Evaluation of Western immunoblotting technique in the serological diagnosis of human syphilitic infections

Purified human syphilitic antibodies against both 15.5 Kd and 45 Kd treponemel antigens appear T. pallidum specific and do not cross react with antigens possessed by other treponemes (T. phagedenis, T. hyodysenteriae and a human intestinal treponeme).By using Western immunoblotting technique, 107 out of 110 syphilitic patients and 291 out of 294 subjects with serologically positive diagnostic tests for syphilis were found to have in their sera antibodies against a 15.5 Kd specific antigen of T. pallidum. These antibodies were present in 100% of the patient with secondary or early latent syphilis, both untreated and treated, in 98.24% of those with late latent treated syphilis and in 100% of patients with neurosyphilis.On the contrary, they were absent in 47 patients with false positive reactions for syphilis and in 121 healthy blood donors. For these reasons, the demonstration of these kind of antibodies in a patient's serum can be considered of high value in differentiating syphilitic patients from non infected individuals.Corresponding author.     

Electron microscopy studies of human intestinal spirochetes

The ultrastructure of twenty human intestinal spirochetes was analyzed using the electron microscope. Negatively stained cells were generally found to be loosely and irregularly waved. The isolates had cell dimensions ranging from 0.12–0.35 m in width and from 3.9–14.2 m in length. Twin bundles of flagella were present in the space between the cytoplasmic membrane and the outer membrane. The majority of isolates had five flagella inserted sub-terminally at each cell end. Human intestinal spirochetes divide by binary fission. They are morphologically similar to swine intestinal treponemes, both pathogenic (Treponema hyodysenteriae) and non pathogenic (Treponema innocens), and different from Treponema pallidum, Treponema phagedenis and Borrelia burgdorferi.Following treatment with sodium deoxycolate, no bundles of cytoplasmic microtubules were observed in cells obtained from cultures of human and swine intestinal spirochetes or from cells of B. burgdorferi, while these structures were present in similarly treated cells of T. pallidum and T. phagedenis.Corresponding author.     

Morphology of Treponema pallidum

In recent years many investigations have been carried out on the morphology of Treponema pallidum by means of the electron microscope, and the use of ultra-thin sections has shown up a number of structural details. However, there is still need for much more evidence before the internal structure of treponemes can be elucidated fully and the functions of the structures interpreted. To provide such evidence, the authors have examined under the electron microscope negative-stained treponemes and ultra-thin sections, using both cultivated strains and treponemes obtained direct from syphilids in people suffering from fresh secondary syphilis. It has been shown that treponemes have a complex structure. T. pallidum has a two-layered outer wall, a cytoplasmic membrane proper, cytoplasm and a bunch of fibrils following a different path in different places on the treponeme. The sites of insertion of the fibrils (the basal granules) were investigated; structures similar to mesosomes and nucleoids were found. Cysts and granular forms are described.     

In vitro cultivation of Treponema pallidum: a review

In vitro cultivation of Treponema pallidum would facilitate many different aspects of syphilis research. This review summarizes developments in this field that have been published since 1975. Findings are discussed in terms of treponemes and the oxygen question, treponemal metabolism involving proteins, nucleic acids, and fatty acids, and treponemal interaction with tissue culture cells. Suggested future approaches and potential problem areas pertinent to successful cultivation are discussed.     

Antigen-specific cytolysis of infected cells in murine candidiasis

Immune L3T4+ and Lyt-2+ lymphocytes play an important role in the acquired resistance of mice to challenge with virulent Candida albicans, and release macrophage-activating cytokines in response to yeast cells in vitro. To determine whether antigen (Ag)-specific cytotoxic T lymphocytes are generated during fungal infection, purified L3T4+ and Lyt-2+ lymphocytes from immunized mice were cultured in the presence of syngeneic accessory cells, Candida Ag, and IL-2. Yeast-infected bone marrow macrophages and peritoneal exudate neutrophils were used as target cells in a standard ⁵¹Cr release assay. Ag-specific, MHC-unrestricted lysis of infected macrophages was evident with immune Lyt-2+ cells after 5–10 days in culture. Under the same experimental conditions, the cytotoxic activity of L3T4+ cells was negligible, but its expression could be induced by the addition of anti-CD3 antibody.Culturing immune Lyt-2+ cells for shorter periods of time (1–2 days) resulted in preferential lysis of infected neutrophils. In addition, at limiting effector cell numbers, Ag-specific MHC-restricted lymphocytes with cytotoxic activity to infected macrophages could be identified. We suggest that C. albicans infection stimulates multiple cytotoxic T-cell precursors with varying recognition stringency, wich may have an important role in antifungal resistance in vivo.     

