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PubMed1983

Studies of rabbit testes infected with Treponema pallidum. I Immunopathology.

Rabbit testes were injected with suspensions of Treponema pallidum, washed T pallidum, heat killed T pallidum, or Reiter treponemes. The testes were removed three to 24 days after injection and examined for the number of treponemes, the presence of treponemal antibodies, histopathological changes, and presence of T and B cells. In animals infected with T pallidum a substantial number (10(6)-10(7)/ml) of organisms were still present at day 24 in spite of early local production of antibodies and increasing infiltration with plasma cells, T lymphocytes, and macrophages. In animals infected with washed T pallidum a lower degree of inflammation was observed than in those infected with unmodified T pallidum, and the treponemal antibodies were detected simultaneously in samples of testicular fluid and serum. In the groups injected with heat killed T pallidum and Reiter treponemes no macroscopical or microscopical changes were detected, although in the group injected with heat killed T pallidum treponemal antibodies were detected in the testicular fluid on day 24.

Animals↗
PubMed1983

Studies of rabbit testes infected with Treponema pallidum. II Local synthesis of antibodies.

Samples of serum and testicular fluid from normal rabbits and those infected with Treponema pallidum were examined for total protein, albumin, IgG, and IgM concentrations. Total protein concentrations were similar in testicular fluid from normal and infected animals but were lower than in serum. The concentrations of individual proteins in serum did not differ substantially between infected and non-infected rabbits. In testicular fluid, however, the concentrations of all three proteins, especially IgM, were greater in infected rabbits. A significantly (p less than 0.02) raised testis-IgM index, together with a higher concentration of anti-treponemal IgM in the infected testicular fluid than in autologous serum, suggests local synthesis of IgM antibodies.

Animals↗
PubMed1984

Susceptibility of Treponema pallidum to the toxic products of oxygen reduction and the non-treponemal nature of its catalase.

We examined the sensitivity of Treponema pallidum (Nichols strain) to toxic products of oxygen reduction. T pallidum was sensitive to hydrogen peroxide at concentrations similar to those to which obligate anaerobes are sensitive. Accelerated death of T pallidum occurred at hydrogen peroxide concentrations below 50 mumol/l. Agents protective against hydrogen peroxide and the hydroxyl free radical (catalase, peroxidase, and mannitol) significantly enhanced treponemal survival in vitro under all three conditions of aerobiosis tested--that is, air, 3% oxygen, and 3% oxygen in conjunction with a reduced medium. Superoxide dismutase (which provides protection against superoxide radicals) did not enhance treponemal survival in normal media. When superoxide radicals were generated in the medium by means of a xanthine and xanthine oxidase system, however, the enzyme did protect T pallidum. A possible toxic involvement of singlet oxygen was also indicated by enhanced treponemal survival in air in the presence of histidine. Extracts of T pallidum from infected rabbit testes showed catalase activity which, on polyacrylamide gel electrophoresis, had the same relative mobility as purified rabbit catalase. The treponemal catalase activity was neutralised by anti rabbit catalase antiserum (raised in guinea pigs). This confirmed that the catalase was of rabbit origin and not an endogenous enzyme of T pallidum. We discuss the relation of these results to the obligate parasitism of T pallidum.

Animals↗
PubMed1984

Treponema pallidum does not synthesise in vitro a capsule containing glycosaminoglycans or proteoglycans.

Treponema pallidum was investigated for its ability to synthesise glycosaminoglycans or proteoglycans in vitro. Isolated viable T pallidum organisms were incubated with radiolabelled precursors of glycosaminoglycans, sodium 35S-sulphate and 3H-glucosamine (tritiated glucosamine). T pallidum failed to incorporate sodium 35S-sulphate but did incorporate 3H-glucosamine into a macromolecule which may be associated with the surface of the treponeme. This macromolecule was resistant to degradation by specific glycosaminoglycanases. We conclude that T pallidum does not synthesise a capsule containing glycosaminoglycans in vitro.

