Chlamydia trachomatis major outer membrane protein variants escape neutralization by both monoclonal antibodies and human immune sera.

We have identified two families of novel Chlamydia trachomatis isolates with amino acid changes within the major outer membrane protein (MOMP) variable domains: one family of Da, D*, and D- and one family of Ia and I-. In order to determine whether these MOMP variants can escape antibody neutralization of infectivity, we tested both the D and I prototype strains and the variants in a complement-independent in vitro neutralization assay. We found that variants can indeed escape neutralization by both monoclonal antibodies and polyclonal human immune sera that neutralize the prototype strain.     

Demonstration of factor H-like protein 1 binding to Treponema denticola, a pathogen associated with periodontal disease in humans

Treponema denticola is an important contributor to periodontal disease. In this study we investigated the ability of T. denticola to bind the complement regulatory proteins factor H and factor H-like protein 1 (FHL-1). The binding of these proteins has been demonstrated to facilitate evasion of the alternative complement cascade and/or to play a role in adherence and invasion. Here we demonstrate that T. denticola specifically binds FHL-1 via a 14-kDa, surface-exposed protein that we designated FhbB. Consistent with its FHL-1 binding specificity, FhbB binds only to factor H recombinant fragments spanning short consensus repeats (SCRs) 1 to 7 (H7 construct) and not to SCR constructs spanning SCRs 8 to 15 and 16 to 20. Binding of H7 to FhbB was inhibited by heparin. The specific involvement of SCR 7 in the interaction was demonstrated using an H7 mutant (H7AB) in which specific charged residues in SCR 7 were replaced by alanine. This construct lost FhbB binding ability. Analyses of the ability of FHL-1 bound to the surface of T. denticola to serve as a cofactor for factor I-mediated cleavage of C3b revealed that C3b is cleaved in an FHL-1/factor I-independent manner, perhaps by an unidentified protease. Based on the data presented here, we hypothesize that the primary function of FHL-1 binding by T. denticola might be to facilitate adherence to FHL-1 present on anchorage-dependent cells and in the extracellular matrix.     

Cytotoxicity of Hemolytic, Cytotoxic Necrotizing Factor 1-Positive and -Negative Escherichia coli to Human T24 Bladder Cells

Approximately one-half of Escherichia coli isolates from patients with cystitis or pyelonephritis produce the pore-forming cytotoxin hemolysin, a molecule with the capacity to lyse erythrocytes and a range of nucleated cell types. A second toxin, cytotoxic necrotizing factor 1 (CNF1), is found in approximately 70% of hemolytic, but rarely in nonhemolytic, isolates. To evaluate the potential interplay of these two toxins, we used epidemiological and molecular biologic techniques to compare the cytotoxicity of hemolytic, CNF1⁺, and CNF1⁻ cystitis strains toward human T24 bladder epithelial cells in vitro. A total of 29 isolates from two collections of cystitis-associated E. coli were evaluated by using methylene blue staining of bladder monolayers at 1-h intervals after inoculation with each strain. Most (20 of 29) isolates damaged or destroyed the T24 monolayer (less than 50% remaining) within 4 h after inoculation. As a group, CNF1⁺ isolates from one collection (11 strains) were less cytotoxic at 4 h than the CNF1⁻ strains in that collection (P = 0.009), but this pattern was not observed among isolates from the second collection (18 strains). To directly evaluate the role of CNF1 in cytotoxicity of hemolytic E. coli without the variables present in multiple clinical isolates, we constructed mutants defective in production of CNF1. Compared to the CNF1⁺ parental isolates, no change in cytotoxicity was detected in these cnf1 mutants. Our results indicate that CNF1 does not have a detectable effect on the ability of hemolytic E. coli to damage human bladder cell monolayers in vitro.     

