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Objective: To determine the kinetic isotypic serum and secretory immune response to Salmonella enteritidis in a cohort of individuals exposed to the organism in a single food source outbreak of dysentery. To determine the clinical outcome and immunogenetics of the exposed cohort and to correlate these features with the immune response., Methods: Following a single point source outbreak of Salmonella enteritidis, a cohort of dysenteric individuals were ascertained using a reactive arthritis screening questionnaire (QUEST). Serum and stimulated saliva samples were obtained at 6, 12, and 24 months following the outbreak of dysentery; examinations were conducted at the same time. Two unexposed control groups were ascertained: (1) general rheumatology clinic patients and (2) well nonarthritic family practice patients. An ELISA to determine quantitative IgA responses to Salmonella enteritidis lipopolysaccharide (LPS) was performed., Results: Eleven of the 84 exposed individuals with dysentery developed reactive arthritis (ReA) of reactive enthesitis (ReE). There was a prolonged salivary IgA anti-LPS response in both the ReA/ReE and DYS (dysentery alone) patients compared with unexposed controls. A ratio of salivary IgA anti-LPS/serum IgA anti-LPS > 1 was associated with a good outcome (remission) of ReA, whereas a ratio < 1 was associated with chronic disease., Conclusions: There is a more prolonged humoral immune response to Salmonella LPS in exposed individuals than hitherto described. A risk factor in the prolongation of ReA is the inability to mount an appropriate specific salivary (secretory) immune response.

An antiviral activity-neutralizing monoclonal antibody (mAb), MIF3037, was developed by the immunization of BALB/c mice with recombinant human interferon-gamma (rhuIFN-gamma). Its neutralizing activity suggests that its epitope may be at or adjacent to a functional domain on the huIFN-gamma. MIF3037 was compared with representative mAb of previously identified epitope-specific groups in a competitive binding assay. In an attempt to determine if there are other functional epitopes recognized by mAb developed with different preparations of huIFN-gamma or different hybridoma screening methods, 14 additional mAb contributed by five other laboratories were similarly analysed. Based on their ability to bind to huIFN-gamma, all the neutralizing mAb except MIF3037 may be classified into three previously defined groups: E1, E2 and E1/E2. Monoclonal antibodies of the E1 group do not compete with those of the E2 group for huIFN-gamma binding, indicating that the E1 and E2 epitopes are distinct domains on the huIFN-gamma important for the antiviral function. Monoclonal antibodies of the E1/E2 group compete with some of the mAb of E1 and/or E2 groups and may bind to regions of the huIFN-gamma that partially overlap the E1 and E2 epitopes. MIF3037 demonstrated no competitive binding inhibition with mAb of the previously identified epitope specificity groups and, therefore, must represent a distinct functional epitope, E3. The huIFN-gamma, therefore, must have at least three distinct functional domains; none of these appeared to be responsible for cell surface receptor binding. Based on this finding, the epitope typing scheme must be extended to include the E3 epitope. The epitope specificity relationships of 13 neutralizing mAb developed by five other laboratories were established which allows correlation of results obtained with these mAb by different laboratories. The location of the epitopes of four widely studied mAb, 69B, 73A, 113B and 220A12, have been deduced based on their competition with the E1 mAb which have recently been mapped.

Serodiagnosis of chancroid is limited by the cross-reactivity of Haemophilus ducreyi with Haemophilus influenzae and Haemophilus parainfluenzae. This research describes an adsorption enzyme immunoassay (EIA) that assesses the humoral immune response of North Americans and Africans to H. ducreyi. Adsorption effectively removed anti-H. influenzae and anti-H. parainfluenzae antibodies, revealing that North American control sera had no residual anti-H. ducreyi reactivity. However, African control sera still had a residual anti-H. ducreyi response. Assessment of the duration of the humoral immune response in sera from African patients with chancroid showed that the humoral antibodies persisted for up to 8 months after the diagnosis. This may explain the lack of specificity of the adsorption EIA in areas where chancroid is endemic. The detection of the humoral immune response was affected by the strain of H. ducreyi used, with indigent strains being most useful. Using H. ducreyi 35000 for Canadian sera, the sensitivity of the adsorption EIA was 100% and the specificity was 88%. For African sera, H. ducreyi strain R018 was used, and the adsorption EIA had a sensitivity of 81% and a specificity of only 23%. These data reveal that the existing humoral response in a country where chancroid is endemic differs from that in a country where it is not, and that care must be used interpreting unadsorbed humoral immune responses. The adsorption EIA approach may prove useful as an epidemiologic tool for definition of existing (past and present) levels of exposure to H. ducreyi.

