Biomedical subjects

J F Alderete

Publications and source records attributed to J F Alderete.

At least 73 records · Page 4Linked to original sources

PubMed1988

Identification and properties of Trichomonas vaginalis proteins involved in cytadherence.

Trichomonas vaginalis NYH286 surface proteins which are candidates for mediating parasite cytadherence (adhesins) were identified. At least four trichomonad protein ligands ranging in relative molecular mass from 65 to less than or equal to 21 kilodaltons were found to selectively bind to chemically stabilized HeLa cells. The proteins were present on the surfaces of 10 different isolates of T. vaginalis examined; however, the nonpathogenic trichomonad T. tenax did not possess similar HeLa cell-binding proteins under identical experimental conditions, suggesting that these proteins are unique to the pathogenic human trichomonads. The surface nature of the candidate adhesins was confirmed by the ability of the proteins on intact, live organisms to be radioiodinated and to be removed with trypsin treatment. Rabbit antiserum (immunoglobulin G fraction) generated against adhesin proteins electroeluted from acrylamide preparations inhibited cytadherence compared with control immunoglobulin G. An adherence-negative subpopulation of T. vaginalis NYH286 organisms was also isolated. These nonadherent trichomonads did not synthesize the adhesin proteins. Interestingly, absence of adhesins from these parasites paralleled expression of a major immunogen known to undergo phenotypic variation. Revertant organisms derived from the adherence-minus subpopulation synthesized the adhesins and attached to HeLa cells. The emergence of revertant adherent T. vaginalis organisms also corresponded with the appearance of parasites which were without the major immunogen on their surface. Finally, it was determined that only those parasites lacking the major surface immunogen were capable of adherence and toxicity to HeLa cells.

Animals↗

PubMed1988

Specific parasitism of purified vaginal epithelial cells by Trichomonas vaginalis.

Human vaginal epithelial cells (VECs) from vaginal swabs obtained from normal women or from patients with trichomoniasis were purified, and VEC parasitism by Trichomonas vaginalis was examined. Trichomonads bound equally well to live or dead VECs, and up to 20% of VECs were parasitized. Trichomonal cytadherence of human VECs was time, temperature, and pH dependent. Saturation binding levels of live trichomonads to VECs gave approximately 2 organisms adherent to parasitized VEC. No differences in cytadherence levels were detected by different isolates to VECs from the same patient compared with adherence to VECs from normal individuals. Trypsinized, live T. vaginalis organisms failed to recognize VECs. A ligand assay identified four adhesin candidates, and only organisms without a prominent immunogen on the surface (negative phenotype) cytadhered to VECs and synthesized the adhesins, confirming the results of a recently published report by us on adherence to HeLa cell monolayers (J. F. Alderete and G. E. Garza, Infect. Immun. 56:28-33, 1988). These data show the ability of T. vaginalis to parasitize human vaginal epithelial cells in a specific receptor-ligand manner.

Animals↗

PubMed1988

Monoclonal-antibody-based enzyme-linked immunosorbent assay for Trichomonas vaginalis.

Trichomonas vaginalis is estimated to infect 4 million women per year in the United States. The diagnosis of trichomoniasis is predominantly achieved by direct microscopic examination of vaginal exudates. This subjective diagnostic procedure is reported to be 75% sensitive under ideal circumstances. We have developed an enzyme-linked immunosorbent assay (ELISA) for the detection of T. vaginalis directly from vaginal exudates. The ELISA employs a monoclonal antibody specific for a 65-kilodalton surface polypeptide of T. vaginalis as the capture antibody in a sandwich format. A polyclonal rabbit anti-T. vaginalis antibody labeled with horseradish peroxidase serves as the probe. An evaluation of vaginal specimens from women attending clinics revealed a sensitivity and specificity of the ELISA of 89 and 97%, respectively, versus the culture technique. These results indicate the usefulness of this ELISA as an alternative to microscopic and culture methods for the detection of T. vaginalis in vaginal exudates.

Animals↗

PubMed1988

Does lactobacillus vaccine for trichomoniasis, Solco Trichovac, induce antibody reactive with Trichomonas vaginalis?