Chromatography, Gel↗
PubMed1984

Evaluation of an enzyme immunoassay for IgM antibodies to Treponema pallidum in syphilis in man.

An enzyme immunoassay (EIA) for the detection of immunoglobulin M (IgM) antibodies to Treponema pallidum was investigated for specificity and sensitivity. Using the results in serum from 1192 patients with successfully treated syphilis, the assay was calculated to be about 97% specific. As in any other IgM enzyme linked immunosorbent assay (ELISA), rheumatoid factor played an important part in causing false positive results. Pre-absorption of serum with aggregated IgG was therefore necessary to perform the test. Evaluation of the results in serum from 385 patients with untreated primary, secondary, and latent syphilis as well as patients with untreated reinfections showed that the sensitivity of the assay depended on the stage of infection and varied between 98% and 93%. IgM antibody titres were about ten times higher in the EIA than in the indirect immunofluorescence assay using the IgM fractions of serum. From the results it may be concluded that the EIA is an appropriate technique not only for rapid and sensitive measurement of IgM antibodies in most patients with untreated syphilis but also for selecting treponemal IgM non-reactive patients.

Antibody Specificity↗
PubMed1984

Attachment of Treponema pallidum to fibronectin, laminin, collagen IV, and collagen I, and blockage of attachment by immune rabbit IgG.

As shown by scanning electron and phase contrast microscopy, Treponema pallidum attached in vitro to basement membranes purified from kidney cortex tissues or from retinal vessels. This organism also attached to the extracellular matrix remaining after cultured cells had been solubilised with Triton X. Fibronectin, laminin, collagen, IV, collagen I, and hyaluronic acid are structural components of basement membranes and extracellular matrices. Experiments were performed to investigate the in vitro attachment of T pallidum to each of these components. Viable or heat inactivated treponemes were added to glass coverslips precoated with different concentrations of each component. After various times of incubation, coverslips were washed and the attached organisms were counted. Large numbers of viable organisms attached to each of these five components. In contrast, heat inactivation sharply reduced numbers of attached organisms. The IgG fractions of immune and non-immune rabbit serum samples were affinity purified using protein A. T pallidum was preincubated with both fractions, then incubated with either intact cultured cells or with coverslips coated with the five tissue components. The IgG from immune serum blocked treponemal attachment to the cultured cells and to fibronectin, laminin, collagen IV, and collagen I, but not to hyaluronic acid. These results are discussed in terms of attachment mechanisms of T pallidum and potential applications to in vivo infection.

Adhesiveness↗
PubMed1984

Treponema pallidum specific IgM haemagglutination test for serodiagnosis of syphilis.

The Treponema pallidum specific IgM haemagglutination (TP-IgM-HA) test uses erythrocytes sensitised with antiserum to human IgM to separate IgM from IgG in serum. Specific antitreponemal IgM captured in this way is detected by adding a second reagent comprising erythrocytes sensitised with T pallidum antigen. Eighty two serum samples from 82 patients with untreated syphilis, 521 samples from 73 patients with treated syphilis, and 1872 samples from people who did not have syphilis were examined by the 19S(IgM)-TPHA (T pallidum haemagglutination), IgM-FTA-ABS (fluorescent treponemal antibody absorbed), TP-IgM-ELISA (enzyme linked immunosorbent assay), and TP-IgM-HA tests for the presence of 19S(IgM) antibodies specific to treponemes. The sensitivity of the TP-IgM-HA test was 97.6% and the specificity was 99.7%. We also traced IgM specific to treponemes in untreated patients with primary syphilis by four different tests. The TP-IgM-HA test results clearly reflected the effect of the treatment.

Antibodies, Bacterial↗
PubMed1985

Differences in susceptibility to infection with Treponema pallidum (Nichols) between five strains of guinea pig.

