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A detailed investigation of the binding of secretory component to immunoglobulin A (IgA) in human secretory IgA2 (S-IgA2) was made possible by the development of a new method of purifying S-IgA1, S-IgA2 and free secretory component from human colostrum using thiophilic gel chromatography and chromatography on Jacalin-agarose. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of unreduced pure S-IgA2 revealed that, unlike in S-IgA1, a significant proportion of the secretory component was bound non-covalently in S-IgA2. When S-IgA1 was incubated with a protease purified from Proteus mirabilis the secretory component, but not the alpha-chain, was cleaved. This is in contrast to serum IgA1, in which the alpha-chain was cleaved under the same conditions - direct evidence that secretory component does protect the alpha-chain from proteolytic cleavage in S-IgA. Comparisons between the products of cleavage with P. mirabilis protease of free secretory component and bound secretory component in S-IgA1 and S-IgA2 also indicated that, contrary to the general assumption, the binding of secretory component to IgA is different in S-IgA2 from that in S-IgA1.

The influence of regions, other than the hinge, on the susceptibility of human IgA1 to cleavage by diverse bacterial IgA1 proteases, was examined using IgA1 mutants bearing amino acid deletions, substitutions, and domain swaps. IgA1 lacking the tailpiece retained its susceptibility to cleavage by all of the IgA1 proteases. The domain swap molecule alpha1alpha2gamma3, in which the CH3 domain of IgA1 was exchanged for that of human IgG1, was resistant to cleavage with the type 1 and 2 serine IgA1 proteases of Neisseria meningitidis, Neisseria gonorrhoeae, and Haemophilus influenzae, but remained sensitive to cleavage with the metallo-IgA1 proteases of Streptococcus pneumoniae, Streptococcus oralis, Streptococcus sanguis, and Streptococcus mitis. Substitution of the IgA1 Calpha3 domain motif Pro440 -Phe443 into the corresponding position in the Cgamma3 domain of alpha1alpha2gamma3 resulted now in sensitivity to the type 2 IgA1 protease of N. meningitidis, indicating the possible requirement of these amino acids for sensitivity to this protease. For the H. influenzae type 2 protease, resistance of an IgA1 mutant in which the CH3 domain residues 399-409 were exchanged with those from IgG1, but sensitivity of mutant HuBovalpha3 in which the Calpha3 domain of bovine IgA replaces that of human IgA1, suggests that CH3 domain residues Glu403, Gln406, and Thr409 influence sensitivity to this enzyme. Hence, unlike the situation with the metallo-IgA1 proteases of Streptococcus spp., the sensitivity of human IgA1 to cleavage with the serine IgA1 proteases of Neisseria and Haemophilus involves their binding to different sites specifically in the CH3 domain.

Objective: Antibodies to Proteus mirabilis were previously detected in patients with established rheumatoid arthritis (RA). We examined the prevalence of antibodies to P. mirabilis and their associations with RA in early synovitis patients., Methods: Two hundred and forty-six patients with inflammatory arthritis for less than 1 yr were prospectively evaluated for 1 yr. Of these patients, 30% had rheumatoid factor (RF)-positive RA, 16% RF-negative RA, 17% a spondyloarthropathy and 37% undifferentiated arthritis. Serum antibodies to P. mirabilis, Escherichia coli and other potentially arthritogenic organisms (Chlamydia, Salmonella, Shigella, Campylobacter, Yersinia and parvovirus B19) and for antibodies specific for immunoglobulin (Ig) G damaged with advanced glycation end-products (anti-IgG-AGE) were measured., Results: IgM and IgA anti-Proteus antibodies were significantly higher in patients with RF-positive RA compared with all other patient groups (P < 0.0005 and P < 0.005). Anti-P. mirabilis IgG, and IgG, IgA, and IgM antibodies to other potentially arthritogenic pathogens did not differ in the patient groups. IgM antibodies to E. coli were elevated in RF-positive RA patients. Anti-P. mirabilis IgM and IgA results were not explained by false-positive reactions, because after absorption of RF there was no decrease in antibodies to Proteus in 10 of 12 patients. Proteus and E. coli antibodies were highest in patients positive for both RF and anti-IgG-AGE antibodies (P<0.001). Patients with erosions tended to have higher IgA anti-Proteus titres, but no association with the shared HLA epitope or treatment was detected., Conclusion: Anti-P. mirabilis IgM and IgA and anti-E. coli IgM antibody elevations are associated with early seropositive RA and the presence of anti-IgG-AGE antibodies. The role that P. mirabilis or E. coli plays in early RF-positive RA requires further investigation.

The influences of IgA hinge length and composition on its susceptibility to cleavage by bacterial IgA1 proteases were examined using a panel of IgA hinge mutants. The IgA1 proteases of Streptococcus pneumoniae, Streptococcus sanguis strains SK4 and SK49, Neisseria meningitidis, Neisseria gonorrhoeae, and Haemophilus influenzae cleaved IgA2-IgA1 half hinge, an Ab featuring half of the IgA1 hinge incorporated into the equivalent site in IgA1 protease-resistant IgA2, whereas those of Streptococcus mitis, Streptococcus oralis, and S. sanguis strain SK1 did not. Hinge length reduction by removal of two of the four C-terminal proline residues rendered IgA2-IgA1 half hinge resistant to all streptococcal IgA1 metalloproteinases but it remained sensitive to cleavage by the serine-type IgA1 proteases of Neisseria and Haemophilus spp. The four C-terminal proline residues could be substituted by alanine residues or transferred to the N-terminal extremity of the hinge without affect on the susceptibility of the Ab to cleavage by serine-type IgA1 proteases. However, their removal rendered the Ab resistant to cleavage by all the IgA1 proteases. We conclude that the serine-type IgA1 proteases of Neisseria and Haemophilus require the Fab and Fc regions to be separated by at least ten (or in the case of N. gonorrhoeae type I protease, nine) amino acids between Val(222) and Cys(241) (IgA1 numbering) for efficient access and cleavage. By contrast, the streptococcal IgA1 metalloproteinases require 12 or more appropriate amino acids between the Fab and Fc to maintain a minimum critical distance between the scissile bond and the start of the Fc.

