Histamine and TNF-alpha release by rat peritoneal mast cells stimulated with Trichomonas vaginalis

Mast cells have been reported to be predominant in the vaginal smears of patients infected with T. vaginalis. In this study, we investigated whether T. vaginalis could induce mast cells to migrate and to produce TNF-alpha and histamine. Rat peritoneal mast cells (RPMC), a primary mast cell, were used for the study. T. vaginalis induced an increase in chemotactic migration of the mast cells toward excretory and secretory product (ESP) of T. vaginalis, and the mast cells activated with T. vaginalis showed an increased release of histamine and TNF-alpha. Therefore, mast cells may be involved in the inflammatory response caused by T. vaginalis.     

Trichomonas vaginalis: microtubule cytoskeleton distribution using fluorescent taxoid

Trichomonas vaginalis is a flagellated parasitic protist of the human urogenital tract. The parasite has a poorly known cytoskeleton formed by an axostyle and a pelta, which are formed by stable structures such as microtubules, essential for the maintenance of cell shape and organization. FLUTAX-2 is an active fluorescent derivative of Taxol, binds to alphabeta-tubulin dimer polymerized. In this paper we present the analysis of microtubule distribution in living trophozoites of T. vaginalis using FLUTAX-2.     

The interaction of Trichomonas vaginalis with epithelial cells, polymorphonuclear leucocytes and erythrocytes on vaginal smears: light microscopic observation

In this study, vaginal smears taken from 400 patients were examined cytologically using the Papanicolaou technique. Twenty of the 400 patients were detected as harbouring Trichomonas vaginalis. The interactions of T. vaginalis with epithelial cells, polymorphonuclear leucocytes (PMNLs) and erythrocytes were determined at light microscopic level. It was observed that T. vaginalis were juxtaposed to the epithelial cells and changed shape according to the contours of the epithelial cell revealing the cytopathic effect of trichomonads on epithelial cells. Trichomonads attached to PMNLs produced pseudopodia to phagocytose the cells. Occasionally an amoeboid shaped T. vaginalis organism was seen trailed by a row of PMNLs. This light microscopic study supports the production by trichomonads of a chemotactic factor for PMNLs. Phagocytosed erythrocytes were also seen in the cytoplasm of T. vaginalis, suggesting the need for the patient to be tested for anaemia.     

Trichomonas vaginalis and trichomoniasis in the Republic of Korea

Vaginal trichomoniasis, caused by Trichomonas vaginalis, is the most common sexually transmitted disease. More than 170 million people worldwide are annually infected by this protozoan. In the Republic of Korea, 10.4% of women complaining of vaginal symptoms and signs were found to be infected with T. vaginalis. However, despite its high prevalence, the pathogenesis of T. vaginalis infection has not been clearly characterized although neutrophil infiltration is considered to be primarily responsible for the cytologic changes associated with this infection. We hypothesized that trichomonads in the vagina sometime after an acute infection secrete proteins like excretory-secretory product that have a chemotactic effect on neutrophils, and that these neutrophils are further stimulated by T. vaginalis to produce chemokines like IL-8 and GRO-alpha, which further promote neutrophil recruitment and chemotaxis. Thus, neutrophil accumulation is believed to maintain or aggravate inflammation. However, enhanced neutrophil apoptosis induced by live T. vaginalis could contribute to resolution of inflammation. Macrophages may constitute an important component of host defense against T. vaginalis infection. For example, mouse macrophages alone and those activated by lymphokines or nitric oxide are known to be involved in the extracellular killing of T. vaginalis. In the host, T. vaginalis uses a capping phenomenon to cleave host immunoglobulins with proteinases and thus escape from host immune responses. Recently, we developed a highly sensitive and specific diagnostic polymerase chain reaction (PCR) technique using primers based on a repetitive sequence cloned from T. vaginalis (TV-E650), and found that the method enables the detection of T. vaginalis at concentrations as low as 1 cell per PCR mixture.     

