Humoral and T cell-mediated immune response against trichomoniasis.

Trichomonas vaginalis (T. vaginalis) infection leads to the synthesis of specific antibodies in the serum and local secretions. The profile of T. vaginalis-specific antibodies and T cell-mediated immune responses may influence the outcome of infection, towards parasite elimination, persistence or pathological reactions. Studies have indicated that Th1-, Th17- and Th22 cell-related cytokines may be protective or pathogenic, whereas Th2- and Treg cell-related cytokines can exert anti-inflammatory effects during T. vaginalis infection. A number of T. vaginalis-related components such as lipophosphoglycan (TvLPG), α-actinin, migration inhibitory factor (TvMIF), pyruvate:ferredoxin oxidoreductase (PFO), legumain-1 (TvLEGU-1), adhesins and cysteine proteases lead to the induction of specific antibodies. T. vaginalis has acquired several strategies to evade the humoral immune responses such as degradation of immunoglobulins by cysteine proteases, antigenic variation and killing of antibody-producing B cells. The characterization of the T. vaginalis-specific antibodies to significant immunogenic molecules and formulation of strategies to promote their induction in vaginal mucosa may reveal their potential protective effects against trichomoniasis. In this review, we discuss the current understanding of antibody and T cell-mediated immune responses to T. vaginalis and highlight novel insights into the possible role of immune responses in protection against parasite.

Role of α-Actinin 2 in Cytoadherence and Cytotoxicity of Trichomonas vaginalis.

Trichomonas vaginalis is a pathogen that triggers severe immune responses in hosts. T. vaginalis α-actinin 2 (Tvα-actinin 2) has been used to diagnose trichomoniasis. Tvα-actinin 2 was dissected into three parts; the N-terminal, central, and C-terminal portions of the protein (#1, #2, and #3, respectively). Western blot of these Tvα-actinin 2 proteins with pooled patients' sera indicated that #2 and #3, but not #1, reacted with those sera. Immunofluorescence assays of two different forms of T. vaginalis (trophozoites and amoeboid forms), using anti-Tvα-actinin 2 antibodies, showed localization of Tvα-actinin 2 close to the plasma membranes of the amoeboid form. Fractionation experiments indicated the presence of Tvα-actinin 2 in cytoplasmic, membrane, and secreted proteins of T. vaginalis. Binding of fluorescence-labeled Trichomonas to vaginal epithelial cells and prostate cells was decreased in the antibody blocking experiment using anti-Tvα-actinin 2 antibodies. Pretreatment of T. vaginalis with anti-rTvα-actinin 2 antibodies also resulted in reduction in its cytotoxicity. Flow cytometry, ligand-binding immunoblotting assay, and observation by fluorescence microscopy were used to detect the binding of recombinant Tvα-actinin 2 to human epithelial cell lines. Specifically, the truncated N-terminal portion of Tvα-actinin 2, Tvα-actinin 2 #1, was shown to bind directly to vaginal epithelial cells. These data suggest that α-actinin 2 is one of the virulence factors responsible for the pathogenesis of T. vaginalis by serving as an adhesin to the host cells.

Trichomonas vaginalis infection and risk of prostate cancer: associations by disease aggressiveness and race/ethnicity in the PLCO Trial.

PURPOSE: Results from previous sero-epidemiologic studies of Trichomonas vaginalis infection and prostate cancer (PCa) support a positive association between this sexually transmitted infection and aggressive PCa. However, findings from previous studies are not entirely consistent, and only one has investigated the possible relation between T. vaginalis seropositivity and PCa in African-American men who are at highest risk of both infection and PCa. Therefore, we examined this possible relation in the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial, including separate analyses for aggressive PCa and African-American men. METHODS: We included a sample of participants from a previous nested case-control study of PCa, as well as all additional Caucasian, aggressive, and African-American cases diagnosed since the previous study (total n = 438 Gleason 7 Caucasian cases, 487 more advanced Caucasian cases (≥Gleason 8 or stage III/IV), 201 African-American cases, and 1216 controls). We tested baseline sera for T. vaginalis antibodies. RESULTS: No associations were observed for risk of Gleason 7 (odds ratio (OR) = 0.87, 95% confidence interval (CI) 0.55-1.37) or more advanced (OR = 0.90, 95% CI 0.58-1.38) PCa in Caucasian men, or for risk of any PCa (OR = 1.06, 95% CI 0.67-1.68) in African-American men. CONCLUSIONS: Our findings do not support an association between T. vaginalis infection and PCa.