Components of the human immunoglobulin A1 (IgA1) hinge governing sensitivity to cleavage by bacterial IgA1 proteases were investigated. Recombinant antibodies with distinct hinge mutations were constructed from a hybrid comprised of human IgA2 bearing half of the human IgA1 hinge region. This hybrid antibody and all the mutant antibodies derived from it were resistant to cleavage by the IgA1 proteases from Streptococcus oralis and Streptococcus mitis biovar 1 strains but were cleaved to various degrees by those of Streptococcus pneumoniae, some Streptococcus sanguis strains, and the type 1 and 2 IgA1 proteases of Haemophilus influenzae, Neisseria meningitidis, and Neisseria gonorrhoeae. Remarkably, those proteases that cleave a Pro-Ser peptide bond in the wild-type IgA1 hinge were able to cleave mutant antibodies lacking a Pro-Ser peptide bond in the hinge, and those that cleave a Pro-Thr peptide bond in the wild-type IgA1 hinge were able to cleave mutant antibodies devoid of a Pro-Thr peptide bond in the hinge. Thus, the enzymes can cleave alternatives to their preferred postproline peptide bond when such a bond is unavailable. Peptide sequence analysis of a representative antibody digestion product confirmed this conclusion. The presence of a cleavable peptide bond near the CH2 end of the hinge appeared to result in greater cleavage than if the scissile bond was at the CH1 end of the hinge. Proline-to-serine substitution at residue 230 in a hinge containing potentially cleavable Pro-Ser and Pro-Thr peptide bonds increased the resistance of the antibody to cleavage by many IgA1 proteases.

The cleavage of human serum monomeric immunoglobulin A1 (IgA1) and human secretory IgA1 (S-IgA1) by IgA1 proteinase of Neisseria meningitidis and cleavage by the proteinase from Proteus mirabilis have been compared. For serum IgA1, both proteinases cleaved only the alpha chain. N. meningitidis proteinase cleaved only in the hinge. P. mirabilis proteinase sequentially removed the tailpiece, the CH3 domain, and the CH2 domain. The cleavage of S-IgA1 by N. meningitidis proteinase occurred only in the hinge and was as rapid as that of serum IgA1. P. mirabilis proteinase predominantly cleaved the secretory component (SC) of S-IgA1. The SC of S-IgA1, whether cleaved or not, appeared to protect the alpha1 chain. Purified Fc fragment derived from the cleavage of serum IgA1 by N. meningitidis proteinase stimulated a respiratory burst in neutrophils through Fcalpha receptors, whereas the (Fcalpha1)(2)-SC fragment from digested S-IgA1 did not. The loss of the tailpiece from serum IgA1 treated with P. mirabilis proteinase had little effect, but the loss of the CH3 domain was concurrent with a rapid loss in the ability to bind to Fcalpha receptors. S-IgA1 treated with P. mirabilis proteinase under the same conditions retained the ability to bind to Fcalpha receptors. The results are consistent with the Fcalpha receptor binding site being at the CH2-CH3 interface. These data shed further light on the structure of S-IgA1 and indicate that the binding site for the Fcalpha receptor in S-IgA is protected by SC, thus prolonging its ability to activate phagocytic cells at the mucosal surface.

The amino acid sequence requirements in the hinge of human immunoglobulin A1 (IgA1) for cleavage by IgA1 proteases of different species of Streptococcus were investigated. Recombinant IgA1 antibodies were generated with point mutations at proline 227 and threonine 228, the residues lying on either side of the peptide bond at which all streptococcal IgA1 proteases cleave wild-type human IgA1. The amino acid substitutions produced no major effect upon the structure of the mutant IgA1 antibodies or their functional ability to bind to Fcalpha receptors. However, the substitutions had a substantial effect upon sensitivity to cleavage with some streptococcal IgA1 proteases, with, in some cases, a single point mutation rendering the antibody resistant to a particular IgA1 protease. This effect was least marked with the IgA1 protease from Streptococcus pneumoniae, which showed no absolute requirement for either proline or threonine at residues 227 to 228. By contrast, the IgA1 proteases of Streptococcus oralis, Streptococcus sanguis, and Streptococcus mitis had an absolute requirement for proline at 227 but not for threonine at 228, which could be replaced by valine. There was evidence in S. mitis that proteases from different strains may have different amino acid requirements for cleavage. Remarkably, some streptococcal proteases appeared able to cleave the hinge at a distant alternative site if substitution prevented efficient cleavage of the original site. Hence, this study has identified key residues required for the recognition of the IgA1 hinge as a substrate by streptococcal IgA1 proteases, and it marks a preliminary step towards development of specific enzyme inhibitors.

All the IgA1 proteases of the different pathogenic species of Streptococcus cleave the hinge of the alpha chain of human IgA1 only at one proline-threonine peptide bond. In order to study the importance of these amino acids for cleavage, several hinge mutant recombinant IgA1 antibodies were constructed. The mutations were found to be without major effect upon the structure or functional abilities of the antibodies. However, they had a major effect upon their sensitivity to cleavage by some of the IgA1 proteases.

The hormone human chorionic gonadotropin (hCG) serves to maintain the fetus during early pregnancy and regulate the onset of labor in late pregnancy. hCG also prevents Neisseria gonorrhoeae from developing invasive characteristics. Part of the beta subunit of hCG has an amino acid sequence similar to that of the hinge of human IgA1, which is the site of action of IgA1 proteases. This study examined the sensitivity of hCG to gonococcal IgA1 proteases, by means of autoradiography, immunoblotting, and RIA. hCG was cleaved in the beta subunit by the type 1 but not the type 2 IgA1 proteases of N. gonorrhoeae. hCG cleavage by gonococcal IgA1 proteases in vivo may increase the invasiveness of the pathogen and destroy its natural biologic activity, with major consequences for the fetus and the pregnancy.