An ectonucleotide ATP-diphosphohydrolase activity in Trichomonas vaginalis stimulated by galactose and its possible role in virulence

This work describes the ability of living Trichomonas vaginalis to hydrolyze extracellular ATP (164.0 +/- 13.9 nmol Pi/h x 10(7) cells). This ecto-enzyme was stimulated by ZnCl2, CaCl2 and MgCl2, was insensitive to several ATPase and phosphatase inhibitors and was able to hydrolyze several nucleotides besides ATP. The activity was linear with cell density and with time for at least 60 min. The optimum pH for the T. vaginalis ecto-ATPase lies in the alkaline range. D-galactose, known to be involved in adhesion of T. vaginalis to host cells, stimulated this enzyme by more than 90%. A comparison between two strains of T. vaginalis showed that the ecto-ATPase activity of a fresh isolate was twice as much as that of a strain axenically maintained in culture, through daily passages, for several years. The results suggest a possible role for this ecto-ATPase in adhesion of T. vaginalis to host cells and in its pathogenicity.     

Evidence for Gardnerella vaginalis uptake and internalization by squamous vaginal epithelial cells: implications for the pathogenesis of bacterial vaginosis

Bacterial vaginosis (BV), a common condition seen in premenopausal women, is associated with preterm labor, pelvic inflammatory disease, and delivery of low birth weight infants. Gardnerella vaginalis is the predominant bacterial species associated with BV, although its exact role in the pathology of BV is unknown. Using immunofluorescence, confocal and transmission electron microscopy, we found that VK2 vaginal epithelial cells take up G. vaginalis after exposure to the bacteria. Confocal microscopy also indicated the presence of internalized G. vaginalis within vaginal epithelial cells obtained from a subject with BV. Using VK2 cells and (35)S labeled bacteria in an invasion assay, we found that a 1 h uptake of G. vaginalis was 21.8-fold higher than heat-killed G. vaginalis, 84-fold compared to Lactobacillus acidophilus and 6.6-fold compared to Lactobacillus crispatus. Internalization was inhibited by pre-exposure of cells to cytochalasin-D. In addition, the cytoskeletal protein vimentin was upregulated in VK2 cells exposed to G. vaginalis, but there was no change in actin cytoskeletal polymerization/rearrangements or vimentin subcellular relocalization post exposure. Cytoskeletal protein modifications could represent a potential mechanism for G. vaginalis mediated internalization by vaginal epithelial cells. Finally, understanding vaginal bacteria/host interactions will allow us to better understand the underlying mechanisms of BV pathogenesis.     

Detection of microtubules of the flagellate Trichomonas vaginalis by monoclonal antibodies specific for beta-tubulin

Monoclonal antibodies specific for mammalian beta-tubulin recognized the microtubule cytoskeleton of the flagellated protozoon Trichomonas vaginalis. Of seven antibodies, two demonstrated the axostyle, costa, recurrent flagellum, and anterior flagella by indirect immunofluorescence microscopy. The remaining five stained a hazy reticular pattern in the cytoplasm of formaldehydefixed, detergent-extracted organisms. Western immunoblots of whole T. vaginalis extracts treated with protease inhibitors and electrophoresed on polyacrylamide gels containing sodium dodecyl sulfate showed a major band at molecular weight 50,000 when probed with only one of the antibodies which stained the axial cytoskeleton. The antibodies which stained only the cytoplasm showed a different western blot pattern with a major doublet band at MW 58,000-60,000. Another antibody, which stained both the axial cytoskeleton and the reticular cytoplasmic pattern showed major bands at MW 58,000-60,000 and also at MW 40,000-42,000. The recognition of microtubule populations in T. vaginalis by these monoclonal antibodies was different than we found earlier with Leishmania donovani and Toxoplasma gondii, where all seven antibodies recognize cytoskeletal microtubules and produce western blots characteristic of tubulin. Only one of these seven antibodies recognizes tubulin in T. vaginalis by immunoblot. The microtubules of T. vaginalis do not demonstrate all epitopes recognized by monoclonal antibodies specific for mammalian beta-tubulin; one of the antibodies appears to recognize an epitope which is morphologically associated with microtubules but does not have the characteristic MW of tubulin.     