Cellular and extracellular protein antigens of Treponema pallidum synthesized during in vitro incubation of freshly extracted organisms

A new medium that permits radiolabeling of freshly extracted cells of Treponema pallidum with [35S]methionine very efficiently has been devised. Although treponemes were not purified free of contaminating rabbit tissue, label was incorporated exclusively into treponemal protein in a linear manner for at least the first 16 h of in vitro incubation. Throughout this period, virtually a full complement of treponemal proteins was synthesized, based on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis comparison of the radiolabeled protein profile with the Coomassie blue-stained profile of gradient-purified treponemes. The radiolabeled protein profiles obtained with three pathogenic strains were very similar but not identical. Using solubilized treponemal extracts and a sensitive radioimmunoprecipitation procedure, we identified the protein antigens of T. pallidum that were recognized by immunoglobulin G antibodies in various rabbit and human syphilitic sera. A simple fractionation procedure has been used to separate soluble and membrane-bound treponemal proteins. A number of the membrane proteins are exposed on the cell surface, since intact radiolabeled treponemes bound antibodies directed against these proteins. In addition, a unique class of low-molecular-weight extracellular treponemal proteins has been identified. The cell surface-exposed proteins were among the earliest proteins recognized by immunoglobulin G antibodies after experimental infection of rabbits with T. pallidum.     

Impact of patient characteristics on performance of nucleic acid amplification tests and DNA probe for detection of Chlamydia trachomatis in women with genital infections

The performance of nucleic acid amplified tests (NAAT) for Chlamydia trachomatis at the cervix and in urine was examined in 3,551 women, and the impacts of clinical findings (age, endocervical and urethral inflammation, menses, and gonococcal coinfection) were assessed. Ligase chain reaction (LCR) and first-generation uniplex PCR were studied relative to an unamplified DNA probe (PACE2) and to an expanded, independent diagnostic reference standard. Relative to the expanded standard, cervical or urine LCR was generally the most sensitive test in most subgroups. Increased detection by NAAT of cervical C. trachomatis over PACE2 was highest among women without mucopurulent endocervical discharge versus those with (relative increase in positivity with cervical LCR, 46%) and among women > or =20 years old versus younger women (relative increase in positivity with cervical LCR, 45%). The sensitivity of cervical PCR was highest when mucopurulent endocervical discharge was present (84%) and highest for cervical LCR when cervical gonococcal coinfection was detected (91%). Urethral inflammation was associated with higher sensitivities of urine LCR (86 compared to 70% when inflammation was absent) and PCR (82 compared to 62% when inflammation was absent). Menses had no effect on test performance. The effects of patient characteristics on test specificities were less pronounced and were closely related to observed sensitivities. These findings support expanded use of NAAT for screening and diagnosis of C. trachomatis in diverse clinical populations of women.     

Up-regulation of endothelial nitric oxide synthase inhibits pulmonary leukocyte migration following lung ischemia-reperfusion in mice

Endogenous nitric oxide (NO) is known to modulate post-ischemic inflammatory response in various organs. However, the role of nitric oxide synthase isoforms (NOS) in mediating pulmonary post-ischemic inflammatory response is poorly understood. We therefore studied post-ischemic endothelial adhesion molecule expression and leukocyte migration in endothelial NOS knockout (eNOS-KO) mice subjected to pulmonary ischemia and reperfusion in vivo. Under anesthesia and mechanical ventilation, the left pulmonary hilum in wild-type (WT) and eNOS-KO mice was clamped for 1 hour, followed by reperfusion for up to 24 hours. In WT mice, we observed a selective up-regulation of both eNOS mRNA and protein in lung tissue, while inducible NOS (iNOS) and neuronal NOS (nNOS) remained unchanged. Survival in eNOS-KO mice was reduced due to severe pulmonary edema, underlining an increased susceptibility to ischemia-reperfusion (I/R) injury. Interstitial tissue infiltration by CD18- and CD11a-positive white blood cells as well as lung tissue water content peaked at 5 hours of reperfusion and were found significantly higher than in WT mice. Enhanced leukocyte-endothelial interaction was associated with pronounced up-regulation of vascular cell adhesion molecule (VCAM) in eNOS-KO mice during post-ischemic reperfusion. We conclude that eNOS attenuates post-ischemic inflammatory injury to the lung most probably via inhibition of endothelial adhesion molecule expression.     