To understand more about the factors influencing the cleavage of immunoglobulin A1 (IgA1) by microbial IgA1 proteases, a recombinant human IgA2/IgA1 hybrid molecule was generated. In the hybrid, termed IgA2/A1 half hinge, a seven-amino-acid sequence corresponding to one half of the duplicated sequence making up the IgA1 hinge was incorporated into the equivalent site in IgA2. Insertion of the IgA1 half hinge into IgA2 did not affect antigen binding capacity or the functional activity of the hybrid molecule, as judged by its ability to bind to IgA Fcalpha receptors and trigger respiratory bursts in neutrophils. Although the IgA2/A1 hybrid contained only half of the IgA1 hinge, it was found to be cleaved by a variety of different bacterial IgA1 proteases, including representatives of those that cleave IgA1 in the different duplicated halves of the hinge, namely, those of Prevotella melaninogenica, Streptococcus pneumoniae, S. sanguis, Neisseria meningitidis types 1 and 2, N. gonorrhoeae types 1 and 2, and Haemophilus influenzae type 2. Thus, for these enzymes the recognition site for IgA1 cleavage is contained within half of the IgA1 hinge region; additional distal elements, if required, are provided by either an IgA1 or an IgA2 framework. In contrast, the IgA2/A1 hybrid appeared to be resistant to cleavage with S. oralis and some H. influenzae type 1 IgA1 proteases, suggesting these enzymes require additional determinants for efficient substrate recognition.

Background: Much has been published on the efficacy and cost effectiveness of Helicobacter pylori eradication treatment as an alternative to histamine H2-receptor antagonist maintenance treatment in peptic ulcer disease. However, most studies have analysed and emphasised H. pylori eradication rates rather than management/control of symptoms and the associated cost savings. Although H. pylori eradication therapy is very successful in clearing the infection, dyspeptic symptoms may persist and management of these can be expensive., Objective: The aim of this study was to assess the cost implications in controlling symptoms using either H2-receptor antagonist maintenance therapy or H. pylori eradication therapy in patients with duodenal ulcer disease., Design: This was a non-blind, prospective, randomised, parallel-group study comparing maintenance H2-receptor antagonist treatment using ranitidine with H. pylori eradication therapy, with a 1-year follow-up., Setting: This was a study of outpatients from general practices in Dundee, Scotland, or the Ninewells Hospital, Dundee, gastroenterology clinic., Patients and Participants: 119 patients with confirmed duodenal ulcer, free from active ulceration at study entry but positive for H. pylori infection, who were receiving maintenance H2-receptor antagonist therapy., Interventions: Patients were randomised to receive either continuing maintenance therapy with ranitidine (initially 150 mg daily; 58 patients) or H. pylori eradication therapy using an omeprazole/amoxicillin/metronidazole regimen (or omeprazole/clarithromycin if allergic to penicillin)., Main Outcome Measures and Results: Overall, H. pylori eradication rates were 100% per protocol and 95.1% intention-to-treat. At completion of 1 year of follow-up, 12 of the 61 (19.7%) patients successfully eradicated of H. pylori were still dependent on acid suppression for symptom relief. H. pylori eradication treatment was the least-cost strategy in managing/controlling symptoms at 1 year (168 Pounds vs 210 Pounds per patient; 1996 values). However, over time, post-eradication treatment costs were greater than H2-receptor antagonist therapy costs. Any potential savings were directly related to the proportion of patients needing further treatment post-eradication, the cost of endoscopy and the urea breath test., Conclusions: If dyspepsia persists long term, H. pylori eradication treatment may not be the least-cost option for patients with duodenal ulcer.

Many diverse clinical isolates of Proteus mirabilis (48 strains), P. penneri (25), P. vulgaris biogroup 2 (48) and P. vulgaris biogroup 3 (21) from man were examined for their ability to produce proteolytic enzymes and the nature and characteristics of the proteases were studied. All the P. penneri isolates, most (94-90%) of the P. mirabilis and P. vulgaris biogroup 2 isolates, but only 71% of the P. vulgaris biogroup 3 isolates, secreted proteolytic enzymes. These were detected most readily at pH 8 with gelatin as substrate. A strong correlation was found between the ability of a strain to form swarming growth and its ability to secrete proteases. Non-swarming isolates invariably appeared to be non-proteolytic. However, some isolates, particularly of P. vulgaris biogroup 3, were non-proteolytic even when they formed swarming growth. Analysis of the secreted enzymes of the different Proteus spp. on polyacrylamide-gelatin gels under various constraints of pH and other factors showed that they were all EDTA-sensitive metalloproteinases. Analysis of the kinetics of production of the proteases revealed the formation of an additional protease of undefined type and function that was cell-associated and formed before the others were secreted. The secreted protease was subsequently modified to two isoforms whose mass (53-46 kDa) varied with the Proteus spp. and the strain. There was no evidence that the secreted proteases of strains of Proteus spp. were of types other than metalloproteinases.

A total of 59 isolates of different Haemophilus spp., mostly from clinical specimens, was characterised, biotyped and examined for production of type 1 or type 2 IgA1 protease. IgA1 protease activity was not found in any isolate of a species with no or low virulence for man including H. parainfluenzae, H. haemolyticus, H. aphrophilus, H. paraphrophilus, H. segnis, H. paraphrohaemolyticus and H. haemoglobinophilus. IgA1 protease was produced by all isolates of H. influenzae and H. aegyptius and by some isolates of H. parahaemolyticus. The type of IgA1 protease appeared to be independent of the biotype of the isolate in H. influenzae. For the first time some isolates of H. aegyptius were found that produced type 2 IgA1 protease. IgA1 protease production in H. parahaemolyticus may be associated with the virulence of the isolate.