Groups of 10 young male guinea pigs of inbred strains 2 and 13 and outbred strains Hartley A, Hartley B, and one deficient in the fourth component of complement (C4D) were infected intradermally with 80 X 10(6) Treponema pallidum (Nichols). The course of infection and production of antitreponemal antibody were examined. Strain C4D guinea pigs were the most susceptible to infection (100%); inbred strains 2 and 13 and outbred strain Hartley B showed 80-90% symptomatic infection; and the Hartley A strain was the least susceptible to infection (10%). Strain 13 animals responded with the highest antitreponemal antibody activity, and the Hartley A strain with the lowest. The results suggest that genetic factors or complement, or both, may influence the degree of susceptibility to infection with T pallidum in guinea pigs.

Animals↗
PubMed1985

Mucopolysaccharides in suspensions of Treponema pallidum extracted from infected rabbit testes.

The amount and nature of mucopolysaccharides present in extraction fluids routinely obtained in the isolation procedure of Treponema pallidum from infected rabbit testes was investigated. The mean quantity of mucopolysaccharides extracted from both testes of groups of 10 rabbits was 3.09 mg after infection for seven days and 26.88 mg after infection for 12 days, while from the testes of uninfected rabbits a mean of 0.42 mg was obtained. On electrophoresis the isolated mucopolysaccharides showed only one single band with the migration characteristics of hyaluronic acid. This band disappeared completely after pretreatment with hyaluronidase from bovine testes, which showed that during infection with T pallidum increasing amounts of hyaluronic acid accumulate. They can, at least in part, be extracted by a gentle extraction procedure, suggesting that this material binds loosely. The amount of hyaluronic acid isolated 12 days after infection showed positive correlations with the wet weight of testes as well as the number of treponemes isolated; seven days after infection such correlations were not present.

Animals↗
PubMed1985

Experimental infection of inbred guinea pigs with Treponema pallidum: development of lesions and formation of antibodies.

Inbred strain 2 and strain 13 guinea pigs were infected intradermally in the hind legs with different numbers of the virulent Nichols strain of Treponema pallidum. About 91% of the animals developed clearly visible lesions after being injected with 5 X 10(6) to 10 X 10(6) treponemes. T pallidum organisms could be isolated from skin lesions at various stages after infection. Infected animals were monitored for the production of specific treponemal and non-specific cardiolipin antibodies by the fluorescent treponemal antibody (FTA) and microhaemagglutination (MHA-TP) tests and the rapid plasma reagin (RPR) card test. Low levels of specific antibodies could be detected by both FTA and MHA-TP tests from three to four weeks after infection. Maximum titres of treponemal antibody generally occurred after week 6 and persisted for several more months. These peak titres ranged from 1/40 to 1/80 in the FTA test and 1/160 to 1/320 in the MHA-TP test. During the same period infected guinea pigs, unlike rabbits with syphilis, did not produce detectable quantities of antibodies against cardiolipin.

Animals↗
PubMed1985

Interactions of fibronectin with Treponema pallidum.

The adhesive or opsonic glycoprotein, fibronectin, is associated with the surface of Treponema pallidum as shown by immunofluorescence. A quantitative assay using iodine-125 (125I) showed that T pallidum harvested seven days after infection bound more fibronectin than T pallidum harvested 14 days after infection. This increased binding by "younger" organisms was confirmed by radioimmunoassay techniques. Fibronectin appears to have a role in treponemal attachment. Preincubation of T pallidum with goat or rabbit antibody to fibronectin blocked treponemal attachment to cultured cells and to isolated capillaries and inhibited treponemal virulence. Treponemes were incubated in glass wool columns pretreated with fibronectin and were then eluted from the columns. This technique yielded a population of T pallidum that failed to bind to fibronectin. Compared with treponemes eluted from control ovalbumin columns, organisms eluted from fibronectin columns attached to cultured cells in larger numbers but did not survive as long and were not as virulent. Findings are discussed in terms of the relevance of interaction between treponemes and fibronectin in the pathogenesis of T pallidum.

Animals↗
PubMed1986

Effect of four serum components on survival of Treponema pallidum and its attachment to rabbit cells in vitro.