Host-Protozoan Interactions Protect from Mucosal Infections through Activation of the Inflammasome.

While conventional pathogenic protists have been extensively studied, there is an underappreciated constitutive protist microbiota that is an integral part of the vertebrate microbiome. The impact of these species on the host and their potential contributions to mucosal immune homeostasis remain poorly studied. Here, we show that the protozoan Tritrichomonas musculis activates the host epithelial inflammasome to induce IL-18 release. Epithelial-derived IL-18 promotes dendritic cell-driven Th1 and Th17 immunity and confers dramatic protection from mucosal bacterial infections. Along with its role as a "protistic" antibiotic, colonization with T. musculis exacerbates the development of T-cell-driven colitis and sporadic colorectal tumors. Our findings demonstrate a novel mutualistic host-protozoan interaction that increases mucosal host defenses at the cost of an increased risk of inflammatory disease.

Signalling pathways associated with IL-6 production and epithelial-mesenchymal transition induction in prostate epithelial cells stimulated with Trichomonas vaginalis.

Trichomonas vaginalis (Tv) has been found in patient tissue of benign prostatic hyperplasia (BPH), and suggested to cause chronic prostatitis. IL-6 is known as one of the important factors of chronic inflammation in prostate cancer. Patients with chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) had higher levels of IL-6 in seminal plasma. Furthermore, inflammatory conditions induced by pathogen infections have been shown to promote epithelial-mesenchymal transition (EMT). Here, we investigated the signals involved in IL-6 production by human prostate epithelial cells (PECs) stimulated with Tv and examined whether Tv induces EMT in PECs. We found that PECs stimulated with Tv increased the production of IL-6, as well as the expression of TLR2, TLR4, MAPKs (p38, JNK, ERK), NF-κB and JAK2/STAT3, and levels of ROS. Inhibition of TLR2 or TLR4 reduced IL-6 production as well as expression of these other factors, and agents inhibiting ROS, MAPKs, NF-κB and JAK reduced IL-6 production. However, when PECs were stimulated with Tv, transcripts of mesenchymal cell markers increased, and epithelial cell markers decreased. In addition, the induction of EMT was suppressed by inhibitors of JAK or NF-κB. These findings are the first evidence that Tv infection of prostate epithelial cells may induce EMT.

Inflammatory Responses in a Benign Prostatic Hyperplasia Epithelial Cell Line (BPH-1) Infected with Trichomonas vaginalis.

Trichomonas vaginalis causes the most prevalent sexually transmitted infection worldwide. Trichomonads have been detected in prostatic tissues from prostatitis, benign prostatic hyperplasia (BPH), and prostate cancer. Chronic prostatic inflammation is known as a risk factor for prostate enlargement, benign prostatic hyperplasia symptoms, and acute urinary retention. Our aim was to investigate whether T. vaginalis could induce inflammatory responses in cells of a benign prostatic hyperplasia epithelial cell line (BPH-1). When BPH-1 cells were infected with T. vaginalis, the protein and mRNA of inflammatory cytokines, such as CXCL8, CCL2, IL-1ß, and IL-6, were increased. The activities of TLR4, ROS, MAPK, JAK2/STAT3, and NF-κB were also increased, whereas inhibitors of ROS, MAPK, PI3K, NF-κB, and anti-TLR4 antibody decreased the production of the 4 cytokines although the extent of inhibition differed. However, a JAK2 inhibitor inhibited only IL-6 production. Culture supernatants of the BPH-1 cells that had been incubated with live T. vaginalis (trichomonad-conditioned medium, TCM) contained the 4 cytokines and induced the migration of human monocytes (THP-1 cells) and mast cells (HMC-1 cells). TCM conditioned by BPH-1 cells pretreated with NF-κB inhibitor showed decreased levels of cytokines and induced less migration. Therefore, it is suggested that these cytokines are involved in migration of inflammatory cells. These results suggest that T. vaginalis infection of BPH patients may cause inflammation, which may induce lower urinary tract symptoms (LUTS).