Solco Trichovac is a vaccine of Lactobacillus acidophilus developed for treating trichomoniasis. The efficacy of the vaccine is reportedly due to cross reacting antibody being produced in people immunised with the lactobacillus bacteria. Several techniques, including enzyme linked immunosorbent, indirect immunofluorescence, immunoblot, and radioimmunoprecipitation assays were used to assess the extent of antigenic cross reactivity between Lactobacillus acidophilus and Trichomonas vaginalis. Data show a lack of antigenic relatedness between the Solco Trichovac lactobacilli and several strains of T vaginalis. Furthermore, antiserum to L acidophilus failed to inhibit trichomonad cytadherence or host cell killing, as is suggested by the producers of the Solco Trichovac vaccine.

Animals↗

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↗

PubMed1987

Trichomonas vaginalis phenotypic variation occurs only among trichomonads infected with the double-stranded RNA virus.

Trichomonas vaginalis isolates were examined for the presence of viral double-stranded RNA (dsRNA) and the property of phenotypic variation. Only the heterogeneous isolates composed of mAb-reactive and -nonreactive organisms, as determined by indirect immunofluorescence and flow cytofluorometry, and capable of phenotypic variation possessed the dsRNA. Both the positive and negative phenotype subpopulations separated from the heterogeneous parent contained equal amounts of the dsRNA. Loss of the dsRNA upon prolonged in vitro cultivation always correlated with the lack of expression of the major immunogen. The data indicate a relationship between the presence of the dsRNA and the ability of the pathogenic human trichomonads to express immunogens on their surfaces and to undergo phenotypic variation.

Animals↗

PubMed1987

Phenotypes and protein-epitope phenotypic variation among fresh isolates of Trichomonas vaginalis.

Fresh isolates of Trichomonas vaginalis were examined for reactions to a panel of five monoclonal antibodies (MAbs). Four MAbs (C20A3, DM126, DM116, and C55) were to distinct surface immunogens and one MAb (L64) was to a cytoplasmic component. The fresh isolates were evaluated by indirect immunofluorescence (IF), immunoblotting, and radioimmunoprecipitation. IF assay with C20A3 MAb gave isolates which were homogeneous nonstaining (negative [Neg] phenotype) and isolates which were heterogeneous staining and nonstaining (positive [Pos] and Neg phenotype, respectively) organisms. Immunoblotting and radioimmunoprecipitation assays revealed that surface phenotypic heterogeneity among isolates with C20A3 MAb was due to the presence or absence of the immunogen from the parasite surface. IF assay with DM126 MAb also gave Pos and Neg phenotypes among parasites of some isolates. All of the isolates were always Neg phenotype with DM116 and C55 MAbs. The occurrence of Neg phenotype organisms with DM126, DM116, and C55 was due to epitope inaccessibility to their respective MAbs and not to the absence of the immunogen from trichomonal membranes. All isolates possessed the cytoplasmic protein recognized by L64 MAb. Paired isolates (taken 5 to 6 days apart) from 24 women were also studied. Four of the 24 paired isolates (16%) had different phenotype distributions at the two timepoints for C20A3. Fresh isolates also underwent phenotypic variation during in vitro growth and multiplication, as determined with C20A3. Also, 7 of the 24 paired isolates demonstrated dramatic changes in the accessibility of DM126 MAb to epitope binding. Lastly, 55 (90%) of 60 serum samples from patients with trichomoniasis evaluated in this study possessed antibody to the C20A3 reactive molecule. The data show that the fresh T. vaginalis isolates were predominantly Neg phenotype and confirm the occurrence of protein and epitope phenotypic variation for major immunogens among fresh isolates of the pathogenic human trichomonads.

Antigens, Protozoan↗

PubMed1987

Trichomonas vaginalis NYH286 phenotypic variation may be coordinated for a repertoire of trichomonad surface immunogens.