Transferrin and, to a lesser extent, insulin were two components of serum that enhanced the survival of Treponema pallidum in microaerophilic, acellular medium. These two components also enhanced the attachment of T pallidum to baby rabbit genital organ (BRGO) cells in both aerobic and microaerophilic tissue culture systems. Growth hormone and epidermal growth factor marginally enhanced the attachment of T pallidum to BRGO cells.

Animals↗
PubMed1987

Cloning structural genes for Treponema pallidum immunogens and characterisation of recombinant treponemal surface protein, P2 (P2 star).

A genomic library consisting of partially digested 10 to 20 kilobase pair fragments of Treponema pallidum deoxyribonucleic acid (DNA) was constructed using bacteriophage lambda EMBL-3 as the vector. Positive clones expressing T pallidum antigens were detected with sera from experimentally infected rabbits. Treponemal proteins ranging in molecular weight from 37,000 daltons to 120,000 daltons were identified by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and immunoblotting of phage lysate proteins. One recombinant phage was examined further and contained an insert encoding a prominent treponemal 37,000 dalton protein. The recombinant protein was not recognised by antiserum directed against a fibronectin binding treponemal adhesion that contained the same electrophoretic mobility. Neither did antibody to the recombinant 37,000 dalton protein react with any treponemal proteins purified by fibronectin affinity chromatography. The recombinant protein in Escherichia coli lysates was labelled P2 (P2 star) to differentiate it from the comigrating adhesin protein called P2. Native P2 protein was present on T pallidum surfaces as shown by radioimmunoprecipitation assays with extrinsically labelled organisms. A cross reactive molecule like P2 was not synthesised by the avirulent spirochaete, T phagedenis biotype Reiter, which indicated that P2 is a protein specific to virulent T pallidum organisms. Finally, only sera of patients with primary syphilis possessed appreciable concentrations of antibody to recombinant P2 protein.

Antibodies, Bacterial↗
PubMed1987

Light and electron microscopy of rabbit testes infected with Treponema pallidum (Nichols strain): nature of deposited mucopolysaccharides and localisation of treponemes.

The mucopolysaccharide nature of the material deposited in rabbit testes infected with Treponema pallidum was confirmed by histochemical staining with alcian blue. Differential staining of mucopolysaccharides showed the presence of sulphated mucopolysaccharides as an almost constant feature, whereas in little more than half of the orchitic testes studied variable deposits of hyaluronic acid were seen. The treponemes were almost exclusively present in the areas rich in mucopolysaccharide. A combination staining with the Warthin-Starry method and alcian blue showed treponemes in close association with pre-existing fibrils and cells contained in these fibrils. The latter findings were confirmed by electron microscopy, and the fibroblasts to which treponemes adhered displayed the characteristics of activated cells. The close parallel between the histopathological changes observed here and their descriptions in published reports shows that our specific strain still behaves the same as the original Nichols pathogenic strain of T pallidum.

Animals↗
PubMed1987

Molecular cloning of Treponema pallidum outer envelope fibronectin binding proteins, P1 and P2.

Phages directing the synthesis of Treponema pallidum fibronectin binding adhesin proteins, P1 and P2, were isolated from an EMBL-3 bacteriophage lambda library of T pallidum deoxyribonucleic acid (DNA). The recombinant phages were identified using antisera generated to treponemal proteins purified in fibronectin-Sepharose. Recombinant P1 and P2 proteins possessed the same relative molecular weights as the native surface polypeptides of spirochaetes. The structural genes for these proteins were subcloned into the plasmid vector pUC19, and transformed Escherichia coli expressed and translocated recombinant P1 and P2 to their outer membranes. Finally, the recombinant adhesin proteins, P1 and P2, were purified from detergent solubilised E coli outer membrane preparations using fibronectin-Sepharose affinity chromatography, which confirmed that the fibronectin binding properties of the cloned proteins were retained.