Ginkgo biloba modulates glutathione redox cycle in vascular endothelial cells

We recently reported that Ginkgo biloba extract (GBE) suppressed oxidative burst in macrophages and protected bovine pulmonary artery endothelial cells (PAEC) from oxidant injury. In this study the effects of GBE on glutathione (GSH) redox cycle and activity of antioxidant enzymes were investigated. Confluent monolayers of PAEC were incubated with GBE for 8–48 h, washed, and then lysed with Triton X-100. Biochemical assays were performed with the lysate. GBE caused both dose- and time-dependent increase in GSH level and glutathione disulfide (GSSG) reductase activity while GSH peroxidase and superoxide dismutase activity remained unaffected. Exposure of PAEC to an organic oxidant tert-butyl hydroperoxide (tBHP) resulted in decreased GSH level, increased lipid peroxidation, and elevated leakage of intracellular lactate dehydrogenase. Preincubation or simultaneous treatment of PAEC with GBE prevented these changes induced by tBHP. Our data suggest that the antioxidant effect of GBE may be due to its modulation of the GSH redox cycle in PAEC as well as direct scavenging of the oxidant.     

Histidine-rich glycoprotein derived peptides affect endometrial angiogenesis in vitro but has no effect on embryo development

Histidine-rich glycoprotein (HRG) is an abundant plasma protein involved in multiple biological processes including immunology, vascularisation, and coagulation. These processes are of importance in regulating embryo development and implantation. A specific polymorphism in the HRG gene, HRG C633T, has an impact on various aspects of fertility, such as oocyte quality, endometrial receptivity, and possibly the capacity of the embryo itself to implant. To further examine the potential role of the HRG C633T polymorphism in regulating endometrial angiogenesis and on embryo development, two HRG peptides were constructed. These HRG peptides correspond to the amino acids 169-203 of the protein which, in turn, reflects the C633T polymorphism in the gene. The HRG proline or serine peptides were added to cultures of primary human endometrial endothelial (HEE) cells and to human embryos in vitro. The HRG peptides inhibited vascular endothelial growth factor (VEGF) induced proliferation and migration and promoted tube formation of HEE cells. The embryos were monitored using a time-lapse system (EmbryoScope®). Except for a prolonged time from first cleavage after thawing to development of the morula, no difference in embryo morphokinetics or embryo quality was noted in human embryos cultured in the presence of the HRG proline peptide. Taken together, these results suggest that treatment with a specific HRG peptide might prime the endometrium for implantation and be beneficial for adequate placentation. However, addition of a specific HRG proline peptide to human embryos has no beneficial effects in terms of embryo development.

Abbreviations: HRG: histidine-rich glycoprotein; HEE: human endometrial endothelial; VEGF: vascular endothelial growth factor; TSP: thrombospondin; SNP; single nucleotide polymorphism; IVF: in vitro fertilization; CLESH-1: CD36 LIMPII Emp structural homology domain-1; ECM: endothelial cell medium; FBS: fetal bovine serum; cDNA: complementary DNA     

The protease from Vibrio Cholerae nicks arginine at position 192 from the N-terminus of the heat-labile enterotoxin a subunit from enterotoxigenic Escherichia Coli

It was examined where a protease purified from Vibrio cholerae might nick the heat-labile enterotoxin (LT) A subunit from enterotoxigenicEscherichia coli.LT was digested by the protease and contained a fragment which had the same mobility on SDS-PAGE as that of the Al fragment of LT digested by trypsin. The biological activity of LT by this protease was also identical to that of LT by trypsin. The amino acid sequence of the N-terminus of the A2-like fragment was Thr-Ser-Thr-Gly, which corresponded to the sequence from 193 to 196 of the A subunit.These data suggest that this protease, like trypsin, nicks arginine at position 192 from the N-terminus of the A subunit and that the biological activation of LT by this protease is similar to that by trypsin.Corresponding author.     