PCR for diagnosis of male Trichomonas vaginalis infection with chronic prostatitis and urethritis

The aim of this study was to assess the usefulness of PCR for diagnosis of Trichomonas vaginalis infection among male patients with chronic recurrent prostatitis and urethritis. Between June 2001 and December 2003, a total of 33 patients visited the Department of Urology, Hanyang University Guri Hospital and were examined for T. vaginalis infection by PCR and culture in TYM medium. For the PCR, we used primers based on a repetitive sequence cloned from T. vaginalis (TV-E650). Voided bladder urine (VB1 and VB3) was sampled from 33 men with symptoms of lower urinary tract infection (urethral charge, residual urine sensation, and frequency). Culture failed to detect any T. vaginalis infection whereas PCR identified 7 cases of trichomoniasis (21.2%). Five of the 7 cases had been diagnosed with prostatitis and 2 with urethritis. PCR for the 5 prostatitis cases yielded a positive 330 bp band from bothVB1 and VB3, whereas positive results were only obtained from VB1 for the 2 urethritis patients. We showed that the PCR method could detect T. vaginalis when there was only 1 T. vaginalis cell per PCR mixture. Our results strongly support the usefulness of PCR on urine samples for detecting T. vaginalis in chronic prostatitis and urethritis patients.     

Trichomonas vaginalis lipophosphoglycan mutants have reduced adherence and cytotoxicity to human ectocervical cells

The extracellular human pathogen Trichomonas vaginalis is covered by a dense glycocalyx thought to play a role in host-parasite interactions. The main component of the glycocalyx is lipophosphoglycan (LPG), a polysaccharide anchored in the plasma membrane by inositol phosphoceramide. To study the role of LPG in trichomonads, we produced T. vaginalis LPG mutants by chemical mutagenesis and lectin selection and characterized them using morphological, biochemical, and functional assays. Two independently selected LPG mutants, with growth rates comparable to that of the wild-type (parent) strain, lost the ability to bind the lectins Ricinnus comunis agglutinin I (RCA120) and wheat germ agglutinin, indicating alterations in surface galactose and glucosamine residues. LPG isolated from mutants migrated faster than parent strain LPG on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting the mutants had shorter LPG molecules. Dionex high-performance anion exchange chromatography with pulsed amperometric detection analyses revealed galactosamine, glucosamine, galactose, glucose, mannose/xylose, and rhamnose as the main monosaccharides of T. vaginalis parent strain LPG. LPG from both mutants showed a reduction of galactose and glucosamine, corresponding with the reduced size of their LPG molecules and inability to bind the lectins RCA120 and wheat germ agglutinin. Mutant parasites were defective in attachment to plastic, a characteristic associated with avirulent strains of T. vaginalis. Moreover, the mutants were less adherent and less cytotoxic to human vaginal ectocervical cells in vitro than the parental strain. Finally, while parent strain LPG could inhibit the attachment of parent strain parasites to vaginal cells, LPG from either mutant could not inhibit attachment. These combined results demonstrate that T. vaginalis adherence to host cells is LPG mediated and that an altered LPG leads to reduced adherence and cytotoxicity of this parasite.     

Interactions between Trichomonas vaginalis and vaginal flora in a mouse model.