Trichomonas vaginalis isolate NYH286 was fractionated with immunoglobulin G of sera from patients with trichomoniasis. Subpopulations of trichomonads with phenotypes of either patient serum-immunoglobulin G reactive (PS+) or nonreactive (PS-) were obtained. Flow cytofluorometry of PS+ and PS- subpopulations with a monoclonal antibody called C20A3 which reacts with a high-molecular-weight immunogen of T. vaginalis gave corresponding fluorescent (positive) and nonfluorescent (negative) phenotypes. No relationship was seen between PS+ and PS- phenotypes and binding of soybean agglutinin, wheat germ agglutinin, and concanavalin A, showing that PS- organisms still possessed carbohydrate moieties on their surfaces based on lectin binding. Phenotypic variation among the PS+ and PS- trichomonads was observed during in vitro growth. A positive-to-negative phenotype shift was also recorded for parasites obtained from lesions of mice subcutaneously infected with PS+ trichomonads. The involvement of surface proteins in the differential PS+ and PS- reactions was supported by soluble antigen and whole cell radioimmunoprecipitation assays. Finally, enhanced parasitism and killing of HeLa cells in monolayer cultures were observed for PS- subpopulations as compared with PS+ counterparts. The data support the idea that phenotypic variation for T. vaginalis may be coordinated for a repertoire of trichomonad immunogens and that such membrane dynamics influence expression of virulence determinants for these sexually transmitted disease agents.

Antibodies, Monoclonal↗

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

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↗

PubMed1986

Isolation of a Treponema pallidum gene encoding immunodominant outer envelope protein P6, which reacts with sera from patients at different stages of syphilis.

A phage directing the synthesis of an abundant 45-kD Treponema pallidum surface protein was isolated from an EMBL-4 bacteriophage lambda library of T. pallidum DNA. The recombinant phage was identified using an mAb that was directed toward an immunodominant, outer envelope T. pallidum protein designated P6. The recombinant P6 protein possessed the same mol mass as the native treponemal antigen detected from total T. pallidum protein preparations, confirming the cloning of the structural gene for this molecule. Furthermore, E. coli was transformed by a 4.5-kb Eco RI lambda insert fragment subcloned into the plasmid vector pUC19. These transformed cells expressed and translocated the 45-kD protein to their outer membranes. Finally, all sera from patients with different stages of syphilis (primary, secondary, and latent) contained antibody reactive to this protein.

Bacterial Outer Membrane Proteins↗

PubMed1986

Localization of the ganglioside-binding site of fibronectin.

It has been demonstrated via biological assays that fibronectin possesses a receptor for gangliosides that is involved in cell adhesion and restoration of the normal morphology of transformed cells. In this study, fluorescence polarization has been employed to monitor the binding of ganglioside oligosaccharide to fibronectin. Parameters involved in ganglioside oligosaccharide binding to fibronectin are described and compared to the interaction of heparin with fibronectin. A Kd of 1.4 X 10(-8) mol/liter has been calculated, and it is demonstrated that labeled ganglioside oligosaccharides can be eluted from fibronectin with either unlabeled ganglioside oligosaccharides or 4 M urea. Using the fluorescence polarization assay developed in this study for measurement of ganglioside binding to fibronectin, it is demonstrated that gangliosides bind to the 31,000-dalton amino terminal heparin-binding domain of fibronectin. A ganglioside-Sepharose affinity column has been constructed which specifically binds the 31,000-dalton amino terminal fragment of fibronectin. The localization of the ganglioside receptor to the amino terminal domain of fibronectin indicates that the ganglioside receptor is distinct from the putative fibronectin cell surface receptor which is located near the center of the fibronectin molecule.

Binding Sites↗

PubMed1986

Trichomonas vaginalis: electrophoretic analysis and heterogeneity among isolates due to high-molecular-weight trichomonad proteins.