Specific primers for PCR amplification of the ITS1 (ribosomal DNA) of Trypanosoma lewisi

Trypanosoma lewisi is a mild or non-pathogenic parasite of the sub-genus Herpetosoma transmitted by fleas to rats. In a previous study we described pan-trypanosome specific primers TRYP1 which amplify the ITS1 of ribosomal DNA by hybridizing in highly conserved regions of 18S and 5.8S genes. These primers proved to be useful for detecting T. lewisi DNA in laboratory rats, but a recent large scale survey in wild rodents demonstrated a lack of specificity. In the present study, we designed and evaluated mono-specific primers LEW1S and LEW1R, for the detection and identification of T. lewisi by a single-step PCR. These primers were designed inside the highly variable region of the ITS1 sequence of T. lewisi ribosomal DNA. The product size of 220 bp is specific to T. lewisi. The sensitivity limit was estimated between 0.055 and 0.55 pg of DNA per reaction, equivalent to 1–10 organisms per reaction. All the PCR products obtained from 6 different T. lewisi isolates were more than 98% similar with each other and similar to the sequences of T. lewisi already published in Genbank. All DNA of 7 T. lewisi stocks from China gave the specific 220 bp product. We showed that LEW1S and LEW1R primers enabled sensitive detection and identification of T. lewisi infection in laboratory and wild rats. This assay is recommended for monitoring T. lewisi infections in rat colonies or for studying infections in the wild fauna. An absence of cross reaction with human DNA means that these primers can be used to investigate atypical trypanosome infections in humans. Given the risk of T. lewisi infection in human, we believe that these primers will be beneficial for public health diagnosis and rodents investigation programmes.     

The hemolysin of escherichia coli

Many strains of E. coli elaborate a hemolysin which is responsible for the zone of -hemolysis surrounding bacterial colonies on blood agar. The significance of this cytolysin as a determinant of bacterial pathogenicity has been established in animal models with the use of genetically engineered, isogenic bacterial strains. An analogous role in human infections has been inferred from the high association of hemolysin production with disease. Studies at a molecular genetical level have defined 4 genes that are required for the synthesis, post,translational modification and secretion of the hemolysin. The structural gene hlyA encodes for a 107-110 000 polypeptide, which must be modified in an unknown manner to its active form by the product of the neighboring hlyC gene. Genes hlyb and hlyD encode for proteins that export the molecule to the extracellular medium. The signal for secretion is contained in the C-terminal portion of the toxin molecule. The secreted hemolysin attacks plasma membranes of target mammalian cells by inserting as a monomer into the bilayer and generating hydrophilic transmembrane pores of approximately 2 nm effective diameter. The pores display a marked selectivity for cations over anions and pore-opening is dependent on the presence of a correct transmembrane potential. Binding to a membrane target does not require the presence of a specific receptor, and pores may be generated in planar lipid membranes consisting solely of phosphatidylcholine. Pore formation in nucleated cells can trigger secondary reactions such as stimulation of arachidonate metabolism with release of lipid mediators, probably initiated by passive influx of extracellular Ca²⁺. Perfusion of subcytolytic doses through isolated and ventilated rabbit lungs induces lung edema that is at least partially due to such secondary events. The capacity of E. coli hemolysin to damage human tissues via primary and secondary processes is consistent with the concept of its pathogenic role in human infections.[/p]Corresponding author.     