To study the role of vaginal flora and pH in the pathogenesis of Trichomonas vaginalis, an intravaginal mouse model of infection was established. By employing this model, the vaginal flora and pH of mice could be monitored for changes caused by the parasite. As a baseline, the endemic vaginal flora of BALB/c mice was examined first and found to consist mainly of Staphylococcus aureus and Enterococcus species (32-76%). Lactobacilli and enteric bacilli were moderate (16-32%) in their frequency of isolation, and the prevalence of both anaerobic species and coagulase-negative staphylococci was low (4-16%). Vaginal pH was recorded at 6.5 +/- 0.3. Estrogenization, which was required for a sustained T. vaginalis infection, did not significantly alter vaginal flora; however, a slight rise in the number of bacterial species isolated per mouse and a drop in vaginal pH (6.2 +/- 0.5) were observed. Trichomonas vaginalis-infected mice did not appear to show significant changes in vaginal flora although vaginal pH was slightly increased. This mouse model could have applications in both immunologic and pathogenic studies of T. vaginalis and, with further modifications, aid in the study of protist-bacterial interactions.     

Interaction of Trichomonas vaginalis and Tritrichomonas foetus with keratin: an important role in parasite infection

Trichomonas vaginalis and Tritrichomonas foetus are human and bovine parasites, respectively, that provoke the sexually transmitted disease trichomoniasis. These extracellular parasites adhere to the host epithelial cell surface. Although mucinases and proteases have been described as important proteins for parasite adhesion to epithelial cells, no studies have examined the role of the keratin molecules that cornify the vaginal epithelium. Here, we investigated the interaction of T. vaginalis and T. foetus with human keratin in vitro; additionally, adherence assays were performed in cattle with T. foetus to elucidate whether trichomonads were able to interact with keratin in vivo. We demonstrated that both T. vaginalisand T. foetusinteracted directly with keratin. Additionally, the trichomonads ingested and digested keratin, shedding new light on the Trichomonas infection process.     

Characterization, cloning and immunolocalization of a coronin homologue in Trichomonas vaginalis

On adhesion to host cells the flagellate Trichomonas vaginalis switches to an amoeboid form rich in actin microfilaments. We have undertaken the identification of actin-associated proteins that regulate actin dynamics. A monoclonal antibody 4C12 raised against a cytoskeletal fraction of T. vaginalis labeled a protein doublet at circa 50 kDa. These two bands were recognized by the antibody against Dictyostelium discoideum coronin. During cell extraction and actin polymerization, T. vaginalis coronin cosedimented with F-actin. By two-dimensional gel electrophoresis, the protein doublet was separated into two sets of isoforms covering two Ip zones around 6 and 7. By screening a T. vaginalis library with 4C12, two clones Cor 1 and Cor 2 were isolated. This gene duplicity is a particularity among unicellular organisms examined. The complete sequence of the gene Cor 1 encodes a 435-residue protein with a calculated molecular mass of 48 kDa and Ip of 5.58. The incomplete sequence Cor 2 was very similar but with a more basic calculated Ip than Cor 1 on the same region. T. vaginalis coronin had 50% similarity with the coronin family, possessing the five WD-repeats and a leucine zipper in its C-terminal part. Double immunofluorescence labeling showed that coronin mainly colocalized with actin at the periphery of the adherent amoeboid cells. However, coronin labeling displayed patches within a reticular array. Immunogold electron microscopy confirmed the coronin labeling in the actin-rich microfilamentous fringe beneath the plasma membrane, with accumulation in phagocytic zones and pseudopodial extensions. In T. vaginalis, one of the first emerging lineage of eukaryotes, coronin seems to play an important role in actin dynamics and may be a downstream target of a signaling mechanism for the cytoskeleton reorganization.     