Persistence of Trichomonas vaginalis serostatus in men over time.

PURPOSE: Previous epidemiologic studies have observed positive associations between Trichomonas vaginalis (Tv) serostatus and both prostate cancer (PCa) risk and mortality. However, only a few small older studies have examined Tv antibody persistence over time, all of which were composed mainly of female patients. Therefore, we examined Tv antibody persistence over time, as well as intra-individual variability, among middle- to older-aged men in the Southern Community Cohort Study (SCCS). METHODS: We tested baseline and repeat plasma specimens (collected 1-3 years later) from 248 male participants for Tv antibodies. We used the same enzyme-linked immunosorbent assay as in previous studies of Tv serostatus and PCa. RESULTS: At baseline, 46 (18.5 %) participants were seropositive for Tv infection. Seventy-six percent of these men were still seropositive 1-3 years later. A similar proportion of men "seroconverted" (4.0 %) as "seroreverted" (4.4 %), all of whom had absorbance values near the cutoff point for seropositivity. Overall, substantial agreement was observed between baseline and repeat serostatus (κ = 0.72, 95 % confidence interval 0.60-0.83). CONCLUSION: Tv seropositivity was largely persistent between plasma specimens collected 1-3 years apart from middle- to older-aged men. These high levels of persistence are similar to those observed for other sexually transmitted infections frequently investigated in relation to PCa.

Trichomonas vaginalis homolog of macrophage migration inhibitory factor induces prostate cell growth, invasiveness, and inflammatory responses.

The human-infective parasite Trichomonas vaginalis causes the most prevalent nonviral sexually transmitted infection worldwide. Infections in men may result in colonization of the prostate and are correlated with increased risk of aggressive prostate cancer. We have found that T. vaginalis secretes a protein, T. vaginalis macrophage migration inhibitory factor (TvMIF), that is 47% similar to human macrophage migration inhibitory factor (HuMIF), a proinflammatory cytokine. Because HuMIF is reported to be elevated in prostate cancer and inflammation plays an important role in the initiation and progression of cancers, we have explored a role for TvMIF in prostate cancer. Here, we show that TvMIF has tautomerase activity, inhibits macrophage migration, and is proinflammatory. We also demonstrate that TvMIF binds the human CD74 MIF receptor with high affinity, comparable to that of HuMIF, which triggers activation of ERK, Akt, and Bcl-2-associated death promoter phosphorylation at a physiologically relevant concentration (1 ng/mL, 80 pM). TvMIF increases the in vitro growth and invasion through Matrigel of benign and prostate cancer cells. Sera from patients infected with T. vaginalis are reactive to TvMIF, especially in males. The presence of anti-TvMIF antibodies indicates that TvMIF is released by the parasite and elicits host immune responses during infection. Together, these data indicate that chronic T. vaginalis infections may result in TvMIF-driven inflammation and cell proliferation, thus triggering pathways that contribute to the promotion and progression of prostate cancer.

Prospective study of effect modification by Toll-like receptor 4 variation on the association between Trichomonas vaginalis serostatus and prostate cancer.

PURPOSE: In previous studies, we observed a positive association between Trichomonas vaginalis serostatus and risk of prostate cancer, particularly aggressive cancer, which we hypothesized might be due to T. vaginalis-mediated intraprostatic inflammation and cell damage. To explore this hypothesis further, we investigated effect modification by Toll-like receptor 4 (TLR4) variation on this association. We hypothesized that TLR4 variation might serve a marker of the anti-trichomonad immune response because T. vaginalis has been shown to elicit inflammation through this receptor. METHODS: We previously genotyped the non-synonymous TLR4 single nucleotide polymorphism (SNP), rs4986790, and determined T. vaginalis serostatus for 690 incident prostate cancer cases and 692 controls in a nested case-control study within the Health Professionals Follow-up Study. RESULTS: A non-significant suggestion of effect modification was observed by rs4986790 carrier status on the association between T. vaginalis serostatus and prostate cancer risk (p interaction = 0.07). While no association was observed among men homozygous wildtype for this SNP (odds ratio (OR) = 1.23, 95 % confidence interval (CI): 0.86-1.77), a positive association was observed among variant carriers (OR = 4.16, 95 % CI: 1.32-13.1). CONCLUSIONS: Although not statistically significant, TLR4 variation appeared to influence the association between T. vaginalis serostatus and prostate cancer risk consistent with the hypothesis that inflammation plays a role in this association. Larger studies will be necessary to explore this possible effect modification further.