Detection of Chlamydia trachomatis inclusions in Mccoy cell cultures with fluorescein-conjugated monoclonal antibodies.

We compared two methods for identification of Chlamydia trachomatis inclusions in McCoy cell monolayers: conventional iodine staining and immunofluorescence staining with monoclonal antibodies against the species-specific major outer membrane protein antigen of C. trachomatis. Among 878 urethral and cervical specimens tested in parallel, the immunofluorescence method detected eightfold more inclusions per monolayer, identified a higher proportion of positive specimens on first passage (98 versus 62% by iodine staining; P less than 0.01), and improved overall sensitivity (98% of total positive specimens detected versus 84% by iodine staining; P less than 0.01). Improved sensitivity was most evident in specimens with low numbers of inclusions. Compared with conventional iodine staining, immunofluorescence staining with monoclonal antibodies improves sensitivity and offers more rapid detection of chlamydial inclusions in cell culture.     

Large deletions of the MECP2 gene detected by gene dosage analysis in patients with Rett syndrome

The original article to which this Erratum refers was published in Human Mutation 23:234–244 Human Mutation(2004) 23(3) 234–244     

Demonstration of skin-derived antileukoproteinase (skalp) and its target enzyme human leukocyte elastase in squamous cell carcinoma

Skin-derived antileukoproteinase (SKALP), also known as elafin, is a strong and specific inhibitor of elastase and proteinase 3. SKALP is not present in normal epidermis, but is expressed by epidermal keratinocytes under hyperproliferative conditions such as psoriasis, wound healing, and in cell culture. In human epidermal tumours, SKALP is differentially expressed and restricted to tumours with distinct squamous differentiation. We have studied the presence of both SKALP and one of its known target enzymes, leukocyte elastase, in 41 squamous cell carcinomas of the skin. SKALP expression correlated with the degree of differentiation: strong expression was seen in well-differentiated cells and expression was absent in poorly differentiated tumour cells. Most of the squamous cell carcinomas showed elastase-positive cells in the tumour stroma and also within the tumour cell nests. SKALP may interfere with the proteoloytic activity of infiltrating inflammatory cells or with hitherto unknown proteinases from the tumour cells. We hypothesize that in squamous cell carcinoma progressive loss of SKALP expression could facilitate tumour spread.     

Diagnosis of Chlamydia trachomatis urethritis in men by polymerase chain reaction assay of first-catch urine.

To determine the accuracy of a recently developed polymerase chain reaction (PCR) urine assay to detect Chlamydia trachomatis urethral infection in men, we obtained urethral swabs and first-catch urine from 365 men attending a sexually transmitted diseases clinic. Thirty-three (9%) of the 365 men were infected with C. trachomatis as defined by urethral culture. Thirty-two of the 33 men with culture-positive urethral swabs also had PCR-positive urine assays. Of 332 patients with culture-negative urethral swabs, 325 had PCR-negative urine. Compared with chlamydia culture of urethral specimens, PCR assay of urine samples thus had a sensitivity of 97% and a specificity of 98%. The positive predictive value of the urine PCR assay was 82%, and the negative predictive value was 99%. Analysis of discrepant results indicated that six of seven PCR-positive, urethral culture-negative patients probably had chlamydial urethritis. All six patients had symptoms of urethritis and had either a positive urethral swab PCR or a positive urine PCR with a different amplification target. After resolution of discrepant results, (defining true positives as the 33 culture-positive patients and the 6 PCR-positive, culture-negative patients just described), the sensitivity and specificity of culture were 85% (33 of 39) and 100% (326 of 326), respectively. The revised sensitivity and specificity of PCR were 97% (38 of 39) and 99.7% (325 of 326), respectively. We conclude that this urine PCR assay provides a highly sensitive, noninvasive alternative method for the detection of C. trachomatis urethral infection in high-risk men attending a sexually transmitted diseases clinic. This assay could greatly facilitate the testing of larger numbers of male patients for chlamydial infection and should be studied in other settings.