Objectives: patients with rheumatoid arthritis (RA) are reported to have in their sera raised levels of antibody specific to Proteus mirabilis. The aim of the study was to verify this and to determine an explanation for it by investigating the frequency of P. mirabilis urinary tract infection in RA patients and matched controls., Methods: freshly voided urine was examined for the presence, number and identity of infecting bacteria. The levels of antibody in blood and in urine of the IgM, IgA and IgG classes to the common O serotypes of P. mirabilis and the antigens to which they reacted were determined by enzyme-linked immunosorbent assay (ELISA) and immunoblotting., Results: analysis of urine from 76 patients with RA and 48 age- and gender-matched healthy controls showed that only two (4%) of the control urines but 25 (33%) of those from the RA patients were infected. The commonest infecting organism in the RA patients' urine was Proteus mirabilis which occurred twice as frequently as Escherichia coli. Proteus mirabilis was found in 52% of the infected urines of the RA patients and was always detected as a pure growth and usually in insignificant (< 10(4)/ml) numbers. It is highly improbable that this finding was the outcome of differences in age, physical ability or medication between the RA and control patient groups. Comparison of antibody levels to P. mirabilis by ELISA showed RA patients had raised (P < 0.0001, P = 0.001, P = 0.0063) levels of IgA, IgG and IgM respectively in their sera and raised (P < 0.0001, P < 0.0001, P = 0.0001) levels of IgG, IgM and IgA respectively in their urine compared with the control group. It was not possible to detect an antibody reacting to a P. mirabilis antigen that was specific to the RA patients., Conclusion: the results confirm that RA patients have raised levels of antibody to P. mirabilis not only in blood but also in urine and suggest that this arises because RA patients have an asymptomatic, non-significant P. mirabilis bacteriuria more frequently or more prolonged than control patients. This may be the trigger for their RA condition.

Background: Few outcome studies directly compare Helicobacter pylori eradication therapy with maintenance H2-antagonist therapy in duodenal ulcer disease., Aim: To examine prospectively the efficacy of H. pylori eradication therapy with ranitidine maintenance therapy over 1 year in patients with confirmed chronic duodenal ulcer., Methods: One hundred and nineteen patients with active H. pylori infection were randomized to receive ranitidine, 150 mg/day initially (58 patients), or omeprazole, 40 mg/day, amoxycillin 2 g/day and metronidazole 1.2 g/day for 14 days, or omeprazole 40 mg/day and clarithromycin 1.5 g/day, for 14 days (if penicillin-allergic). Symptoms were assessed using the Gastrointestinal System Rating Scale (GSRS) and SF36 quality of life index., Results: 13C urea breath testing confirmed overall treatment success in 100% of patients (58/58) per protocol and 95.1% (58/61) on an intention-to-treat basis. At 4 and 12 months there were no differences in any GSRS symptoms between treatment groups. SF36 analysis showed a perceived health improvement at 4 and 12 months in patients who received H. pylori eradication. However, despite successful H. pylori eradication, one-fifth of patients still required antisecretory therapy., Conclusion: Following successful H. pylori eradication, chronic duodenal ulcer patients were at least as well symptomatically as when taking maintenance ranitidine. They perceived that their health had improved, but a subgroup was still acid-suppression dependent.

Urinary isolates of Proteus mirabilis, obtained from 49 RA patients and 44 healthy controls, were tested for susceptibility to antibiotics by the disc diffusion method. In addition, P. mirabilis isolates were also tested for proticine production and sensitivity (p/s) typing by the inhibition of growth of each test isolate against 13 reference strains of P. mirabilis. The P. mirabilis isolates from both RA patients and healthy controls were highly susceptible to norfloxacin, ciprofloxacin and trimethoprim, but less to minocycline. The urine of RA patients contained fewer different types of P. mirabilis strains than those isolated from healthy controls. All of the strains found in the RA patients were proticine producers (P < 0.001), mostly of proticine 3 (P < 0.005). The presence of such strains provides evidence of a sub-clinical upper urinary tract infection with P. mirabilis in some RA patients. Therapeutic intervention in RA with relevant antibiotics requires evaluation.

Human immunoglobulin A (IgA) is found in serum and secretions in several different forms (1), Serum IgA is produced by the bone marrow and is pre-dominately monomeric (M ( r )= 160 kDa). Most of the IgA in secretions (sIgA) is polymeric, predominantly dimeric, comprising two monomer subunits linked together by intersubunit disulfide bonds and a cysteine-rich polypeptide termed J-chain (M ( r )= 16 kDa). Polymeric IgA in secretions, but not in serum, contains an additional heavily glycosylated protein called secretory component (SC) (M ( r )= 70 kDa).

Fluoride was found to affect the production of the bacterial IgA1 protease but to have no effect on IgA1 protease activity. The concentrations of fluoride that do affect Streptococcus sanguis growth and IgA1 protease production are higher than those normally seen in vivo under normal circumstances. The concentrations of fluoride in dental plaque following use of a fluoride rinse or dentifrice would be sufficient to reduce Strep. sanguis IgA1 protease production.

A medium (PAM: Providencia alcalifaciens medium) is described that enables the presence of the enteropathogen P. alcalifaciens in faeces to be detected with ease and simplicity. This organism is probably the only oxidase-negative organism likely to be present in tetrathionate broth cultures of faeces that is unable to ferment the mannitol, xylose or galactose present in the medium. Thus the red colonies of P. alcalifaciens appeared quite distinct from the lemon-yellow acid-forming colonies of all the other bacteria that ferment one or more of these sugars. Extensive tests showed the medium to be both highly specific and sensitive in detecting P. alcalifaciens. Two additional media are described that enable the identity of presumptive P. alcalifaciens isolates to be confirmed unequivocally and with ease.

