Monoclonal antibodies define genus-specific, species-specific, and cross-reactive epitopes of the chlamydial 60-kilodalton heat shock protein (hsp60): specific immunodetection and purification of chlamydial hsp60.

Ocular and urogenital tract infections with Chlamydia trachomatis can progress to chronic inflammatory diseases that produce blindness and tubal infertility. The pathophysiology of these chronic disease conditions is thought to be immunologically mediated, and the chlamydial 60-kDa heat shock protein (hsp60) has been implicated as a major target antigen that stimulates the immunopathological response. The lack of chlamydial hsp60 antibodies and purified hsp60 has severely restricted studies to define more thoroughly the role of this protein in the immunopathogenesis of chlamydial disease. We produced a panel of antichlamydial hsp60 monoclonal antibodies (MAbs) and defined their specificities by immunoblotting against lysates of C. trachomatis, C. psittaci, and six other genera of bacteria. Three patterns of anti-hsp60 immunoreactivity were observed: chlamydial species specific, chlamydial genus specific, and cross-reactive. The epitopes recognized by these MAbs were localized within the primary amino acid sequence of hsp60 by immunoblotting against recombinant amino-terminal truncated hsp60 fusion polypeptides and then precisely mapped by use of overlapping synthetic peptides. The majority of the MAbs mapped to either the amino or the carboxyl termini of hsp60. Epitopes defining all three MAb reactivities mapped within amino-terminal residues 6 to 16. Genus-specific hsp60 MAbs mapped to epitopes located within this region and to residues 17 to 28 and 177 to 189. Antichlamydial hsp60 MAbs stained inclusions as effectively as MAbs specific for the major outer membrane protein. Homogeneous preparations of full-length recombinant chlamydial hsp60 and amino-terminal truncated recombinant hsp60 polypeptides were obtained by immunoabsorption chromatography with an hsp60 MAb reactive to the carboxyl terminus of the protein. Thus, the antichlamydial MAbs described here should be extremely useful for the specific immunodetection of hsp60 in tissues from individuals having different disease manifestations and for the purification of hsp60 or truncated hsp60 polypeptides for use in serologic and lymphocyte proliferation assays. The availability of these MAbs will facilitate studies to define more precisely the role of hsp60 in the immunopathogenesis of chlamydial disease.     

Immunological Markers of the R4 Protein of Streptococcus agalactiae

This study focuses on immunological markers of R4, an important Streptococcus group B (GBS) protein. The results obtained by using rabbit antisera and purified proteins for antigens in enzyme-linked immunosorbent assay-based experiments provided evidence that R4 possesses two antigenic determinants. One of the determinants is shared with the alpha-like protein 3 (Alp3) of GBS, was named R4/Alp3 common, and was expressed by GBS, which possessed the Alp3-encoding gene alp3 or the R4-encoding gene rib. The other antigenic determinant was detected only in rib-positive GBS organisms and was named R4 specific. This determinant probably is an immunological marker unique to the R4 protein. Neither of the antigenic R4 determinants showed serological cross-reactivity with the GBS proteins Cα, Cβ, and R3 or with alpha-like protein 2. Of 60 clinical serotype III GBS strains, 56 (93%) isolates possessed the rib gene and 50 (89%) of the rib-positive isolates expressed levels of R4 detectable by antibody-based tests, consistent with R4 expression failure or low-level expression in ~10% of rib-positive GBS. alp3 was not detected in type III GBS but was possessed by six of eight type V strains and six of six type VIII strains. All alp3-positive strains were recognized by the R4/Alp3 common antibodies, but none of them were recognized by the R4-specific antibodies. NCTC 9828, a reference strain for R3 and R4, expressed the determinant R4/Alp3 common but not R4 specific. A monoclonal R4 antibody, previously considered to be R4 specific and used in GBS serotyping, targeted R4/Alp3 common and is thus not R4 specific. The results show that failure to discriminate between R4 specific and R4/Alp3 common by antisera designed for GBS serotyping can result in the false identification of Alp3 as R4 or vice versa, whereas anti-R4 antibodies targeting only the determinant R4 specific will detect only R4. Both R4 and Alp3 need further evaluation with respect to the immunobiological function of each distinct antigenic determinant, for instance, with regard to their potential as GBS vaccine components.     