High detection rate of Trichomonas vaginalis in benign hyperplastic prostatic tissue.

While Trichomonas vaginalis, a protozoan parasite, is a well-investigated pathogen in the female population, there is little awareness of its significance in the male uro-genital tract. The presence of T. vaginalis in the prostate gland has only been scarcely investigated and has never been attested in conditions other than clinical prostatitis. Still, by some authors, this organ is regarded as ecologic niche for T. vaginalis. Since normal prostate tissue of sufficient quality is hard to come by, we investigated samples from 86 patients (mean age 68.7 ± 7.6 years) suffering from benign prostatic hyperplasia (BPH), a medical condition currently ranked as noninfectious, but characterized by chronic inflammatory tissue infiltrates of unknown etiology. Applying two different PCR protocols and sequence analysis of the respective amplicons, we detected T. vaginalis DNA in 29/86 (34%) BPH tissue samples, whereas in only 2/86 (2.3%) cases T. vaginalis grew in culture. Detection of T. vaginalis DNA correlated significantly (P < 0.01) with elevated peripheral blood monocytic cell counts, appearing along with protozoan infections. Given the unexpected high prevalence of T. vaginalis in BPH tissue of a nonselected, elderly study population from Austria, further epidemiological studies have to confirm this finding. Potential interactions of T. vaginalis in its prostatic habitat may be investigated with respect to their possible contribution to the inflammatory pathogenesis of BPH, since inflammatory cytokines have been shown to sustain prostatic hyperplastic growth.

Leukotriene B4 receptor BLT-mediated phosphorylation of NF-κB and CREB is involved in IL-8 production in human mast cells induced by Trichomonas vaginalis-derived secretory products.

Trichomonas vaginalis is a protozoan parasite that causes acute tissue inflammation in vaginal trichomoniasis. In this study, we investigated the signaling mechanisms through which T. vaginalis-derived secretory products (TvSP) induce chemokine IL-8 production in human mast cells. Stimulation with TvSP induced up-regulation of IL-8 protein secretion in HMC-1 cells. In addition, TvSP induced phosphorylation of transcription factors NF-κB and CREB in HMC-1 cells. Pretreatment of TvSP with lipase, but not heat or proteinase K strongly abolished the stimulatory effect on IL-8 production. Moreover, TvSP-induced IL-8 production and phosphorylation of NF-κB or CREB were inhibited when HMC-1 cells were stimulated with modified TvSP collected from 5-lipooxygenase inhibitor-treated trichomonads. Indeed, T. vaginalis-secreted lipid mediator LTB(4) (700pg/ml) from 1×10(7) trichomonads. Furthermore, pretreatment of HMC-1 cells with antagonists for LTB(4) receptors BLT1 or BLT2 abolished the stimulatory effects of TvSP. Finally, TvSP-induced IL-8 production was inhibited by pretreatment with IκB or CREB inhibitors. These results suggest that T. vaginalis-derived secretory lipid mediator LTB(4) induces IL-8 production in mast cells via BLT-dependent activation of NF-κB and CREB.

Impact of T. vaginalis infection on innate immune responses and reproductive outcome.

Trichomonas vaginalis is the most common non-viral sexually transmitted pathogen. The infection is prevalent in reproductive age women and is associated with vaginitis, endometritis, adnexitis, pyosalpinx, infertility, preterm birth, low birth weight, bacterial vaginosis, and increased risk of cervical cancer, HPV, and HIV infection. In men, its complications include urethritis, prostatitis, epididymitis, and infertility through inflammatory damage or interference with the sperm function. The infection is often asymptomatic and recurrent despite the presence of specific antibodies, suggesting the importance of the innate immune defense. T. vaginalis adhesion proteins, cysteine proteases, and the major parasite lipophosphoglycan (LPG) play distinct roles in the pathogenesis and evasion of host immunity. LPG plays a key role in the parasite adherence and signaling to human vaginal and cervical epithelial cells, which is at least in part mediated by galectins. The epithelial cells respond to T. vaginalis infection and purified LPG by selective upregulation of proinflammatory mediators. At the same time, T. vaginalis triggers an immunosuppressive response in monocytes, macrophages, and dendritic cells. The molecular mechanisms underlying reproductive complications and epidemiologic risks associated with T. vaginalis infection remain to be elucidated.