Proteus mirabilis strains frequently infect the upper urinary tract and some progress to invade the bloodstream. In an attempt to determine if these were features only of particular strains of P. mirabilis, isolates from the blood of 38 unrelated patients, many of whom had an underlying urinary tract infection, and eight from different sources and sites within the upper urinary tract, were analysed by proticine production/proticine sensitivity (p/s) typing. Twenty-six different p/s types were found among the 38 isolates from blood and seven among the eight isolates from the upper urinary tract. The p/s types found previously to be associated frequently with virulence for the upper urinary tract were not found. Analyses of the frequency of occurrence of the different p/s types and of their distribution showed no evidence that the p/s characteristics of a P. mirabilis strain conferred any advantage on its ability to infect the upper urinary tract or invade the bloodstream.

Unlabelled: It has been suggested that establishing Helicobacter pylori infection status is irrelevant prior to H. pylori eradication treatment in chronic duodenal ulcer, as virtually all may benefit from therapy., Objective: The aim of the present study was (i) to determine the prevalence of active H. pylori infection in patients with proven chronic duodenal ulcer on long-term H2-antagonist prophylactic treatment and whether knowledge of this would influence the use of eradication therapy and (ii) to assess other factors which might influence the clinical diagnosis or H. pylori status, such as non-steroidal and antibiotic use., Methods: One hundred and forty-five patients receiving long-term H2-antagonists for chronic duodenal ulcer were recruited. Their case records and a prescribing database were reviewed. Patients underwent endoscopy with biopsies for rapid urease test, histology and H. pylori culture. Serum was immunoblotted and an enzyme-linked immunosorbent assay for H. pylori was performed., Results: Of the 145 patients, 128 (88%) were H. pylori biopsy positive. Twelve of the 17 H. pylori biopsy-negative patients had anti-H. pylori immunoglobulin G (IgG) antibodies and 10 of the 17 H. pylori-negative patients had previously received antibiotics for other indications. Nine patients were exposed to non-aspirin non-steroidal anti-inflammatory drugs (NSAIDs) and one had additional aspirin exposure., Conclusion: Only 11.7% of patients on maintenance treatment for chronic duodenal ulcer had no current infection with H. pylori, although more than 70% of these had serological evidence of previous infection. Confirmation of active infection may be indicated where there is a history of NSAID or antibiotic exposure and may result in more precise targeting of eradication therapy, thus avoiding unnecessary and potentially hazardous treatment.

Several important and diverse human pathogens are found in the tribe Proteeae. By identifying and concentrating on key biochemical reactions, it has been possible to devise six simple media that permit the identification of all the important members of the tribe with ease, speed and accuracy. This was confirmed by optional additional confirmatory media and tests.

An analysis by ELISA of 100 rheumatoid factor (RF)-positive sera selected at random from a collection of sera from patients with various auto-immune diseases and joint pains, and 100 RF-negative sera from the same collection matched by patient age and gender, showed that the RF-positive sera had highly significantly (p < 0.0001) raised levels of IgM antibody, but not IgG antibody, to Proteus mirabilis over those of the RF-negative sera. This response was subsequently found to be associated with sera from patients who clinically had rheumatoid arthritis (RA). Sera from the RA patients had significantly greater amounts (p = 0.026) of IgM antibody to P. mirabilis than to the other organisms tested and these values were also highly significantly different (p < 0.0001) from P. mirabilis IgM antibody levels in matched RF-negative sera. Sera from RA patients also had significantly greater amounts of IgA to P. mirabilis (p < 0.0001) and greater amounts of IgM to Escherichia coli (p < 0.0001) and Klebsiella pneumoniae (p < 0.0001) than those in matched RF-negative sera. Other classes of antibody to these organisms and all classes of antibody to Pseudomonas aeruginosa were not raised in the sera of RA patients over those of RF-negative controls. The IgM response in RA patients was not specific for only one O serotype of P. mirabilis but was associated with all 11 different O serotypes of P. mirabilis tested and those of other Proteus spp. Moreover, the IgM antibodies to Proteus spp. appeared to be independent from C-reactive protein and RF.(ABSTRACT TRUNCATED AT 250 WORDS)

Twelve diverse strains of Proteus penneri of clinical origin all produced a calcium-dependent haemolysin, unlike most other Proteus spp. In most strains the haemolysin was secreted into the medium during early exponential growth and lysed not only of a variety of erythrocyte types from several animals including man, but also human neutrophils and human embryo lung fibroblasts. The haemolysin was a protein of 107 kDa, the same size as Escherichia coli HlyA, and it reacted with antiserum to E. coli HlyA. Because of its similarity in size, antigenicity and range of action to the HlyA virulence factor of E. coli, P. penneri HlyA is believed to be an important virulence factor for this organism. It was degradable by an EDTA-sensitive protease--probably the IgA protease--to inactive fragments. The interaction of P. penneri HlyA and IgA protease in vivo and the origin of HlyA, which has now been found in many diverse bacteria, are discussed.

Eighteen different strains of Proteus mirabilis were all shown to produce an EDTA-sensitive proteinase of c. 50 kDa that cleaved the heavy chain, but not the light chain, of IgG. Digestion of pure IgG with small amounts of pure P. mirabilis proteinase generated Fabc'2 and Fab'2 fragments; greater amounts generated Fab and Fc fragments that were comparable in size to those generated by pepsin and papain, respectively. Incubation of neutrophils with IgG digested with P. mirabilis proteinase or papain resulted in a marked decrease in the respiratory burst activity of the neutrophils that coincided with cleavage of the IgG into Fab and Fc fragments. Analysis of urine from patients with P. mirabilis urinary tract infection revealed in many the presence of Fab and Fc fragments of IgG indistinguishable in size from those generated by P. mirabilis proteinase. These results indicate that, in P. mirabilis urinary tract infections, the proteinase is secreted and cleaves IgG to fragments that have defective immune effector functions, thereby limiting the effectiveness of the immune response.