Antigenic Determinants of Alpha-Like Proteins of Streptococcus agalactiae

The majority of group B streptococcus (GBS) isolates express one or more of a family of surface-anchored proteins that vary by strain and that form ladder-like patterns on Western blotting due to large repeat units. These proteins, which are important as GBS serotype markers and as inducers of protective antibodies, include the alpha C (Cα) and R4 proteins and the recently described alpha-like protein 2 (Alp2), encoded by alp2, and Alp3, encoded by alp3. In this study, we examined antigenic determinants possessed by Alp2 and Alp3 by testing of antibodies raised in rabbits, mainly by using enzyme-linked immunosorbent assays (ELISA) and an ELISA absorption test. The results showed that Alp2 and Alp3 shared an antigenic determinant, which may be a unique immunological marker of the Alp variants of GBS proteins. Alp2, in addition, possessed an antigenic determinant which showed specificity for Alp2 and a third determinant which showed serological cross-reactivity with Cα. Alp3, in addition to the determinant common to Alp2 and Alp3, harbored an antigenic site which also was present in the R4 protein, whereas no Alp3-specific antigenic site was detected. These ELISA-based results were confirmed by Western blotting and a fluorescent-antibody test. The results are consistent with highly complex antigenic structures of the alpha-like proteins in a fashion which is in agreement with the recently described structural mosaicism of the alp2 and alp3 genes. The results are expected to influence GBS serotyping, immunoprotection studies, and GBS vaccine developments.     

Extracorporeal shockwave lithotripsy of 2000 urinary calculi with the modulith SL-20: success and failure according to size and location of stones

PURPOSE: We analyzed the successes and failures of SWL in the treatment of 2016 urinary calculi stratified according to size and position in the urinary tract. METHODS: All the patients were treated with a Modulith SL-20 (Storz Medical). RESULTS: The overall stone-free rate, regardless of the size and position of the stone, was 87.4%. The rate was different for kidney and ureteral stones. The stone-free rate observed for the different positions of the calculi within the kidney was upper calix 89.2%, middle calix 90.5% lower calix 84.8%, and renal pelvis 86.0%. For staghorn calculi, the stone-free rate was 60.0%. The stone-free rate for the different positions of calculi within the ureter was: upper ureter 84.3%, iliac ureter 82.4%, and pelvic ureter 91.0%. For calculi >24 mm, the retreatment rate increased, and the success rate dropped sharply. CONCLUSION: Extracorporal shockwave lithotripsy should be the first therapeutic option for urinary calculi of <24 mm regardless of their position in the urinary tract.     

Increased mucosal inflammatory cytokines in children with Helicobacter pylori-associated gastritis

The concentrations of tumour necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and IL-1-β in tissue homogenates of gastric mucosal biopsy specimens, and in gastric juice samples from Helicobacter pylori-positive and -negative children, were determined. The study population comprised 30 children with recurrent abdominal pain attending upper gastrointestinal endoscopy. Of these patients 18 were infected with H. pylori. Cytokine concentrations in gastric biopsy homogenate supernatants and in gastric juice were measured by enzyme-linked immunosorbent assay (ELISA). TNF-α levels in gastric juice and in gastric biopsy homogenate supernatants in patients with H. pylori-positive gastritis were found to be significantly higher than those in children without H. pylori infection. IL-6 levels were also higher in H. pylori -infected subjects, but the difference in IL-6 concentrations measured in gastric juice and biopsy homogenate supernatants did not reach statistical significance. IL-1-β concentrations in both specimens showed no significant difference between the two groups of children. It was suggested that increased levels of inflammatory cytokines, especially TNF-α and IL-6 generated locally within the gastric mucosa might be implicated in the pathogenesis of H. pylori-associated gastritis in childhood.