Proinflammatory cytokine and nitric oxide production by human macrophages stimulated with Trichomonas vaginalis.

Trichomonas vaginalis commonly causes vaginitis and perhaps cervicitis in women and urethritis in men and women. Macrophages are important immune cells in response to T. vaginalis infection. In this study, we investigated whether human macrophages could be involved in inflammation induced by T. vaginalis. Human monocyte-derived macrophages (HMDM) were co-cultured with T. vaginalis. Live, opsonized-live trichomonads, and T. vaginalis lysates increased proinflammatory cytokines, such as TNF-alpha, IL-1beta, and IL-6 by HMDM. The involvement of nuclear factor (NF)-kappaB signaling pathway in cytokine production induced by T. vaginalis was confirmed by phosphorylation and nuclear translocation of p65 NF-kappaB. In addition, stimulation with live T. vaginalis induced marked augmentation of nitric oxide (NO) production and expression of inducible NO synthase (iNOS) levels in HMDM. However, trichomonad-induced NF-kappaB activation and TNF-alpha production in macrophages were significantly inhibited by inhibition of iNOS levels with L-NMMA (NO synthase inhibitor). Moreover, pretreatment with NF-kappaB inhibitors (PDTC or Bay11-7082) caused human macrophages to produce less TNF-alpha. These results suggest that T. vaginalis stimulates human macrophages to produce proinflammatory cytokines, such as IL-1, IL-6, and TNF-alpha, and NO. In particular, we showed that T. vaginalis induced TNF-alpha production in macrophages through NO-dependent activation of NF-kappaB, which might be closely involved in inflammation caused by T. vaginalis.

[Cloning, expression and immunogenicity analysis of cysteine proteinase 3 of Trichomonas vaginalis].

OBJECTIVE: To study the humoral antibody response of mice to the recombinant Trichomonas vaginalis cysteine proteinase (TvCP3) in order to investigate the function of the proteinase and its application in diagnosis. METHODS: T. vaginalis cysteine proteinase gene 3 (TvCP3) was cloned by using PCR, and was inserted into the expression vector pET28b. The recombinant plasmid pET28b-TvCP3 or pET28b-TvCP3C (a matured and pre-matured enzyme fragment) was then transformed to Escherichia coli BL21(DE3). The recombinant protein was expressed in E. coli, purified by IMAC (immobilized metal affinity chromatography), and was used to immune BALB/c mice. The mice were divided into groups TvCP3, TvCP3C and control, 6 in each group. The first and second injections for each mouse were administered with 25 microg purified TvCPs which was emulsified with an equal volume of Freund's complete and incomplete adjuvants, respectively. Two more injections were done using 12.5 microg purified antigens without adjuvants, with 2 weeks interval between the first three injections and one week interval between the 3rd and 4th injections. The murine serum samples were detected one week post the 4th injection. The specific IgG antibody in the serum against the recombinant protein was evaluated by ELISA and Western blotting. RESULTS: The expression level of TvCP3 and TvCP3C reached to more than 25% of the total amount of proteins expressed by the bacteria respectively, and the purity in both of them was more than 80% after purified by cobalt-based IMAC resin. ELISA showed that both purified recombinant TvCP3 and TvCP3C induced a high titer of antibodies in the immunized mice (1/204,800 and 1/102,400, respectively). Western blotting analysis indicated that the antibodies reacted with a specific band of the whole T. vaginalis antigens or soluble fractions from T. vaginalis cultures. CONCLUSION: The recombinant TvCP3 and TvCP3C proteins have been highly expressed in E. coli BL21 (DE3) and the expressed products can induce high level of antibodies in BALB/c mice, which recognized a specific band of proteins from T. vaginalis soluble fractions and cultures in vitro.

Dependence on p38 MAPK signalling in the up-regulation of TLR2, TLR4 and TLR9 gene expression in Trichomonas vaginalis-treated HeLa cells.