The proteinases secreted by pathogenic strains of Proteus mirabilis, P. vulgaris biotype 2, P. vulgaris biotype 3, and P. penneri were purified with almost 100% recovery by affinity chromatography on phenyl-Sepharose followed by anion-exchange chromatography. The proteinase purified from the urinary tract pathogen P. mirabilis, which we had previously shown to degrade immunoglobulins A and G, appeared as a composite of a single band and a double band (53 and 50 kDa, respectively) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The other Proteus proteinases had similar patterns but slightly different mobilities. In each case all proteinase activity in culture supernatants was demonstrated by gelatin-sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be associated with only the triple-band complex; all three bands were proteolytically active. The P. mirabilis proteinase was resistant to inhibitors of both serine and thiol proteinases but strongly inhibited by metal chelators, although it was not affected by phosphoramidon, an inhibitor of the thermolysin group of bacterial metalloproteinases. Active proteinase was detected in urine samples from P. mirabilis-infected patients; this is consistent with our detection of immunoglobulin A fragments of a size suggestive of P. mirabilis proteinase activity.

Aims: To assess the efficacy of a bacteriology service in respect of the time interval between collection of specimen and receipt of final report using the number and type of incoming telephone enquiries as a proxy measure., Methods: For three months, all incoming telephone enquiries regarding the results of bacteriology specimens were monitored. Specimen type, date of sampling, the sender's location and the reason for making the telephone enquiry were recorded., Results: The number of telephone enquiries made during the study was 1170, about 5% of the total number of samples received. Most enquiries related to results of urine cultures. These accounted for 33.9% of the calls, but only 5% of the total number of urines cultured. Enquiries relating to blood cultures were the next largest group accounting for 14.9% of calls, but 11% of the blood cultures received resulted in a telephone enquiry. The most frequent reason for making the telephone call was that the sender had not received the final report., Conclusions: Clinicians may have unrealistic expectations of the time taken to examine a specimen. A requirement for reporting sterile blood cultures after a shorter incubation period was found, and the value to patient management of earlier reporting of negative urine samples was identified.

Immunoblotting of urine from 21 patients of both sexes and of wide age range who had a Proteus mirabilis urinary tract infection (UTI) showed that 14 (64%) specimens contained immunoglobulin A (IgA). In nine (64%) of these the IgA heavy chain had been degraded to fragments of a size identical to those formed when purified IgA was degraded by pure P. mirabilis protease. Urine from patients with clinical evidence of upper UTI contained fragmented IgA and in some of these urine samples P. mirabilis protease activity was detectable. Urine infected with a non-proteolytic strain contained only intact IgA. It is concluded that P. mirabilis IgA protease is produced and is active during infections of the urinary tract.

A method is described for the simultaneous purification of IgA1 and IgA2 from human serum. Ammonium sulphate precipitation, gel filtration and ion-exchange chromatography on DEAE-Sephacel yielded a partially purified IgA preparation which was separated quantitatively into IgA1 and IgA2 by affinity chromatography on jacalin-Sepharose. The IgA1 which bound to the jacalin was eluted with 0.8 M D-galactose. The IgA1 preparation was apparently homogeneous by SDS-PAGE but contained a trace of C1-inhibitor and a second protein detected by immunoelectrophoresis. The IgA2 which did not bind to the jacalin was purified to apparent homogeneity by chromatography on columns of Protein G-Sepharose, Fastflow-S Sepharose and Superose 6. Typical yields were 95% and 58% for IgA1 and IgA2 respectively or 253 mg and 24 mg per 100 ml serum. The IgA1 and IgA2 were characterised by their reactivity with isotype specific monoclonal antibodies and sensitivity to bacterial proteinases. The IgA2 preparation apparently contained both allotypes, IgA2m(1) and IgA2m(2).

A new, simple and stable method for typing Morganella morganii strains is described. The 150 strains examined, principally from faeces, contained haemolytic and non-haemolytic representatives of diverse O serogroup, bacteriocin type and biotype. Among the biotypes were some trehalose-fermenting, tetracycline-resistant strains and some non-motile, tetracycline-sensitive, glycerol fermenters. After analysis of cell lysates by sodium dodecyl sulphate-discontinuous polyacrylamide gel electrophoresis, strains could be differentiated into 21 types on the basis of outer membrane proteins (OMP) of 35-40 Kda. The OMP profile was not altered by culture on various common media and was unrelated to either O antigen or morganocin p-type. The finest strain recognition in M. morganii can be achieved by application of all three distinct typing methods.

Proteus mirabilis strains associated with human urinary tract infections have previously been shown to secrete an extracellular metalloproteinase which cleaves serum immunoglobulin A (IgA). The enzyme has now been purified to apparent homogeneity from culture supernatants of P. mirabilis 64676. The protease activity is associated with a 50-kilodalton (kDa) protein. Unlike that of the classic IgA1 proteases, the substrate specificity of the P. mirabilis protease has been found to extend to both sublcasses of IgA, IgG, and the nonimmunoglobulin substrates, secretory component and casein. Cleavage of IgA1 and IgA2 by the P. mirabilis protease yielded fragments on sodium dodecyl sulfate-polyacrylamide gel electrophoresis whose sizes were consistent with a cleavage site outside the hinge region. Both secretory IgA1 and IgA2 were also cleaved by P. mirabilis protease, although the secretory IgA2 molecule was less readily cleaved than secretory IgA1. Free and IgA-bound secretory components were degraded to some extent by P. mirabilis protease. Cleavage of IgG, however, occurred at the hinge region as a two-stage process. The first stage was pepsinlike and generated an F(ab')2 fragment of 120 kDa and a small pFc fragment detected on nonreduced polyacrylamide gels. In the second stage, the F(ab')2 product was cleaved to yield papainlike Fab and Fc fragments, visualized as a diffuse band of 40 to 50 kDa.

