Reactivity tests for supplementary cementitious materials: RILEM TC 267-TRM phase 1

A primary aim of RILEM TC 267-TRM: “Tests for Reactivity of Supplementary Cementitious Materials (SCMs)” is to compare and evaluate the performance of conventional and novel SCM reactivity test methods across a wide range of SCMs. To this purpose, a round robin campaign was organized to investigate 10 different tests for reactivity and 11 SCMs covering the main classes of materials in use, such as granulated blast furnace slag, fly ash, natural pozzolan and calcined clays. The methods were evaluated based on the correlation to the 28 days relative compressive strength of standard mortar bars containing 30% of SCM as cement replacement and the interlaboratory reproducibility of the test results. It was found that only a few test methods showed acceptable correlation to the 28 days relative strength over the whole range of SCMs. The methods that showed the best reproducibility and gave good correlations used the R3 model system of the SCM and Ca(OH)2, supplemented with alkali sulfate/carbonate. The use of this simplified model system isolates the reaction of the SCM and the reactivity can be easily quantified from the heat release or bound water content. Later age (90 days) strength results also correlated well with the results of the IS 1727 (Indian standard) reactivity test, an accelerated strength test using an SCM/Ca(OH)2-based model system. The current standardized tests did not show acceptable correlations across all SCMs, although they performed better when latently hydraulic materials (blast furnace slag) were excluded. However, the Frattini test, Chapelle and modified Chapelle test showed poor interlaboratory reproducibility, demonstrating experimental difficulties. The TC 267-TRM will pursue the development of test protocols based on the R3 model systems. Acceleration and improvement of the reproducibility of the IS 1727 test will be attempted as well

Advancing Prevention of STIs by Developing Specific Serodiagnostic Targets: Trichomonas vginalis as a Model

Point-of-Care (POC) serum antibody screening of large cohorts of women and men at risk for the sexually transmitted infection (STI) caused by Trichomonas vaginalis requires the availability of targets with high specificity. Such targets should comprise epitopes unique to T. vaginalis immunogenic proteins detected by sera of women and men patients with trichomonosis but not uninfected controls. Three enzymes to which patients make serum IgG antibody were identified as fructose-1,6-bisphosphate aldolase (A), α-enolase (E), and glyceraldehyde-3-phosphate dehydrogenase (G). Epitopes within these proteins were identified that had no sequence identity to enzymes of humans and other pathogens. Therefore, I constructed a chimeric recombinant String-Of-Epitopes (SOE) protein consisting of 15-mer peptides, within which are the epitopes of A, E, and G. This novel protein of ~36-kD is comprised of two epitopes of A, ten epitopes of E, and seven epitopes of G (AEG::SOE2). The AEG::SOE2 protein was detected both by immunoblot and by enzyme-linked immunosorbent assay (ELISA) using highly reactive sera of women and men but not negative serum unreactive to T. vaginalis proteins. Finally, AEG::SOE2 was found to be immunogenic, as evidenced by serum IgG from immunized mice. I discuss how this approach is important in relation to infectious disease diagnostic targets for detection of serum IgG antibody in exposed and/or infected individuals and how such novel targets may have potential as subunit vaccine candidates against microbial pathogens

Point-of-Care Diagnostic for <i>Trichomonas vaginalis</i>, the Most Prevalent, Non-Viral Sexually Transmitted Infection

A point-of-care (POC) diagnostic is needed for both women and men to establish universal screening and surveillance for the number one, non-viral sexually transmitted infection (STI) caused by Trichomonas vaginalis. We developed a POC diagnostic for this STI using the MedMira Rapid Vertical Flow (RVF®) Technology test cartridge with a membrane that includes a Vertical procedural/reagent control line (referred to as CVL) and spotted with 1 µg of a 72.4-kDa truncated version of α-actinin called ACT::SOE3. This protein is a specific diagnostic target for antibody in sera of individuals with trichomoniasis. Serum antibody to ACT::SOE3 is a positive reaction with the test spot. Specificity of ACT::SOE3 was revealed with monoclonal antibodies (MAbs) generated to ACT::SOE3. Addition of negative control serum with MAb 67B reactive to ACT::SOE3 shows detection of both ACT::SOE3 and the CVL. Only positive sera of individuals had antibody reactive with ACT::SOE3 and detected the presence of the spot and the CVL. Negative control sera were unreactive with ACT::SOE3 and only showed the presence of the CVL. Importantly, to show proof-of-principle for POC application, ACT::SOE3 was detected with the positive patient sera spiked with whole blood. Finally, packaged cartridges stored with desiccant packs at 37 °C for one year gave identical results with the positive and negative human sera. Our results show the validity of this new POC serodiagnostic for this STI

