNA filter hybridization using radiolabelled probes and a multisample digital autoradiograph for quantitative monitoring. Hybridization efficiency seemed influenced by the binding capacity of the membrane, availability of target molecules and physiological growth control. For chloramphenicol the absolute hybridization signal remained constant over the experimental period. Only a slight decrease was found in experiments with gentamycin whereas viable counts dropped 10,000-fold. For ampicillin a decrease in viable counts was paralleled by diminishing signal strengths. Relative signal strengths (counts per viable cell) increased in all experiments with antibiotics. In conclusion; (i) RNA probes seem to detect bacteria even after onset of antimicrobial therapy; (ii) DNA/RNA filter hybridization appears not suitable for accurate quantitation of bacteria.
Anti-Bacterial Agents↗
Immunodominant epitopes of the adhesin of Mycoplasma pneumoniae.
Overlapping octapeptides from the amino acid sequence of the adherence protein of Mycoplasma pneumoniae were synthesized and used as the antigen in an enzyme-linked immunosorbent assay with serum samples from M. pneumoniae-infected patients. Of a sequence of 338 amino acids positioned between leucine 801 and leucine 1139, only two regions with immunodominant continuous epitopes were detected. The immunoglobulin G and immunoglobulin M antibodies of child and adult patients reacted especially with the NH2-(810)-W-I-G-N-G-Y-R-Y peptide but also reacted with the NH2-(1124)-F-T-D-F-V-K-P-R peptide.
Adhesins, Bacterial↗
Binding sites of attachment-inhibiting monoclonal antibodies and antibodies from patients on peptide fragments of the Mycoplasma pneumoniae adhesin.
The adherence protein (P1 protein) of Mycoplasma pneumoniae was purified by electroelution and cleaved with cyanogen bromide. The resulting peptides were separated by two-dimensional electrophoresis. Spots reacting in Western immunoblots with two attachment-inhibiting monoclonal antibodies were isolated, and the amino-terminal ends of these peptides were microsequenced. The two monoclonal antibodies had different binding sites. One was associated with the amino-terminal region of the whole P1 protein beginning at amino acid position 237, and the other was associated with amino acid position 702, which was localized approximately in the middle of the P1 amino acid sequence. Serum samples from three M. pneumoniae-infected patients were tested by Western blotting against the cyanogen bromide peptide pattern. All three serum samples reacted with peptide fragments beginning at amino acid position 702, but the serum of only one patient also had antibodies against the oligopeptides beginning at amino acid position 237. These results indicate that the corresponding epitopes of the P1 protein are also immunogenic if they are presented at the surface of the infecting organism.
Adhesins, Bacterial↗
Host reactions to Mycoplasma pneumoniae infections in guinea-pigs preimmunized systemically with the adhesin of this pathogen.
Guinea-pigs developed systemic and local humoral responses after intraperitoneal immunization with the isolated adhesin (168 kDa protein) of Mycoplasma pneumoniae cells. Hilar lymphocytes of these animals showed proliferation after in vitro stimulation with the 168 kDa protein or sonicated M. pneumoniae whole cell antigen. Animals preimmunized and subsequently infected with M. pneumoniae showed increased M. pneumoniae-specific IgG, IgA and adherence inhibiting antibody activities. Nevertheless these animals developed severe lung lesions of lympho-histiocyte infiltrations. Furthermore hilar lymph nodes were depleted of immunocompetent lymphocytes, suggesting a cell transfer of specific stimulable lymphocytes to the inflammation sites.
Adhesins, Bacterial↗
Antibody response of patients against a 120 kDa surface protein of Campylobacter pylori.
Campylobacter pylori strains were isolated and serum samples were obtained from 63 patients. Immunoblots of 52 patients sera using their own isolates as antigen showed a 120 kDa band, which was missing in the other 11 isolates and the respective sera. This band was not detected in other Campylobacter species. Effects of trypsin treatment of bacteria and absorption of sera by live organisms suggest a C. pylori-specific surface protein.
Antibodies, Bacterial↗
Immunological reaction of guinea-pigs following intranasal Mycoplasma pneumoniae infection and immunization with the 168 kDa adherence protein.
Humoral responses to Mycoplasma pneumoniae proteins, especially the 168 kDa protein, were demonstrated by Western blotting in sera and bronchial washings of all groups of infected or immunized guinea-pigs. However, infection was not prevented by these local and systemic antibodies. Hilar lymphocytes of infected and immunized guinea-pigs were stimulated in vitro by sonicated M. pneumoniae antigen and by the 168 kDa protein. Stimulation was significantly lower in animals which had been infected twice or had been preimmunized and challenged by infection. Histologically the most severe lesions were seen in the twice-infected group followed by the preimmunized group which was subsequently infected.
Animals↗
Isolation of the adherence protein of Mycoplasma pneumoniae by fractionated solubilization and size exclusion chromatography.
The 168-kDa adherence protein of M. pneumoniae was solubilized and purified to homogeneity. Optimal yield was obtained by pretreatment of whole M. pneumoniae cells with buffer containing 1% Chaps and subsequent extraction with octylglucosid at a detergent to protein ratio of 5 and at octylglycoside concentrations between 1.5 and 2%. Contaminating membrane proteins with high molecular masses were removed by pretreatment with 1% Chaps and proteins of low molecular masses by size exclusion chromatography.
Adhesins, Bacterial↗
Antibodies in the sera of Mycoplasma pneumoniae-infected patients against proteins of Mycoplasma genitalium and other mycoplasmas of man.
Frequency and pattern of anti-Mycoplasma genitalium antibodies in sera of 50 patients with Mycoplasma pneumoniae-infection were examined by the Western immunoblot method. The sera reacted with several proteins of M. genitalium. However, during the course of infection there was only a moderate increase of antibodies mainly against the bands of 135 and 105 kd in contrast to the more intense increase of numerous bands of M. pneumoniae. If antibodies against the 168 kd-adhesin of M. pneumoniae were isolated by affinity chromatography, only the IgG-, but not the IgM-fraction reacted with the 135 kd protein of M. genitalium. Results with rabbit antisera supported these findings, additionally indicating a lack of substantial cross reactions between M. pneumoniae and other species except M. genitalium. The development of anti M. genitalium antibodies at early age and their relatively constant pattern suggest an early immunization by so far unknown cross reacting microbial antigens. Antigenic cross reactions between proteins of M. pneumoniae and M. genitalium apparently do not play a substantial role in the diagnostic serology of M. pneumoniae disease.
Adult↗
Amino acid sequence and antigenicity of the amino-terminus of the 168 kDa adherence protein of Mycoplasma pneumoniae.
The amino-terminal end of the 168 kDa adherence protein from the membrane of Mycoplasma pneumoniae was sequenced up to 12 amino acids. A synthetic peptide containing nine amino acids of this sequence was used to study the antigenicity of the amino-terminus of the 168 kDa protein and the involvement of the homologous sequence of the protein in the adherence process. The synthetic peptide when coupled to ovalbumin was immunogenic in rabbits. Antibodies against this peptide epitope could be demonstrated in sera taken during natural M. pneumoniae infection in humans. The structural domain of the 168 kDa protein homologous with the synthetic peptide did not appear to be involved in adherence, as the synthetic peptide or its homologous antibody failed to inhibit adherence of M. pneumoniae.
Amino Acid Sequence↗
[Epidemiologic significance and diagnosis of chlamydias].
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Chlamydia Infections↗
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↗
Commercial kit for preliminary identification of Staphylococcus saprophyticus in urine.
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Bacteriuria↗
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↗
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↗
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↗