The protein composition of Trichomonas vaginalis isolates was evaluated using one-dimensional and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. At least two hundred intrinsically labeled and about 30 major extrinsically labeled proteins of molecular weights less than 120,000 were resolved by isoelectric focusing and electrophoresis. In general, long-term grown and fresh isolates of T. vaginalis gave similar fluorograms of the readily detectable proteins. Only a minor variance in a distinct protein was noted among three of the four fresh isolated tested. Labeling with [35S]methionine or 3H-amino acids gave almost identical profiles, ensuring the efficient radiolabeling of trichomonad proteins that dominate quantitatively. Comparative analysis of radioactivity profiles of one-dimensional gels emphasizing the region of high-molecular-weight proteins known to reside in low copy number revealed the presence or absence of the internally synthesized proteins from surfaces of T. vaginalis isolates. Finally, immunoblotting of two-dimensional gels demonstrated the highly immunogenic nature of proteins corresponding to quantitatively dominant molecular weight regions of intrinsically labeled proteins.

Animals↗

PubMed1986

Monoclonal antibody to a major surface glycoprotein immunogen differentiates isolates and subpopulations of Trichomonas vaginalis.

To produce monoclonal antibodies (MAbs) to highly immunogenic membrane proteins of Trichomonas vaginalis NYH286, the sera of subcutaneously infected BALB/c mice were first monitored for antibody to trichomonad surface proteins. The sera possessed antibody to one major surface protein by 7 days and antibody to numerous other trichomonad membrane proteins by 4 weeks postinfection. A hybridoma was then generated that synthesized an MAb, designated C20A3, which reacted to a parasite-derived glycoprotein possessing a molecular weight of 267,000 (267K glycoprotein). The immunogen corresponded to the single high-molecular-weight immunogenic surface protein recognized by 7-day mouse antisera. The MAb differentiated T. vaginalis isolates by a whole-cell enzyme-linked immunosorbent assay and by indirect immunofluorescence, using either fixed or live organisms. All isolates, however, possessed C20A3-reactive material when tested by enzyme-linked immunosorbent assay, using detergent extracts of the isolates incubated with MAb-coated microtiter well plates. The epitope was accessible to antibody binding on live T. vaginalis organisms expressing the major immunogen, and the 267K glycoprotein was readily removed from the parasite membranes by trypsinizing the intact trichomonads. The antigen incorporated radiolabeled glucose, mannose, and acetate. Also, an unlabeled 267K glycoprotein on nitrocellulose blots was detected by 125I-concanavalin A and 125I-wheat germ agglutinin, confirming the glycoprotein nature of the immunogen. Finally, of seven isolates used in this study, one possessed a cross-reactive 170K, rather than 267K, antigen. The data reinforce the idea that antigenic heterogeneity among T. vaginalis isolates may be a function of the presence or absence of high-molecular-weight glycoprotein immunogens from trichomonal membranes.

Animals↗

PubMed1986

Enhanced levels of attachment of fibronectin-primed Treponema pallidum to extracellular matrix.

Freshly extracted Treponema pallidum organisms treated with exogenous human fibronectin (Fn) (Fn-primed treponemes) showed a 6- to 15-fold increased level of attachment to Fn-coated cover slips and to extracellular matrix (ECM) when compared with unprimed treponemes. Treponemes primed with collagen or laminin showed no similar enhanced binding to immobilized Fn or ECM. Preexposure of immobilized Fn and ECM to anti-Fn serum but not to anti-collagen or anti-laminin serum prevented treponemal adherence. Also, the presence of proteoglycanlike molecules such as dextran sulfate or heparan sulfate inhibited Fn-primed treponemal attachment to Fn or ECM. In contrast Fn-primed treponemes did not exhibit elevated levels of attachment to eucaryotic cell monolayers. To understand the increased tropism of Fn-primed T. pallidum organisms for Fn and ECM-like surfaces, we radiolabeled freshly extracted treponemes with [35S]methionine and examined them for the presence of surface immunoreactive Fn. Magnetic protAspheres and glass beads coated with monospecific anti-Fn serum bound only 20 to 30% of radiolabeled treponemes. Nonadherent treponemes failed to bind to gelatin-agarose, further confirming the absence of surface Fn or Fn-like material. Fn-free organisms, however, did attach to Fn-coated cover slips and to cell monolayers like treponemes of the original population. Incubation of Fn-free treponemes with human Fn resulted in almost total binding of organisms to anti-Fn antibody on glass beads and also produced increased attachment to Fn-coated cover slips and ECM. These results suggest that enhanced interactions between T. pallidum and the host are dependent on the presence of Fn on syphilis spirochetes and the specific location and orientation of Fn in vivo.

