Wolfgang Bredt

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.

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.

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.

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

The attachment protein ofMycoplasma 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 withMycoplasma pneumoniaeinfection (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.

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.

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.

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.

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.

Simulation and prevention of retrovirus--specific reactions by mycoplasmas.

From human mycosis fungoides tumor-derived cell lines,Mycoplasma hyorhinis was isolated. This mycoplasma shared the following characteristics with retroviruses: uptake of3H-uridine, but not of3H-thymidine in cell culture; banding at 1.16 g/ml sucrose density and partial shift to retrovirus core density position (≅1.24 g/ml) after detergent treatment; incorporation of3H-TMP into high molecular weight material in standard reverse transcriptase assays with the template-primer poly (A) · (dT)12. On the other hand, the specific reverse transcriptase reaction of retroviruses with poly(A) · (dT)12 and poly(C)·(dG)∼16 was almost completely abolished in the presence of the mycoplasma. Thus,M. hyorhinis may interfere with identification and isolation procedures for retroviruses.

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.