Response of Gardnerella vaginalis biofilm to 5 days of moxifloxacin treatment

Polymicrobial communities are often recalcitrant to antibiotics. We tested whether the polymicrobial Gardnerella vaginalis biofilm can be eradicated with moxifloxacin. Twenty women with bacterial vaginosis were treated with 400 mg moxifloxacin for 5 days. The changes in the occurrence and proportions of Gardnerella, Atopobium and Lactobacillus spp. were assessed using FISH. The bacterial biofilm was investigated using desquamated epithelial cells of spontaneously voided urine and sections of vaginal biopsies. Fifteen of 20 women showed a significant and sustained clinical response to moxifloxacin according to Amsel and Nugent criteria. The concentrations of adherent bacteria decreased significantly. The incidence and proportion of Atopobium declined sustainably. The proportions of Lactobacillus in the biofilm mass increased following therapy. Initially, Gardnerella was the main component of the polymicrobial biofilm. Following treatment, Gardnerella was not accessible to FISH in the urine and vaginal samples of 75% of all women. Ten to 12 weeks after the end of therapy, Gardnerella biofilm was cumulatively present in 40%. This was not due to newly acquired disease, but due to reactivation of the persisting, but biochemically inactive biofilm. Despite clear clinical efficacy, and initially definite suppression of the biofilm, moxifloxacin was, similar to metronidazole, not able to eradicate the Gardnerella vaginalis biofilm in all patients.     

Phagocytosis by Trichomonas vaginalis: new insights

BACKGROUND INFORMATION: The parasitic protozoan Trichomonas vaginalis is the causative agent of trichomoniasis, a sexually transmitted disease. The phagocytic activity of this parasite has not been completely elucidated. In order to better understand the mechanisms of trichomonal phagocytosis, we have studied the in vitro capacity of T. vaginalis to phagocytose and degrade Saccharomyces cerevisiae cells. RESULTS AND CONCLUSIONS: To analyse the phagocytic ability and capacity, two isolates of T. vaginalis presenting different virulence grades were used. Complementary techniques, such as fluorescence microscopy, computer-based fluorescence analysis, scanning and transmission electron microscopy and the use of drugs that interfere with the actin microfilaments, were used in order to follow the behaviour of the actin cytoskeleton during phagocytosis of yeast cells by T. vaginalis. It was concluded that: (1) T. vaginalis changes its shape rapidly and engulfs the yeast cells, which are almost as large as the parasite; (2) long-term and fresh cultures are able to phagocytose, although the low-virulence strain JT demonstrated a lower activity when compared with the highly virulent T016 isolate; (3) the T016 strain exhibited an amoeboid morphology during the internalization of yeast cells in contrast with the JT strain; (4) attachment of yeast cells to the parasite occurs via the whole cell surface, including both anterior and recurrent flagella; (5) two forms of phagocytosis were observed: a 'sinking' process without any apparent participation of plasma membrane extensions and the classical phagocytosis where pseudopodia are extended toward the target cell; (6) the internalized S. cerevisiae are digested in lysosomes; (7) competitor sugars D-mannose or L-fucose inhibit the phagocytosis, and inhibition was 1.67 times higher in long-term cultured JT than that of the parasites from fresh isolate T016; (8) a thick layer of actin microfilaments was present underlying the plasma membrane, and especially in the pseudopodia and around the phagocytosed particles; (9) a dramatic change in the distribution pattern of fibrillar actin occurred during phagocytosis; (10) cytochalasin D depressed the phagocytosis; (11) a non-specific recognition and phagocytosis of yeast cells by T. vaginalis is mediated by a mannose receptor present on the parasite surface; (12) the phagocytic process may occur simultaneously during mitosis of the parasite.     

Intranasal immunisation with a 62 kDa proteinase combined with cholera toxin or CpG adjuvant protects against Trichomonas vaginalis genital tract infections in mice

Trichomonosis, caused by the protozoan parasite Trichomonas vaginalis, is one of the most frequent sexually transmitted diseases and is widely spread in all continents. Trichomonas vaginalis as well as other protozoan organisms have high levels of proteolitic activity mainly of the cysteine-proteinase type. This activity is necessary for recognition and adhesion of the parasite to the superficial epithelial cells of the host. In the present study, we show that intranasal immunisation with a 62 kDa cysteine-proteinase purified from T. vaginalis excretion-secretion products in combination with cholera toxin or with synthetic oligodeoxynucleotides (ODN) that contain unmethylated CpG motifs (CpG-ODN) elicits 62kDa specific IgG and IgA in vaginal lavage fluid and specific IgG in serum. This immunisation protocol resulted in enhanced elimination of parasites following intravaginal challenge of BALB/c mice.     

