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PubMed1969

Effect of the fluorescein to protein ratio on anti-Treponema pallidum conjugates.

The effect of using different labeling techniques and various amounts of fluorescein-isothiocyanate was tested with several pools of antisera to Treponema pallidum. Optimal labeling occurred in all serum pools at a fluorescien to protein ratio of 15. The use of pure dye and the dialysis method of labeling is recommended as the method of choice because of its effectiveness in the reduction of nonspecific staining.

Animals↗
PubMed1970

Separation of Treponema pallidum from tissue debris through continuous-particle electrophoresis.

Treponema pallidum can only be cultured in living animal tissue, such as rabbit testes. However, the extract of these organisms from the testicular material leaves the T. pallidum contaminated with tissue debris. This paper describes the separation of T. pallidum from the debris by continuous-particle electrophoresis. The importance of equilibration time before electrophoresis is discussed.

Animals↗
PubMed1970

Passage of Treponema pallidium through membrane filters of various pore sizes.

The passage of Treponema pallidum through commercially available Millipore membrane filters of various pore sizes was examined. No microscopically detectable organisms passed through a filter with a pore diameter of 0.22 mum. As pore size was increased, progressively more organisms passed through. Motile organisms passed through filters to a greater extent than nonmotile ones; however, 22% of the motile and 50% of the nonmotile T. pallidum organisms did not pass through the largest pore diameter tested (14.0 mum). Filtration of T. pallidum suspensions through membrane filters may offer a way of separating the organisms from larger particles of debris which accompany their extraction from rabbit testicular syphilomas.

Animals↗
PubMed1972

Separation of Treponema pallidum from tissue substances by continuous-flow zonal centrifugation.

The zonal ultracentrifuge was used for separation of Treponema pallidum from large volumes of rabbit testicular syphiloma extracts by continuous-flow centrifugation in a cesium chloride density gradient. The gradient was linear with radius from a density of 1.05 to 1.36 g/ml. Operating speeds were 15,000 rev/min for the continuous-flow phase and 25,000 rev/min for a 30-min banding period. A total of 9 x 10(9) (24.3%) treponemes were recovered from the original extract. Of the treponemes recovered, 88% formed a band at a density of 1.170 to 1.211 g/ml. Within the limits of present methods of assay, these fractions were relatively free from testicular particulates and protein when compared with treponemes recovered after differential centrifugation. Observations of the isolated fractions by dark-field and electron microscopy indicated a lack of gross morphological damage to T. pallidum. Their antigenic characteristics were also retained, as evidenced by their ability to react with syphilitic sera in the indirect fluorescent-antibody procedure.

Animals↗
PubMed1973

Conditions that affect the colorimetric analysis of lipopolysaccharide from Escherichia coli and Treponema pallidum.

Material extracted from the Nichols nonpathogenic strain of Treponema pallidum by phenol-water was analyzed by employing a recently reported colorimetric test for detection of lipopolysaccharide (LPS). The fraction isolated from T. pallidum, in combination with the reagent dye, absorbed maximally at a wavelength in the range reported to be positive for LPS. Comparison of this reaction to that of a commercial preparation of Escherichia coli LPS revealed that time and temperature of incubation of the LPS-dye complexes were important variables which had marked but different effects on the LPS of the two sources. However, with careful control of these parameters, concentration-dependent standard curves were established for LPS of both sources. Our results indicate the cell wall of T. pallidum is similar to that of gram-negative organisms.

Absorption↗
PubMed1994

Flow cytometric analysis of peripheral blood lymphocyte immunophenotypes in persons infected with Treponema pallidum.

To characterize the human immune response to syphilis, we determined the effect of infection with Treponema pallidum on the percentage of the various lymphocyte subpopulations in the peripheral blood of infected and uninfected persons. Monoclonal antibodies labeled with either fluorescein isothiocyanate or phycoerythrin were used to perform dual color analysis on a FACScan with the following markers: CD3 for total T cells, CD4 for T helper cells, CD8 for T suppressor cells, CD19 for B cells, and CD16 plus CD56 for natural killer cells. Lymphocyte immunophenotype results were analyzed by the stage of untreated syphilis and by gender. Although they were within the ranges of the normal distribution of immunophenotypes, the percentages of CD4+ cells were significantly lower (P < 0.001) and those of CD8+ cells were higher (P = 0.03) in patients with syphilis than in the uninfected population. For infected versus uninfected subjects, both women and men, the differences in the mean percentages of CD3+ and CD4+ cells were significant (P < or = 0.05). Significant differences were noted between the sexes in secondary syphilis only in the mean percentages of cells positive for CD3, CD4, CD8, and CD16 plus CD56. Gender had no effect on lymphocyte subpopulations in subjects with primary or latent syphilis. In the control population, significant differences due to gender were observed in the percentages of cells positive for CD3, CD4, and CD16 plus CD56.