A collection of 142 strains of Morganella morganii, principally from unrelated patients' faeces was examined to determine the relationship, if any, between haemolysin production, O antigen and morganocin production/sensitivity type. Only 55 (38.7%) were agglutinable with the existing 44 O antisera. However, when O antisera were raised to some of the non-typable strains 11 new O antigens were found and 126 (88.7%) of the strains were typable. The number of O antigenic groups in M. morganii is now 55. It was confirmed that the O antigenic characteristics of strains were independent from morganocin producer types. An epidemiological retrospective survey showed that finer strain recognition in M. morganii can be achieved by using both methods than either method alone. Approximately 30% of strains were haemolytic. The ability to produce haemolysin was more common in strains of certain O serogroups and morganocin producer types than in others.

Two hundred and four isolates of swarming strains of Proteus species which had been typed for their ability to produce bacteriocin (proticine) and also their proticine sensitivity (P/S typing) were tested in all combinations for their Dienes compatibility with each other. Ninety-eight distinct Dienes compatibility groups were found. Physiological and genetic experiments supported the evidence of typing results that, irrespective of species, both the type of proticine a strain produces (P type) and the sensitivity of the strain to proticine (S type) were determinants of Dienes compatibility. Strains showing compatibility in the Dienes test were of the same P/S type, whereas those of different P/S types were incompatible.

A simple, reliable and highly discriminating scheme for the bacteriocine typing of Proteus has been developed. Strains are typed on MacConkey's agar according to their ability to produce a proticine active against one of 14 indicator strains having a single and specific proticine sensitivity and also according to their sensitivity to the different proticines of 13 proticine-producing strains. This new scheme of combined production and sensitivity typing was formulated after 250 strains of Proteus from clinical material had been examined for the production of proticines active against the 24 indicator strains of Cradock-Watson's proticine typing scheme and for proticine activity and sensitivity towards each other. Three new types of proticinogenic strains were discovered and defined. Strains producing proticines of types 1, 2 and 3 were isolated frequently. These common proticines could be subtyped by their different actions on newly characterised indicator strains. By means of this production/sensitivity (P/S) typing scheme, 250 Proteus strains were differentiated into 90 distinct types, whereas typing by sensitivity alone distinguished only 40 types and typing by production alone distinguished only 20 types (including subtypes).

The ability of Proteus mirabilis to produce the bacteriocin, proticine 3, is found almost exclusively in strains that cause severe infections of the upper urinary tract. Proticine 3 was purified from lysates of mitomycin C-induced cultures. Biological activity was associated with structures resembling bacteriophage tails which, when first produced, were in the form of "nails" with one pointed end and a base plate with appendages at the other end. This form was unstable and changed to a "rocket" form in which the outer sheath contracted and thickened to reveal a protruding, hollow core that often became detached from the sheath. Purified proticine 3 comprises two major and nine minor proteins. Fluorography showed that during production of the proticine, a 58,000 mol. wt protein was synthesized late in the induction process and became the most intensely labelled protein in the culture. Synthesis of this protein coincided with the appearance and increase in titre of biologically-active proticine within the cell and with the appearance of "nail" forms. The protein is believed to be shed when an active "nail" is converted to an inactive "rocket" and to be either the component of proticine 3 associated with its lethal activity, or the protein required for the correct assembly of the constituent components into a biologically-active particle. The role of proticine 3 as a virulence factor is discussed.

The strains of Proteus species found in significant numbers and as pure cultures in urine from 217 individuals were isolated, identified to species level and typed for proticine production (P type) and proticine sensitivity (S type) to give their P/S type. Urinary-tract infections with Proteus, principally P. mirabilis, were associated with the elderly. Ninety seven distinct P/S types were found but three P/S types P3/S1,8, P3/S1,8,13 and P3/S1,13 were isolated at a much higher frequency (14%) then could be explained from their faecal carriage rate. These types were almost without exception restricted to patients with clinical symptoms of urinary-tract infection and it is suggested therefore that they have some special affinity for the urinary tract.

In strains of Proteus mirabilis of urinary origin, no correlation was found between proticine types, reflecting relative affinity for the urinary tract, and the production of haemagglutinins and presence of fimbriae, a measure of adhesiveness.

Type-3 fimbriae isolated from members of five different species of Klebsiella were 4-5 nm in diameter and agglutinated the tannic acid-treated erythrocytes of ox and, in some cases, the untanned erythrocytes of fowl. In sodium dodecyl sulphate-polyacrylamide gel electrophoresis, the type-3 fimbrial proteins had mol. wts in the range 19 500-21 500. Hydrophobic amino acids comprised 39.6% of all the amino acids of the type-3 fimbrial protein of K. oxytoca strain 70/1. The type-1 fimbrial protein of Klebsiella had a mol. wt of c. 18 000 and the type-1 fimbriae were serologically distinct from the type-3 fimbriae. Our results for the type-3 fimbriae of Klebsiella were compared with those of others for morphologically similar and serologically related thin fimbriae of Salmonella and Yersinia.

A collection of 100 strains of Proteeae, in which all species within the tribe were represented, was examined for IgA protease production. The strains were isolated from various clinical specimens from sick and healthy persons in several countries. IgA protease-producing strains were not found amongst species of Providencia and Morganella but were common in Proteus spp. All the strains of P. mirabilis and P. penneri and many of the strains of P. vulgaris examined produced an EDTA-sensitive protease that cleaved the IgA heavy chain outside the hinge region. The proteus enzyme was different in this respect from the EDTA-sensitive, hinge-cutting proteases of other bacteria. The ability to produce IgA protease was unrelated to the O antigenicity, biotype or bacteriocin type of the strain. IgA protease production may be an important virulence mechanism for Proteus strains.