Toll-like receptors (TLRs) are pattern recognition receptors (PRRs) that recognize conserved pathogen-associated molecular patterns (PAMPs) synthesized by micro-organisms. Despite the essential requirement for TLRs in prokaryotic infection, the pattern and regulation of TLR gene expression by Trichomonas vaginalis in the mucocutaneous barrier are still unknown. Our hypothesis is that T. vaginalis-infected epithelial cells are major effector cells in the skin barrier. These cells function as a central regulator of TLR gene expression, thus accelerating the process of barrier dysfunction via increased release of chemokines and proinflammatory cytokines. To test this hypothesis, RT-PCR was performed on TLRs, interleukin (IL)-8 and tumour necrosis factor (TNF)-alpha. Stimulation of HeLa cells by T. vaginalis was observed to up-regulate TLR2, 4 and 9 mRNA expression as well as that of IL-8 and TNF-alpha. To further clarify the molecular mechanism of barrier devastation triggered by these up-regulatory stimuli, we examined the profiles of extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-kappaB activation in HeLa cells using specific inhibitors. Interestingly, pretreatment of HeLa cells with the p38 MAPK inhibitor SB203580 demonstrated inhibition of T. vaginalis-induced up-regulation of TLR2, 4, and 9 mRNA expression. By contrast, inhibition of ERK or NF-kappaB activation failed to block T. vaginalis-induced up-regulation of TLR9 mRNA expression or TLR2 and TLR4 mRNA expression, respectively. In addition, pretreatment with SB203580 reduced epithelium-derived IL-8 and TNF-alpha release evoked by T. vaginalis. Our results show that T. vaginalis infection of the mucocutaneous barrier could up-regulate TLR2, 4 and 9 gene expression via the p38 MAPK signalling pathway in epithelial cells; this process then leads to modulation of p38 MAPK-dependent IL-8 and TNF-alpha release from the epithelium.

Trichomonas vaginalis inhibits proinflammatory cytokine production in macrophages by suppressing NF-kappaB activation.

Activation of NF-kappaB leads to the production of proinflammatory cytokines such as IL-12 and TNF-alpha that are involved in innate and adaptive immunity. We determined whether T. vaginalis-induced inflammatory response in macrophages associated with NF-kappaB. T. vaginalis adhesion led to transient NF-kappaB activation at 6 h but activation declined dramatically by 8 h. Super-shift assays showed that the gel-shifted complexes consisted of p65 (Rel A) and p50 (NF-kappaB1). NF-kappaB activation was accompanied by IkappaB-alpha degradation, and was inhibited by blocking T. vaginalis adhesion, indicating that the early NF-kappaB activation by T. vaginalis depends on IkappaB-alpha degradation. Quantitative real-time RT-PCR analyses revealed that the expression of TNF-alpha and IL-12 mRNA in T. vaginalis-adhesive cells was rapidly suppressed in comparison with LPS stimulation. We also observed that the parasite inhibited the nuclear translocation of NF-kappaB at 8 h, and diminished IL-12 and TNF-alpha production in response to LPS. In addition, inhibition of IkappaB-alpha degradation by MG-132 resulted in apoptosis. These results demonstrate that effects of T. vaginalis on NF-kappaB regulation are critical for cytokine production and the survival of macrophages. We suggest that there exist inhibitory mechanisms induced by T. vaginalis to evade host immunity.

Involvement of MAP kinases in apoptosis of macrophage treated with Trichomonas vaginalis.

A primitive protozoan parasite Trichomonas vaginalis selectively activates the signal transduction pathways in macrophages (RAW264.7). This study evaluated the correlation of these signaling pathways and T. vaginalis-induced cell apoptosis. In macrophages infected with T. vaginalis, apoptosis was assessed on the basis of DNA fragmentation on agarose gel electrophoresis. Infection of macrophages with T. vaginalis induced tyrosine phosphorylation of several proteins. Infected cells with T. vaginalis were shown to associate with phosphorylation of the extracellular signal-regulated (ERK)1/2 kinase, p38, c-Jun N-terminal kinase (JNK) mitogen-activated protein (MAP) kinases on Western blot analysis. The present finding also demonstrated a link between the ERK1/2, JNK and p38 apoptotic pathways that was modulated by T. vaginalis infection.