Observations on Trichomonas vaginalis infections in Zambia

Microscopy and culture of high vaginal swabs from an ante-natal clinic showed that 38.5% of the women harboured Trichomonas vaginalis; the corresponding figure from a gynaecology clinic was 31.4%. The incorporation of the culture method together with direct microscopy as a diagnostic technique in a routine laboratory is emphasized. Urine microscopy in out-patients, in-patients and medical examinees indicates that T. vaginalis in both males and females is not a rare infestation but the prevalence rate of 4.8% in males is considered an underestimate. Asymptomatic and symptomatic trichomoniasis was evident during the study. The age group in which the infection is most common is noted; this evidence suggests that venereal transmission need not be the only source of infection. The role of urinary schistosomiasis as a possible cause of urethritis is discussed.     

Localization of acetylated alpha-tubulin in Tritrichomonas foetus and Trichomonas vaginalis

We used monoclonal antibodies specific for acetylated and nonacetylated alpha-tubulin to detect and to localize microtubules containing acetylated alpha-tubulin (stable microtubules) in the pathogenic protozoa Tritrichomonas foetus and Trichomonas vaginalis. SDS-PAGE analysis showed that tubulin is a major protein of both parasites, being enriched in cytoskeletal preparations of whole cells extracted with Triton X-100. The monoclonal antibodies, which recognize all isoforms of alpha-tubulin (B-5-1-2) and only acetylated alpha-tubulin (6-11B-1), bind to the tubulin of T. foetus and T. vaginalis as seen by immunoblotting. Tubulin-containing structures were localized using immunofluorescence microscopy and transmission electron microscopy of the whole cytoskeleton previously incubated in the presence of the anti-tubulin antibodies and a second antibody-gold complex, and then processed using the negative staining or replica techniques. The results obtained indicate that, in addition to the flagellar microtubules, those which form the peltar-axostyle system represent stable microtubules containing acetylated alpha-tubulin.     

Chlamydia trachomatis infections in women with urogenital symptoms.

Chlamydia trachomatis was isolated from 30 to 100 women attending a family physician''s office with dysuria, frequency or vaginal discharge, compared with 2 of 30 asymptomatic women. Multiple infections were common: C. trachomatis coexisted with Gardnerella vaginalis, Candida albicans, Trichomonas vaginalis or a bacterial cause of urinary tract infection in 15 patients. C. trachomatis was isolated alone from 15 symptomatic women. The source of the positive culture was not always the site of symptoms. C. trachomatis was isolated from both the cervix and the urine of 9 patients, either simultaneously or sequentially. The probability of finding a chlamydial infection was 30% in young women with vaginal discharge alone, 33% in those with dysuria and frequency alone and 53% in those with abdominal or pelvic pain in addition to lower urogenital tract symptoms.     

Bacterial vaginosis among a group of married Jordanian women: occurrence and laboratory diagnosis

A total of 310 vaginal swabs collected from a group of married Jordanian women complaining of vaginal discharge were examined for bacterial vaginosis. The scoring system of Nugent for the interpretation of Gram staining was employed. This system revealed the presence of the condition in 29.7% of patients. Results obtained using the scoring system correlated significantly with the detection of clue cells and the scarcity of white blood cells in the vaginal discharge. An inverse relationship was found between bacterial vaginosis and Lactobacillus morphotypes determined by Gram staining. No definite relationship was detected between bacterial vaginosis and the recovery of Gardnerella vaginalis by culture as this organism was isolated from swabs which according to the Nugent criterion were negative for bacterial vaginosis. Bacterial vaginosis among the women investigated was more prevalent than vaginitis caused by Trichomonas vaginalis or yeasts.