Strains of Proteus mirabilis and P. vulgaris isolated in England, Scotland and Sweden were characterised by proticine production-proticine sensitivity (P-S) typing, O serotyping and Dienes typing methods. The determinants of O antigenicity were independent of those determining proticine production and proticine sensitivity. Because of this independence, the combination of P-S typing and O serotyping for the analysis of the 133 serotypable strains separated them into 81 distinct types whereas P-S typing and O serotyping methods alone separated them into only 56 and 19 types respectively. There was a relationship between the Dienes type and the P-S type; the determinants of Dienes compatibility were the proticine production-proticine sensitivity characters. The determinants of O antigenicity appeared to play no role in the Dienes reaction. Some strains that were indistinguishable by P-S typing and O serotyping methods were distinguished by Dienes typing.

A strain of Salmonella of serotype Salinatis, that produced a mannose-resistant and eluting haemagglutinin (MREHA) when cultured at 37 degrees C but not at 18 degrees C, was examined by electron microscopy after negative staining. Production of this MREHA, previously described as being non-fimbrial, was correlated with the presence of thin fimbriae which had an external diameter of 3.6 nm. The purified Salinatis thin fimbriae had an estimated Mr of 19 kDa. This fimbrial MREHA was not produced by strains of the antigenically related serotypes Duisburg and Sandiego.

During a period of 12 months, 57 strains of Proteus were isolated from the ears of 38 unrelated patients with chronic suppurative otitis media. Each strain was identified, typed for bacteriocin production and sensitivity, and tested for Dienes compatibility. The majority of the strains (79%) were P. mirabilis; all but one of the remainder were P.vulgaris. Although 19 different bacteriocin production/sensitivity types were found, two rare types, P. mirabilis P7/S5,12 and P. vulgaris P0/S9, were associated with 47% of these infections. This was confirmed by Dienes typing. Patients with bilateral ear disease carried a different strain in each ear. There was no evidence that persistence of infection had arisen because of the development of antibiotic resistance. Although there was some evidence that persistence may have been the result of reinfection, the isolation of these rare types of Proteus from so many patients with chronic suppurative otitis media may indicate that they play an important role in the pathogenesis of the disease. Most of the Proteus isolates were of "non-faecal" types and it is believed that infection had arisen by a route other than the faecal-aural one.

A rapid and simple method is described by which colonies of Proteus can be distinguished from those of Salmonella and Shigella and other non-lactose-fermenting organisms growing on MacConkey's agar or desoxycholate citrate agar. The method is based on the ability of Proteus to produce urease constitutively. The enzyme was detected by the degradation of urea by the inoculum, thereby creating an alkaline reaction on pH-indicator paper.

The ureases produced by a large number of strains of different Proteus species, some of which were known to have a special affinity for the urinary tract, were examined by polyacrylamide-gel electrophoresis. Each Proteus strain gave a pattern of urease isoenzymes that was characteristic and unique to its species although strains of P. Mirabilis and P. vulgaris gave isoenzyme patterns that were closely similar. There was some minor variation in the patterns of urease isoenzymes even between strains of the same species. This was most noticeable among P. rettgeri strains and to a lesser extent among P. vulgaris strains. No correlation was found between the types of ureases a strain produced and its pathogenicity for the urinary tract.

Proteus mirabilis CW977 produced high yields of the bacteriocin proticine 3 upon mitomycin C induction of cultures growing at 30 degrees C. The proticine was purified and found to have a relative density of 1.299 and to be composed of 10 proteins assembled into structures resembling contractile phage tails. When induction was performed at 41 degrees C neither proticine particles nor proticine activity was detected, although the growth rate of cells and degree of lysis were indistinguishable from that at 30 degrees C. Failure in proticine production was due to a 41 degrees C sensitive stage occurring between 60 and 90 min after the addition of mitomycin C. During this period at 30 degrees C, two proteins of mol. wt 58 000 and 41 000 were formed. These proteins were associated with events leading to the formation of proticine particles with biological activity. When the production of both proteins was prevented either by chloramphenicol or as a result of mutation or through sampling before they were formed, no proticine particles were found nor proticine activity detected. The synthesis of both proteins was also inhibited at 41 degrees C. Co-electrophoresis of the labelled proteins with unlabelled purified proticine confirmed that the protein of mol. wt 58 000 was a proticine structural protein. The protein of mol. wt 41 000 was not a structural component of proticine and its role, if any, in proticine 3 production is possibly that of an assembly protein.

A typing scheme for Morganella morgani based on bacteriocin (morganocin) production and sensitivity is described. These characteristics were determined by testing 160 strains in all combinations and permitted their differentiation into 90 types. Morganocin production was induced with mitomycin C and morganocin sensitivity determined with a diluted inoculum on Lab-lemco agar at 30 degrees C. Most strains (82.5%) produced morganocins and 49 different types were defined. Most strains (97.5%) were sensitive to morganocins and usually to several different types. The scheme is more discriminating than other reported methods. The finding in an epidemiological survey of the carriage of certain strains in the bowel for several weeks suggests that in practice the method is stable and reproducible.

Strains of Salmonella of serotype Sendai, producing a mannose-resistant and eluting haemagglutinin (MREHA) when cultured at 37 degrees C but not at 18 degrees C, were examined by electronmicroscopy after negative staining. Production of this MREHA, previously thought to be nonfimbrial, was correlated with the presence of thick fimbriae with an external diameter of 13.6 nm. These fimbriae were readily fragmented and, when purified, had an estimated Mr of 28 Kda. Production of fimbrial MREHA by Sendai strains was associated with the ability to adhere to a wide range of substrates and to form a fimbrial pellicle at the surface of liquid media incubated statically in air. The origin of this unusual Sendai fimbrial MREHA is unknown. Thin filamentous structures produced independently of fimbrial MREHA by Sendai strains were also described. Fimbrial MREHA was not produced by strains of the antigenically similar serotype Miami which, however, and unlike Sendai strains, formed mannose-sensitive haemagglutinin and type-1 fimbriae. The ability to differentiate strains of Miami and Sendai (serotype 1,9,12:a: 1,5) by means of their fimbriae is noted.