Enno Jacobs

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.

Seroepidemiological study of the immune response toCampylobacter pylori in potential risk groups

To gain more understanding of the epidemiology ofCampylobacter pylori infection, the immune response to the organism was studied in the following selected potential risk groups: endoscopy staff (n=45), dental staff (n=58), orphanage children (n=24), psychiatric patients (n=58), and family contacts ofCampylobacter pylori-infected patients (n=55). The frequency of an IgG and IgA antibody response in the different groups was determined by the immunoblot method and compared with that in an appropriate control group (n=189). The frequency of a positive antibody response was dependent on age (p<0.0001) but not on sex. When results were corrected for age by logistic regression analysis, all groups, with the exception of dental staff and orphanage children, revealed a significantly raised frequency of an IgA and combined IgG/IgA immune response compared to controls. There was not a significant difference for the IgG response, except in orphanage children. It is concluded that endoscopy staff, family contacts ofCampylobacter pylori-infected patients and people living in closed communities such as psychiatric patients and orphanage children must be considered as risk groups forCampylobacter pylori infection. The findings support the notion that person-to-person spread and a common source are the predominant modes of transmission ofCampylobacter pylori in addition to endoscopes.

Identification of Chlamydia pneumoniae-specific protein antigens in immunoblots.

The immunoblot patterns of 248 sera, all examined previously by the microimmunofluorescence test (MIF) for species-specificChlamydia antibodies, were analyzed. Predominant specific antibody activity was directed to the 54 kDa protein ofChlamydia pneumoniae, which was recognized by 93 % of sera positive forChlamydia pneumoniae by MIF but by only 2 % of sera positive forChlamydia trachomatis and negative forChlamydia pneumoniae and by 3 % of sera negative for bothChlamydia pneumoniae andChlamydia trachomatis. This antigen appears to be specific forChlamydia pneumoniae. OtherChlamydia pneumoniae-specific protein antigens were recognized far less frequently. Absorption analysis indicated that the 54 kDa protein is located on the surface of theChlamydia pneumoniae elementary bodies.

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.

Characterization of the cellular response of spleen cells in BALB/c mice inoculated with Mycoplasma pneumoniae or the P1 protein

BALB/c mice were intranasally infected or intraperitoneally inoculated with Mycoplasma pneumoniae whole cells or were immunized with the isolated adhesin (P1 protein). Spleen cells were isolated and tested in vitro for proliferation activity after stimulation with the P1 protein and sonicated M. pneumoniae whole antigen preparations. In frequency analysis experiments the P1 protein-specific proliferative response of spleen lymphocytes increased from 1/11494 in mice immunized once to 1/3246 in eightfold-inoculated mice, demonstrating that the P1 protein is a prominent immunogen of M. pneumoniae cells. Depletion experiments showed that T and B cells are activated in a 2∶1 relation. Fluorescence-activated cells sorting analysis revealed a shift of the CD4/CD8 ratio from 2∶1 in control mice up to 3∶1 in M. pneumoniae-, and to 3.4∶1 in P1 protein-immunized mice, as well as an increase in interleukin 2 receptor-bearing cells and macrophage cell populations. The results indicate that this animal model is appropriate to study host-M. pneumoniae interactions and vaccination schedules.

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.