Vibrio cholerae 01 can assume a ‘rugose’ survival form that resists killing by chlorine,yet retains virulence

Vibrio cholerae 01 is able to shift between smooth and rugose colonial morphologies. Cultures of smooth V. cholerae strains were inactivated in less than 20 s at a concentration of 1.0 mg l^‐1 free chlorine. In contrast, cultures of rugose variants exposed to this concentration of chlorine showed an initial rapid drop in viable counts, followed by persistence of a protected subpopulation of cells. Viable V. cholerae could still be recovered from rugose cultures even after exposure to 2.0 mg l^‐1 free chlorine for 30 min. Preliminary studies suggest that resistance to killing by chlorine was due to formation of cell aggregates enclosed in a gelatinous mucoid material. Rugose strains appeared to be fully virulent, based on their ability to adhere to Caco‐2 cells and elicit fluid accumulation in rabbit ileal loops. Our data suggest that the V. cholerae rugose phenotype represents a fully virulent survival form of the organism that can persist in the presence of free chlorine.     

Genetic diversity in Treponema pallidum: implications for pathogenesis, evolution and molecular diagnostics of syphilis and yaws

Pathogenic uncultivable treponemes, similar to syphilis-causing Treponema pallidum subspecies pallidum, include T. pallidum ssp. pertenue, T. pallidum ssp. endemicum and Treponema carateum, which cause yaws, bejel and pinta, respectively. Genetic analyses of these pathogens revealed striking similarity among these bacteria and also a high degree of similarity to the rabbit pathogen, Treponema paraluiscuniculi, a treponeme not infectious to humans. Genome comparisons between pallidum and non-pallidum treponemes revealed genes with potential involvement in human infectivity, whereas comparisons between pallidum and pertenue treponemes identified genes possibly involved in the high invasivity of syphilis treponemes. Genetic variability within syphilis strains is considered as the basis of syphilis molecular epidemiology with potential to detect more virulent strains, whereas genetic variability within a single strain is related to its ability to elude the immune system of the host. Genome analyses also shed light on treponemal evolution and on chromosomal targets for molecular diagnostics of treponemal infections.     

A micro-capture ELISA for detecting Mycoplasma pneumoniae IgM: comparison with indirect immunofluorescence and indirect ELISA

The biochemical and biological properties of the flagella of Campylobacter jejuni have been investigated using two variants selected from a flagellate, motile clinical isolate (strain 81116): a flagellate, non-motile variant (SF-1) and an aflagellate variant (SF-2). Phenotypic and biochemical analysis of the strains and amino acid analysis of the isolated flagella suggest that the variants differed from the wild-type strain only in the absence of flagella and/or motility. The aflagellate variant poorly colonized the gastrointestinal tract of infant mice but the flagellate, non-motile variant colonized the mice as successfully as the wild-type strain. 35S-labelled organisms were used to investigate the attachment of the variants to human epithelial cell monolayers in vitro. The flagellate, non-motile strain attached more efficiently to the cells than the wild-type strain or the aflagellate strain. Differences in attachment suggest that an adhesin is intimately associated with flagella of C. jejuni and that active flagella mediate only a tenuous association with host cells. This adhesin attached most efficiently to cells of intestinal epithelial origin and was not specifically inhibited by various sugars.     

Non-pathogenic Borrelia burgdorferi expressing Treponema pallidum TprK and Tp0435 antigens as a novel approach to evaluate syphilis vaccine candidates

Background

Syphilis is resurgent in many developed countries and still prevalent in developing nations. Current and future control campaigns would benefit from the development of a vaccine, but although promising vaccine candidates were identified among the putative surface-exposed integral outer membrane proteins of the syphilis spirochete, immunization experiments in the rabbit model using recombinant antigens have failed to fully protect animals upon infectious challenge. We speculated that such recombinant immunogens, purified under denaturing conditions from Escherichia coli prior to immunization might not necessarily harbor their original structure, and hypothesized that enhanced protection would result from performing similar immunization/challenge experiments with native antigens.

Severe damage of cultured vascular endothelial cell monolayer after simultaneous exposure to cadmium and lead

This study evaluates the toxicity of lead in the presence or absence of cadmium on the maintenance of a monolayer of cultured bovine aortic endothelial cells. It was histologically shown that lead at 50 M or less induced a slight formation of de-endothelialized areas on the monolayer after a 24-h incubation. Although lead accumulated in endothelial cells in a dose-dependent manner, the cytotoxicity (evaluated by the [³H]adenine release assay) was not detected. However, cadmium (1.0 or 2.0 M)-induced de-endothelialized areas on the monolayer was considerably larger in the presence of lead at 10 M; the cytotoxicity of cadmium was significantly enhanced by lead. It was concluded that vascular endothelial cell monolayer is severely destroyed by a greater cytotoxicity of combination of cadmium with lead.     