B-Lymphocytes↗
PubMed1994

Evaluation of a Treponema pallidum enzyme immunoassay as a screening test for syphilis.

The CAPTIA Syphilis-G enzyme immunoassay for the detection of antibodies to Treponema pallidum was evaluated as a screening test for syphilis in comparison with the standard rapid plasma reagin (RPR) test. One thousand samples were tested, and the standard fluorescent treponemal antibody absorption test and the standard microhemmaglutination test were used to confirm the presence of treponemal antibodies. Diagnosis of syphilis was based on traditional standard serology results. Clinical data used in the diagnosis of patients whose samples yielded conflicting results were provided by physicians. Initially, 7 patients whose samples were reactive in the RPR test and 14 patients whose samples yielded positive or equivocal results in the CAPTIA Syphilis-G test were diagnosed as not being infected. After discrepancies due to technical problems were reconciled, samples from six patients remained reactive in the RPR test and that from one patient remained positive in the CAPTIA Syphilis-G test. In addition, seven patients whose samples were nonreactive in the RPR test and two patients whose samples were negative in the CAPTIA Syphilis-G test were diagnosed as having untreated syphilis. After discrepancies were reconciled, samples from five patients remained nonreactive in the RPR test and none remained negative in the CAPTIA Syphilis-G test. Final results indicate that the specificities are 99.4 and 99.9%, respectively. In addition to the improved sensitivity and specificity of the CAPTIA Syphilis-G screen, other potential benefits of this assay lead us to believe that this method could serve as a better screening tool than the RPR test.

Evaluation Studies as Topic↗
PubMed2005

Evaluation of LIAISON Treponema Screen, a novel recombinant antigen-based chemiluminescence immunoassay for laboratory diagnosis of syphilis.

The purpose of this study was to evaluate the diagnostic performance of LIAISON Treponema Screen (DiaSorin, Saluggia, Italy), a new automated chemiluminescence immunoassay (CLIA), in comparison with that of rapid plasma reagin (RPR) and the following currently used treponemal tests: hemagglutination test (TPHA), immunoenzymatic assay (EIA), and Western blot (WB). First, a retrospective study was performed with a panel of 2,494 blood donor sera, a panel of 131 clinical and serologically characterized syphilitic sera, and 96 samples obtained from subjects with potentially interfering diseases or conditions. A prospective study was also performed by testing 1,800 unselected samples submitted to the Microbiology Laboratory of the St. Orsola Hospital in Bologna, Italy, for routine screening for syphilis. As expected, RPR was the least specific method, especially when potentially cross-reacting sera were tested. On the contrary, all of the treponemal tests proved to be very specific (99.9%) and they performed with the following sensitivities: 100% (WB), 99.2% (CLIA), 95.4% (EIA), and 94.7% (TPHA).

Antigens, Bacterial↗
PubMed2001

Western immunoblotting with five Treponema pallidum recombinant antigens for serologic diagnosis of syphilis.

Five immunodominant Treponema pallidum recombinant polypeptides (rTpN47, rTmpA, rTpN37, rTpN17, and rTpN15) were blotted onto strips, and 450 sera (200 from blood donors, 200 from syphilis patients, and 50 potentially cross-reactive) were tested to evaluate the diagnostic performance of recombinant Western blotting (recWB) in comparison with in-house whole-cell lysate antigen-based immunoblotting (wclWB) and T. pallidum hemagglutination (MHA-TP) for the laboratory diagnosis of syphilis. None of the serum specimens from blood donors or from potential cross-reactors gave a positive result when evaluated by recWB, wclWB, or MHA-TP. The evaluation of the immunoglobulin G immune response by recWB in sera from patients with different stages of syphilis showed that rTmpA was the most frequently identified antigen (95%), whereas only 41% of the specimens were reactive to rTpN37. The remaining recombinant polypeptides were recognized as follows: rTpN47, 92.5%; rTpN17, 89.5%; and rTpN15, 67.5%. The agreement between recWB and MHA-TP was 95.0% (100% with sera from patients with latent and late disease), and the concordance between wclWB and MHA-TP was 92.0%. The overall concordance between recWB and wclWB was 97.5% (100% with sera from patients with secondary and late syphilis and 94.6 and 98.6% with sera from patients with primary and latent syphilis, respectively). The overall sensitivity of recWB was 98.8% and the specificity was 97.1% with MHA-TP as the reference method. These values for sensitivity and specificity were slightly superior to those calculated for wclWB (sensitivity, 97.1%, and specificity, 96.1%). With wclWB as the standard test, the sensitivity and specificity of recWB were 98.9 and 99.3%, respectively. These findings suggest that the five recombinant polypeptides used in this study could be used as substitutes for the whole-cell lysate T. pallidum antigens and that this newly developed recWB test is a good, easy-to-use confirmatory method for the detection of syphilis antibodies in serum.