A prospective, quantitative assessment was undertaken of the effect of drying on the bacterial load in duodenoscopes that had been used for endoscopic retrograde cholangiopancreatography procedures. The endoscopes were washed and disinfected using an automatic washer and samples were taken through the suction channel at 2, 24 and 48 h post-disinfection. Twenty-one of the 42 duodenoscopes tested were contaminated. The ratio of Gram-negative bacilli to Gram-positive cocci increased from 70:1 at 2 h up to 4000:1 at 48 h for those duodenoscopes that were contaminated. Pseudomonas species (6 of 12 contaminated endoscopes) and Acinetobacter species (7 of 21 contaminated endoscopes) were the most common isolates. There was visible moisture remaining in the suction channel despite the use of the complete recommended automatic washer cycle. Bacterial concentrations reached as high as 1 x 10(7) colony forming units (cfu) ml-1. An additional 10 min of drying using either an 'in house' air line or the manual machine dry prevented bacterial overgrowth of all 19 endoscopes tested 48 h post-disinfection. If the additional 10 min of drying was used, then no alcohol rinse was required. Although no infections related to use of contaminated endoscopes were reported, it was apparent that Gram-negative bacilli were multiplying to unacceptably high concentrations and that this could be prevented by an additional 10 min of drying. The additional drying was only required at the end of the endoscopy list and not between patients.

Haemophilus ducreyi is the etiological agent of chancroid. The organism shares extensive immunological cross-reactivity with other Haemophilus species. This presents substantial difficulties for the production of specific monoclonal antibodies (MAbs). A competition ELISA was devised for hybridoma screening which allowed the detection of H. ducreyi-specific antibody-producing hybridoma cultures during the initial screening process. With this screening method, seven MAbs specific for H. ducreyi were obtained in a single cell fusion exercise. The specificities of the 7 MAbs were demonstrated by direct ELISA and dot immunobinding assays against several strains each of H. influenzae, H. parainfluenzae and Neisseria gonorrhoeae. Five of the MAbs reacted against all ten strains of H. ducreyi. These MAbs may permit the development of rapid and efficient immunodiagnostics for chancroid. The principle of the competition ELISA for hybridoma screening should be widely applicable to the development of specific MAbs to other organisms in which immunological cross-reactivity is an impediment to hybridoma screening by conventional methods.

A panel of mouse monoclonal antibodies (MAbs) against the recombinant human gamma interferon (HuIFN-gamma) has been produced for the study of the structure-function relationships of this important lymphokine. Enzyme linked immunosorbent assay (ELISA) is the current method of choice to screen hybridomas for specific MAb production. The purity of the antigen used for screening dictates the specificity of the ELISA. As often is the case in many systems, adequately purified biologically active HuIFN-gamma was not readily available for this purpose. A sandwich ELISA which allowed the use of unpurified HuIFN-gamma for hybridoma screening was developed. A rabbit antiserum against the denatured HuIFN-gamma purified by SDS-PAGE was prepared and the nonspecific binding activity was removed by adsorption to control cell proteins immobilized on Sepharose. The adsorbed immunoglobulin fraction was bound to the ELISA plate: (i) to trap HuIFN-gamma specifically from the whole cell lysate, thus providing specificity for MAb detection, and (ii) to avoid direct adsorption of the HuIFN-gamma to the ELISA plate because others have found that this prevented detection of neutralizing MAb. The sandwich ELISA detected both neutralizing and non-neutralizing MAbs with relatively low false positive reactions. This approach to the development of an ELISA method to screen hybridomas without purified antigen should be applicable to the production of MAbs to other proteins.

Studies of the morphology Mycoplasma hominis have shown that the cells resemble those of small coccoid or ovoid bacteria; the microstructure of the organism is compatible with reproduction by a fission process. Although dividing forms with equal-sized lobes are common, fragmenting filaments and replicating buds sometimes occur, perhaps in response to suboptimal growth conditions. Data based on morphometric analysis show that cells of M. hominis ATCC 14027 have average dimensions of 0.27 X 0.74 micron and a mean diameter of 0.42 micron. The volume of a M. hominis cell was only about 60% of that of Ureaplasma urealyticum T960, a species that has a mean cell diameter of 0.5 micron and that is sometimes a pathogen of the human genital tract. There are wide variations in the colonial morphology of M. hominis.