Pseudomonas entomophila and Pseudomonas mendocina: Potential models for studying the bacterial type VI secretion system

A diversity of molecular translocation mechanisms, including various secretion systems, has been elaborated in host–bacterial interactions. The newly described type VI secretion system (T6SS) appears to be involved in bacterial pathogenesis by acting as a nano-syringe, contributing in translocation of several effector-proteins into the eukaryotic host cell cytoplasm. Recent evidences revealed the involvement of T6SS machinery in inter-bacterial interactions.Several Pseudomonas species are found to harbour multiple and well organised T6SS loci, however, their genomic structural similarities as well as phylogenetic divergence suggest an independent evolution. Until now elementary evidence was provided for the presence of T6SS in the genomes of Pseudomonas entomophila (Pen), an aggressive insect pathogen as well as the human opportunistic pathogen Pseudomonas mendocina (Pme).In this report we evidenced by in silico genome mining along with bioinformatic analysis the presence of genes encoding for putative T6SS core components and secreted proteins in the sequenced Pen L48 and Pme ymp, strains and designated their putative promoters, sigma factors binding sites and various regulatory proteins. Moreover, we investigated the phylogenetic relatedness of four T6SS core proteins from these strains with their orthologues from various Pseudomonas species.Our analysis revealed two phylogenetically distinguishable T6SS loci in the genome of Pme that appeared to be highly homologous to Pseudomonas aeruginosa Hcp-Secretion Island-I (HSI-I) and -II. Our findings suggest that Pme could be excellent additional to P. aeruginosa model, for the elucidation of HSI-I and -II biological role(s), avoiding the overlapping activity HSI-III (Lesic et al., 2009), which is missing from Pme's genome.Likewise, our analysis revealed the presence of a unique entire T6SS in Pen genome, which appears to be phylogenetically close to Pme T6SS-II and P. aeruginosa HSI-II. Since Pen lacks the common secretion systems T3SS and T4SS, the single T6SS locus could have an enforced role in the insect–bacterial interactions, providing thus a promising model for studying its biological function.     

Isolation ofCoxiella burnetii and of an unknown rickettsial organism fromIxodes ricinus ticks collected in Austria

Two strains ofCoxiella burnetii and two strains of an unidentified rickettsial organism were isolated for the first time fromIxodes ricinus ticks collected in the Alpine region of Tirol, Austria. TheC. burnetii strains belong to the group of agents causing acute forms of Q fever. The other two strains of isolated rickettsial agent share some antigenic epitopes withC. burnetii andR. prowazekii but they differ from them by their high sensitivity to freezing and refreezing and by poor multiplication in yolk sacs of chick embryos. There is at present no evidence that these organisms cause human illness and no ecological information is available. We suggest they may be some new species of rickettsiae or rickettsia-like organisms.     

A one-day Treponema pallidum immobilization test

The author has previously shown that the addition of egg-white lysozyme to the reaction mixture used in the Treponema pallidum immobilization test (TPI test) can reduce the time required for immobilization from 18 hours to as little as 6 hours. This opened up the possibility of developing a one-day TPI test and of further simplifying the procedure, as the conditions do not need to be so strictly controlled to ensure survival of the treponemes for the shorter time. In this paper it is shown that the standard procedure of incubation under an atmosphere of nitrogen and carbon dioxide can be replaced by incubation under a layer of liquid paraffin. The survival of treponemes is satisfactory under these conditions and the oil technique does not alter the sensitivity of the 6-hour test with added lysozyme. Comparative tests using the standard procedure, the lysozyme-gas technique and the lysozyme-oil technique showed that both modifications give the same degree of sensitivity and specificity after incubation for 6 hours as does the standard method. The feasibility of introducing these modifications into routine laboratory practice is discussed.