Antigens, Bacterial↗
PubMed2006

Quantitative analysis of tpr gene expression in Treponema pallidum isolates: Differences among isolates and correlation with T-cell responsiveness in experimental syphilis.

Transcriptional analysis of the tpr genes in Treponema pallidum subsp. pallidum (referred to here as simply T. pallidum) has been limited to date, and yet the expression of members of this gene family is likely relevant to the pathogenesis of syphilis. Recently, immunological studies and semiquantitative mRNA analysis led to the hypothesis of the modulation of tpr gene transcription during infection and suggested that various strains of T. pallidum might differentially express these genes. In this study we developed a real-time amplification assay to quantify the tpr mRNAs with respect to the 47-kDa lipoprotein message and to compare transcript levels among four different strains of T. pallidum. In addition, we analyzed the lymphocyte responsiveness pattern toward the Tpr antigens in late experimental syphilis to identify tpr genes that had been expressed during the course of infection. The T-cell response has been implicated in clearance of treponemes from early lesions, and some of the Tprs were identified as strong targets of the cellular immune response. We show that message for many of the tpr genes can be detected in treponemes harvested at the peak of early infection. Interestingly, tprK seems to be preferentially expressed in almost every strain, and it is uniformly the target of the strongest cellular immune response. These studies demonstrate the differential expression of certain tpr genes among strains of T. pallidum, and further studies are needed to explore the relationship between tpr gene expression and the clinical course of syphilis in infected individuals.

Animals↗
PubMed2006

Identification of the gene encoding the FhbB protein of Treponema denticola, a highly unique factor H-like protein 1 binding protein.

The gene encoding the Treponema denticola factor H-like protein 1 (FHL-1) binding protein, FhbB, was recovered and characterized. Sequence conservation, expression, and properties of FhbB were analyzed. The identification of FhbB represents an important step in understanding the contribution of FHL-1 binding in T. denticola pathogenesis and in development of periodontal disease.

Bacterial Proteins↗
PubMed2007

Phagocytosis of Borrelia burgdorferi and Treponema pallidum potentiates innate immune activation and induces gamma interferon production.

We examined the interactions of live and lysed spirochetes with innate immune cells. THP-1 monocytoid cells were activated to comparable extents by live Borrelia burgdorferi and by B. burgdorferi and Treponema pallidum lysates but were poorly activated by live T. pallidum. Because THP-1 cells poorly internalized live spirochetes, we turned to an ex vivo peripheral blood mononuclear cell system that would more closely reflect spirochete-mononuclear phagocyte interactions that occur during actual infection. In this system, B. burgdorferi induced significantly greater monocyte activation and inflammatory cytokine production than did borrelial lysates or T. pallidum, and only B. burgdorferi elicited gamma interferon (IFN-gamma) from NK cells. B. burgdorferi was phagocytosed avidly by monocytes, while T. pallidum was not, suggesting that the enhanced response to live B. burgdorferi was due to phagocytosis of the organism. When cytochalasin D was used to block phagocytosis of live B. burgdorferi, cytokine production decreased to levels comparable to those induced by B. burgdorferi lysates, while the IFN-gamma response was abrogated altogether. In the presence of human syphilitic serum, T. pallidum was efficiently internalized and initiated responses resembling those observed with live B. burgdorferi, including the production of IFN-gamma by NK cells. Depletion of monocytes revealed that they were the primary source of inflammatory cytokines, while dendritic cells (DCs) directed IFN-gamma production from innate lymphocytes. Thus, phagocytosis of live spirochetes initiates cell activation programs in monocytes and DCs that differ qualitatively and quantitatively from those induced at the cell surface by lipoprotein-enriched lysates. The greater stimulatory capacity of B. burgdorferi versus T. pallidum appears to be explained by the successful recognition and phagocytosis of B. burgdorferi by host cells and the ability of T. pallidum to avoid detection and uptake by virtue of its denuded outer membrane rather than by differences in surface lipoprotein expression.

Borrelia burgdorferi↗
PubMed1974

Oxygen uptake by Treponema pallidum.

Virulent Treponema pallidum has been shown to consume O(2) at a rate similar to that of the known aerobic spirochaete, Leptospira. Such O(2) uptake is cyanide sensitive, indicating a functioning cytochrome oxidase. Inhibition of O(2) uptake by azide, chlorpromazine, and amytal further suggests a functioning electron transport system for the oxidation of nicotinamide adenine dinucleotide (reduced) to O(2). Evidence is consistent with the probability that this terminal electron-transport system is coupled to oxidative phosphorylation. The potential significance of these findings is discussed.