Adhesins, Bacterial↗
PubMed1988

Affinities of Treponema pallidum for human lactoferrin and transferrin.

The acquisition of lactoferrin and transferrin by live Treponema pallidum organisms was examined. Saturation binding kinetics were obtained for virulent treponemes with increasing amounts of radioiodinated lactoferrin but not with transferrin. Furthermore, lactoferrin bound up to 100 times more effectively than transferrin. Only unlabelled lactoferrin stoichiometrically completed with iodinated lactoferrin binding. Time course kinetics showed maximum lactoferrin acquisition within the first five minutes at 34 degrees C. Optimum iron accumulation, however, was achieved by T pallidum in 30 minutes at 34 degrees C, and amounts of iron were six times greater than the equivalent amount of lactoferrin bound. Interestingly, iron uptake was also detected in the presence of transferrin, despite the minimal amounts of transferrin acquired by live treponemes. These observations reinforce the possibility that the associations of T pallidum with host molecules, such as plasma proteins, are essential for survival of the parasite within host environments.

Humans↗
PubMed1989

Serum lipoprotein binding by Treponema pallidum: possible role for proteoglycans.

Acquisition by the syphilis spirochaete, Treponema pallidum, of radioiodinated total human plasma lipoprotein and lipoprotein subfractions was examined. Time dependent and saturation binding kinetics were observed for total lipoproteins and subfractions, including high density lipoproteins, low density lipoproteins (LDL), and very low density lipoproteins. All subfractions competed equally well in binding iodinated total lipoproteins and individual subfractions, but apoproteins common to all subfractions were ineffective in inhibiting lipoprotein acquisition. The interaction of LDL with T pallidum was studied further and, interestingly, the presence of 17% sulphated dextran sulphate (DS) in the reaction mixture containing treponemes and LDL resulted in up to 172 times more LDL being bound by live treponemes. Biological variability was observed in the extent of increased LDL bound in the presence of 17% sulphated DS by preparations of T pallidum isolated from different infected rabbits. Saturation kinetics of iodinated LDL acquisition was obtained in the presence of 17% sulphated DS but not 1% sulphated DS. Other proteoglycan molecules, such as chondroitin sulphate, hyaluronic acid and heparin, and fibronectin, the extracellular matrix protein targeted by treponemes in parasitism of host cells and tissues neither diminished nor enhanced LDL binding by live treponemes. Only 5% and 10% of associated radioactivity was released from treponemal surfaces after T pallidum was incubated with iodinated LDL and 17% sulphated-DS for 15 and 30 minutes, respectively. These data show binding and possible internalisation of host lipoproteins by T pallidum, which may be mediated by sulphated proteoglycan. Sulphated proteoglycans accumulate during T pallidum infections of host cells.

Dextran Sulfate↗
PubMed1990

Anti-Treponema pallidum IgM, IgA, and IgG subclass antibody responses after treatment in patients with syphilis at various stages: 1. Assessments by enzyme-linked immunosorbent assay.

Patients with syphilis at various stages were followed from the beginning by serial measurements of antibodies against Treponema pallidum that belonged to the classes IgM and IgA, and to the subclasses IgG1 to 4 by an enzyme-linked immunosorbent assay. A marked decrease in levels of antibodies was noted within three months after treatment. In the case of IgM this occurred in 19 of the 31 patients studied (61%), in the case of IgA in 15 patients (48%) and in the case of one or more of the subclasses of IgG in 18 patients (52%). These findings indicate the usefulness of levels of IgM antibodies as markers for evaluating the effects of treatment. There were, nevertheless, 12 patients (39%) whose levels of IgM antibodies remained consistently below the normal limits or failed to decline despite treatment. In five (16%) of these cases, the serum level of IgA antibodies or of one or more subclasses of IgG fell after treatment. The results suggest the potential usefulness of IgA antibodies in the various IgG subclasses as a marker for evaluating the results of treatment in those patients whose levels of IgM antibodies do not change in response to treatment.

Antibodies, Bacterial↗