Trichomonas vaginalis-induced apoptosis in RAW264.7 cells is regulated through Bcl-xL, but not Bcl-2.

The purpose of this study was to determine whether anti-apoptotic proteins of the Bcl-2 family such as Bcl-2 and Bcl-x(L), proteins that confer resistance to apoptotic death from some stimuli, block apoptotic cell death in RAW264.7 cells upon treatment with Trichomonas vaginalis. In this study, the expression level of Bcl-2 was unchanged throughout the course of apoptotic cell death, and overexpressed Bcl-2 did not prevent release of cytochrome c, the significant change of the membrane potential, activation of caspases, and PARP cleavage in T. vaginalis-treated RAW264.7 cells. On the other hand, Bcl-x(L)expression was decreased after T. vaginalis treatment accompanied with Bax activation. Furthermore, we showed that release of mitochondrial cytochrome c, cleavage of caspase-9 and PARP during apoptosis in T. vaginalis-treated RAW264.7 cells were considerably diminished by transfection with overexpressed Bcl-x(L), and overexpressed Bcl-x(L)could inhibit T. vaginalis-induced apoptosis in RAW264.7 cells. In addition, interestingly, pre-treatment with caspase inhibitors, Boc-D-FMK and Z-DEVD-FMK, significantly abolished T. vaginalis-induced down-regulation of Bcl-x(L), suggesting that caspase-3 may play a pivotal role in the process of apoptosis as well as the down-regulation of Bcl-x(L)by T. vaginalis. Therefore, these results suggest that T. vaginalis-induced apoptosis in RAW264.7 cells can occur via a Bcl-x(L)-dependent apoptotic mechanism.

Evaluation of an IgG cystatin capture enzyme-linked immunosorbent assay for the detection of anti-cysteine proteinase antibodies in asymptomatic trichomoniasis patients.

All isolates of T. vaginalis release cysteine proteinases proteolytic enzymes that are shed into the vagina or culture medium. Cystatin has been used successfully as a capture agent in ELISA to detect cysteine proteinase antibodies without the need for purified proteinases. The ELISA was evaluated in comparison to wet mount microscopy and culture techniques. IgG cystatin capture ELISA proved to be a sensitive and highly specific (100%) assay that could rapidly detect anti-cysteine proteinase antibodies in both vaginal washouts and sera of asymptomatic patients with a sensitivity of 100% and 86.7%, respectively. A defined discrimination between sero-positive and sero-negative individuals was markedly observed for ELISA-vaginal washouts providing a more conclusive diagnosis by this technique. The results demonstrated that in trichomoniasis patients (52 cases) whether symptomatic or asymptomatic. T. vaginalis infection was detected in 19 (36.5%), 49 (94.2%), 50 (96.1%) and 48 (92.3%) by wet mount, culture, cystatin capture ELISA-vaginal washouts and ELISA-sera, respectively. The assay was reliable also as a test of cure with a specificity of 94.4% in the vaginal washouts and 83.3% in sera.

Modulation by Polypodium leucotomos extract of cytokine patterns in experimental trichomoniasis model.

The immunomodulating effects of Anapsos, an aqueous hydrosoluble extract obtained from the rhizomes of the fern Polypodium leucotomos, on both pathogenicity and cytokine levels in serum (IFN-gamma/IL-4) were assayed in a Trichomonas vaginalis experimental model (BALB/c mice infected with 10(7) trichomonads and examined at day 15 after infection). Doses of 20 mg/kg/day administered for 10 days before the infection with the parasite induced a decrease of the experimental pathogenicity approximately 10-20% compared to controls. Gross histopathologic changes at abdominal organs and mortality rate, as a consequence of pathogenicity of the protozoa and the immune response of the host, were evaluated. IFN-gamma and IL-4 cytokines were determined on days -5, 0, 5, 10, and 15 postinfection by indirect ELISA. Treatment with PAL before infection modulates and downregulates the IFN-gamma concentration, while anticipates and upregulates the IL-4 level. The assays performed have showed the utility of the murine model of experimental trichomoniasis for the evaluation of immunomodulatory activity of synthetic or natural products.