Amobarbital↗
PubMed1974

Protein synthesis by Treponema pallidum extracted from infected rabbit tissue.

Virulent Treponema pallidum organisms, extracted from infected rabbit testes, incorporated amino acids into protein. A temperature of 34 C and a pH of 7.6 were optimal for protein synthesis, which was linear during in vitro incubation for 24 h. Selective inhibition of protein synthesis by erythromycin as judged by the incorporation of radiolabeled amino acids and radioautography demonstrated that treponemes were actively synthesizing proteins. Since addition of various sera and ultrafiltrates to the basal incubation mixture did not stimulate the level of protein synthesis, it was uncertain whether treponemes synthesized protein at a maximal or endogenous rate. Based upon the size of the unlabeled amino acid pool contained in infected testicular extract, it appeared that virulent treponemes utilized the majority of amino acids for protein synthesis but at varying efficiency.

Amino Acids↗
PubMed1975

Treponema pallidum (Nichols strain) in tissue cultures: cellular attachment, entry, and survival.

The interaction of Treponema pallidum (Nichols strain) with cultured cells was investigated under aerobic conditions. Cell monolayers derived from rabbit testicular tissue extended the survival of treponemes as indicated by active motility. Large numbers of organisms rapidly attached to cultured cells. Within 3 h, one to twelve actively motile treponemes were attached to 25 to 50% of the cells. In addition, T. pallidum attained intracellularity as early as 30 min after inoculation of the cell monolayers. In sharp contrast, T. phagedenis biotype Reiter and T. denticola did not attach and did not enter cultured cells. Most importantly, intracellular and/or attached T. pallidum retained virulence for at least 24 h. Similar observations of attachment and retention of virulence were detected with ME-180, a cell line derived from a human cervical carcinoma. Preliminary studies with superoxide dismutase indicated that this enzyme prolonged treponemal motility and retention of virulence in the presence of cultured cells. These data provide guidelines for further investigations of in vitro cultivation of T. pallidum.

Animals↗
PubMed1975

Isolation and purification of Treponema pallidum from syphilitic lesions in rabbits.

Treponema pallidum were extracted from testicular syphilomas of corticosteroid-treated rabbits and purified by differential centrifugation. The steroid therapy allowed a longer holding time for infected rabbits, which produced greater treponeme yields, averaging 1.58 x 10(10) treponemes per rabbit. The treatment, which also diminished cellular infiltration and increased the extracellular mucoid material in lesions, produced much cleaner suspensions than preparations from nontreated animals. Most of the treponemes in the purified suspensions were still motile, and none carried demonstrable host immunoglobulin. The preparations were free of recognizable host tissue debris and they contained, on the average, 1.9 x 10(-7) mug of protein per treponeme.

Adrenal Cortex Hormones↗
PubMed1975

Effect macrophage activation on infection with Treponema pallidum.

Infection of rabbits with Treponema pallidum induces nonspecific acquired cellular resistance (ACR) to Listeria monocytogenes. This resistance can be adoptively transferred using thymus-dependent lymphocytes. Since infections that induce ACR are usually brought under control by cellular mechanisms, we sought to determine whether induction of ACR in rabbits stimulates resistance to challenge with T. pallidum. When BCG-infected rabbits which suppressed the growth of Listeria were challenged intravenously with T. pallidum, lesions appeared at the same time and progressed in a fashion similar to that in non-BCG-infected controls. There was a tendency for syphilitic lesions to disseminate more widely in BCG-infected animals and for the lesions to necrose more rapidly in controls. T. pallidum may resist phagocytosis by macrophages, as has been suggested previously, or macrophages may fail to be activated locally in the dermis. Although syphilitic infeciton appears to stimulate ACR, activation of the macrophages may not contribute significantly to the ability of the host to suppress T. pallidum.

Animals↗
PubMed1975

Effect of pretreatment with Mycobacterium bovis (strain BCG) and immune syphilitic serum on rabbit resistance to Treponema pallidum.

Stimulation of the rabbit reticuloendothelial system with viable Mycobacterium bovis (strain BCG), and other agents, had no effect on the development of syphilitic lesions after intradermal or intravenous inoculation with graded doses of Treponema pallidum (virulent Nichol's strain; mean infective doses less than 10). The simultaneous administration of immune syphilitic rabbit serum retarded the development of lesions, but this appeared to be due solely to the immune serum, suggesting no synergism between the activated reticuloendothelial system and the anti-T. pallidum antibodies. The administration of two doses of BCG enhanced syphilitic lesion development in the rabbit.

Animals↗