Surface-Associated Host Proteins on Virulent Treponema pallidum

A surface coat of host serum proteins was detected on virulent Treponema pallidum by sodium dodecyl sulfate-gel electrophoresis. The loosely associated serum proteins could be removed by repeated washings in a protein-free medium. Washed T. pallidum retained the ability to readsorb numerous host proteins from rabbit serum as well as iodinated rabbit or human albumin. In addition, various avidly associated host serum proteins including albumin, α 2 -macroglobulin, transferrin, ceruloplasmin, immunoglobulin G, immunoglobulin M, and C3 were identified on the outer envelope of washed treponemes by an immunoadsorbent technique with protein A-bearing staphylococcus. Hyaluronidase treatment did not remove the avidly associated host proteins from the surface of washed treponemes, whereas trypsin treatment resulted in decreased levels of agglutination. Electrophoretic patterns of trypsin-treated treponemes showed that treponemal proteins as well as adsorbed host proteins were released concurrently by protease digestion. Reacquisition studies involving α 2 -macroglobulin and transferrin suggested the presence of noncompetitive binding sites for serum proteins on the treponemal outer envelope. Finally, differences among the T. pallidum preparations from individual rabbits with respect to incorporation of [ 35 S]methionine, extent of agglutination with antisera, and length of time required for removal of avidly associated host proteins by trypsin treatment indicated biological variability among the treponemal populations

Surface Characterization of Virulent Treponema pallidum

Characterization of the surface of Treponema pallidum was accomplished by [(125)I]lactoperoxidase-catalyzed iodination of intact organisms and sensitive radioimmunoprecipitation and gel electrophoresis technology. At least 11 outer membrane proteins with molecular weights ranging from 89,000 (89K) to 20K were identified, and all elicited high titers of antibody in experimentally infected rabbits. Proteins of 89.5K, 29.5K, and 25.5K previously implicated as ligands involved in attachment (J. B. Baseman and E. C. Hayes, J. Exp. Med. 151:573-586, 1980) were found to reside on the treponemal surface. Low levels of the 89.5K treponemal protein were released by high salt concentrations, whereas the remaining comigrating material was neither radioiodinated nor released with selective detergents. Other lower-molecular-weight (60K, 45K, and 30K) surface proteins were extracted with octyl glucoside detergent, suggesting their hydrophobic interaction with the external membrane. The molecular organization of surface proteins was studied by employing the cross-linker dithiobis(succinimidyl)-propionate, and data suggested the presence of a highly fluid envelope resulting in random collisions by the surface proteins. The biological function of the treponemal outer envelope proteins was evaluated using, as the indicator system, adherence of T. pallidum to monolayer cultures of eucaryotic cells. Trypsin treatment of motile, freshly harvested organisms decreased the extent of surface parasitism to normal rabbit testicular cells, reinforcing the idea of the proteinaceous nature and role of treponemal ligands for attachment. Other data supported functional and antigenic relatedness among the implicated ligands. Finally, brief periodate treatment of human epithelial (HEp-2) and normal rat testicular cells as well as casein-elicited rabbit peritoneal macrophages significantly reduced the extent of treponemal parasitism, suggesting a role of specific host membrane molecules as mediators of attachment

Trichomonas vaginalis adherence mediates differential gene expression in human vaginal epithelial cells

Trichomonas vaginalis, an ancient protist, colonizes the vaginal mucosa causing trichomonosis, a vaginitis that sometimes leads to severe health complications. Preparatory to colonization of the vagina is the adhesion to vaginal epithelial cells (VECs) by trichomonads. We hypothesized that VECs alter the gene expression to form a complex signalling cascade in response to trichomonal adherence. In order to identify the genes that are upregulated, we constructed a subtraction cDNA library after contact with parasites that is enriched for differentially expressed genes from the immortalized MS-74 VECs. Sixty cDNA clones were sequenced and to our knowledge for the first time, differentially regulated genes were identified in response to early trichomonal infection. The identified genes were found to encode functional proteins with specific functions associated with cell structure maintenance and extracellular matrix components, proinflammatory molecules and apoptosis. Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) confirmed expression of selected genes. Further, cyclooxygenase 2 (COX-2) protein expression was analysed using Western blot and immunofluorescence assays. Data suggest that p38 mitogen-activated protein (MAP) kinase and tyrosine kinases play a role in COX-2 induction. Finally, T. vaginalis and Tritrichomonas foetus but not Pentatrichomonas hominis induce expression of COX-2. This is a first attempt at elucidating the basis of interaction of trichomonads with host cells and the corresponding host responses triggered by the parasites