Adhesiveness↗

PubMed1986

Phenotypic variation and diversity among Trichomonas vaginalis isolates and correlation of phenotype with trichomonal virulence determinants.

The extent and nature of heterogeneity among representative Trichomonas vaginalis isolates were evaluated by flow cytofluorometry analysis. Monoclonal antibody and monospecific antiserum to an immunodominant trichomonad surface glycoprotein with a molecular weight of 267,000 (267K glycoprotein) were used to evaluate fresh isolates (JHH and RU375) and long-term grown isolates (NYH286, IR78, and JH31A) of T. vaginalis. Isolates NYH286, JH31A, and JHH were made up of heterogeneous staining (positive [pos] phenotype) and nonstaining (negative [neg] phenotype) populations of trichomonads, whereas RU375 and IR78 were all neg phenotype parasites. Flow cytofluorometric patterns of agar clones derived from single organisms of heterogeneous isolates such as NYH286 showed populations which were either homogeneous pos or neg and also showed clones which were heterogeneous in nature containing both phenotypes. Fluorescence-activated cell sorting was also accomplished, and subpopulations of defined pos or neg phenotype were purified. Flow cytofluorometry evaluation of all isolates for an extended period revealed a phenotypic variation among all heterogeneous isolates and also for all clones and subpopulations derived from the heterogeneous isolates. On the other hand, IR78 and RU375 did not undergo phenotypic variation and have remained neg for greater than 4 years. Parasites which were nonreactive with either monoclonal antibody or monospecific antiserum to the 267K glycoprotein in flow cytofluorometry did not possess the antigen on their surface. This was determined by radioimmunoprecipitation assays using extracts of iodinated trichomonads. Finally, neg phenotype parasites yielded enhanced rates of contact-dependent cytotoxicity of host cell monolayers as compared with the pos phenotype trichomonads.

Antibodies, Monoclonal↗

PubMed1986

Monoclonal antibody to a major glycoprotein immunogen mediates differential complement-independent lysis of Trichomonas vaginalis.

An immunoglobulin G type 2a (IgG2a) monoclonal antibody (MAb), designated C20A3, which reacts with a highly immunogenic trichomonad membrane glycoprotein (approximately 270,000 daltons), produced complement-independent cytolysis of Trichomonas vaginalis organisms. Time- and temperature-dependent lysis of parasites was observed following incubation of washed, live T. vaginalis with certain concentrations of C20A3 IgG. Differential killing of trichomonal isolates and clones of a given isolate by C20A3 was dependent on the presence of the glycoprotein antigen on the parasite surface.

Antibodies, Monoclonal↗

PubMed1985

Immunodiagnostic test for detection of serum antibody to Treponema pallidum (syphilis): fibronectin as a capture vehicle for treponemal adhesins.

Knowledge that Treponema pallidum adhesin proteins bind to host fibronectin (Fn) via ligand-receptor interactions has resulted in development of an ELISA for measuring specific antitreponemal antibodies in sera of syphilitic patients and infected experimental animals. As little as 50 ng of T. pallidum total protein extract added to Fn-coated wells permitted half-maximal levels of ELISA reactivity. Detection of serum antibody from intratesticularly infected rabbits occurred at dilutions greater than 1/100,000. Antibody titers in serum from patients with primary and latent syphilis were positive at 1/1 000 dilutions while serum samples from patients with secondary syphilis were reactive at 1/10,000. Furthermore, the ELISA proved useful for evaluating serum samples from individuals with other treponemal infections. Antibodies raised against the non-pathogenic spirochete, T. phagedenis biotype Reiter, were non-reactive with Fn-T. pallidum complexes. Also, Reiter treponemal proteins did not bind to Fn-coated wells. This ELISA using Fn as a capture vehicle for treponemal adhesin proteins was superior to 3 other routinely used tests for syphilis diagnosis.

Adhesins, Bacterial↗