Production of interleukin-8 by human neutrophils stimulated with Trichomonas vaginalis

Neutrophils are the predominant inflammatory cells found in the vaginal discharges of patients infected with Trichomonas vaginalis. Although chemoattractants, such as leukotriene B(4) and interleukin-8 (IL-8), are found in the vaginal discharges of symptomatic trichomoniasis patients, little is known about the mechanism of how neutrophils accumulate or mediate initial inflammatory response after acute T. vaginalis infection. We examined IL-8 production in neutrophils activated by T. vaginalis and evaluated the factors involved in T. vaginalis adherence that might affect IL-8 production. When human neutrophils were stimulated with live trophozoites, T. vaginalis lysate, or T. vaginalis excretory-secretory products, the live trichomonads induced higher levels of IL-8 production than the lysate or products did. When live trichomonads were pretreated with various inhibitors of proteinase, microtubule, microfilament, or adhesin (which are all known to participate in the adherence of T. vaginalis to vaginal epithelial cells), IL-8 production significantly decreased compared with the untreated controls. Furthermore, an NF-kappaB inhibitor (pyrrolidine dithiocarbamate), a mitogen-activated protein (MAP) kinase (MEK) inhibitor (PD98059), and a p38 MAP kinase inhibitor (SB203580) significantly suppressed IL-8 synthesis in neutrophils. These results suggest that live T. vaginalis, particularly adherent trophozoites, can induce IL-8 production in neutrophils and that this action may be mediated through the NF-kappaB and MAP kinase signaling pathways. In other words, T. vaginalis-induced neutrophil recruitment may be mediated via the IL-8 expressed by neutrophils in response to activation by live T. vaginalis.     

Cytotoxicity of human peripheral blood monocytes against Trichomonas vaginalis.

Human peripheral blood monocytes were purified by adherence on microexudate-coated plastic and detached by exposure to 1 mM ethylenediamine tetracetic acid. Lysis of Trichomonas vaginalis was measured as release of 3H-thymidine from prelabelled protozoa after 48 hr of incubation. Human blood monocytes had appreciable levels of spontaneous cytotoxicity against Trichomonas vaginalis at effector-to-target cell ratios ranging from 3:1 to 25:1. Cytotoxicity was expressed with fetal bovine serum and human AB serum. Unseparated mononuclear cells, containing 10-25% monocytes as assessed by staining with non-specific esterase, were significantly cytotoxic against Trichomonas vaginalis but cytolysis levels were lower than those of purified monocytes. Lymphoid cells depleted of adherent cells by repeated incubations on plastic (less than 2% esterase-positive cells) had little cytotoxicity against these protozoa. In vitro exposure to silica natural 40-75% inhibition of the cytotoxic capacity of monocytic preparations. The natural cytotoxic capacity of human monocytes against Trichomonas vaginalis could represent one line of resistance against these protozoa.     

Antibody to Trichomonas vaginalis in human cervicovaginal secretions.

Human cervicovaginal secretions were obtained from patients at the Gynecology and Obstetrics Clinics at National Taiwan University Hospital and Cathay General Hospital, Republic of China. Among the 500 patients examined, 33 (6.6%) were infected with Trichomonas vaginalis as determined by the culture method. Secretions from 24 of the infected patients and 30 noninfected women were assayed for anti-T. vaginalis immunoglobulins by the indirect immunofluorescent antibody technique. A few serum samples from both infected and noninfected persons were also included in this study. Immunoglobulin G (IgG) antibody against T. vaginalis was detected in 17 (70.8%) secretions from the infected women. Among the 17 positive secretions, anti-parasite IgA was found in two specimens, IgE was found in three, and IgM was found in one. Of the 30 secretions, 7 (23.3%) from noninfected women also contained anti-parasite IgG. Low levels of natural anti-trichomonad IgG and IgM were detected in the sera of normal persons. Infection with T. vaginalis caused an increase in the serum IgG antibody titer. Cross-reaction between T. vaginalis and Pentatrichomonas hominis was also observed.     

Guanosine kinase from Trichomonas vaginalis.

A novel guanosine kinase was partially purified from the parasitic protozoan Trichomonas vaginalis. Unlike nucleoside kinases from other sources, the preferred substrate for this enzyme was guanosine (Vmax/Km = 120). The enzyme also catalyzed the phosphorylation of inosine (Vmax/Km = 3), uridine (2), adenosine (0.5), cytidine (0.2), and 2'-deoxyguanosine (0.1). The 2'-deoxyribonucleosides of adenine, hypoxanthine, uracil, cytosine and thymine were not phosphorylated. The Km for ATP was 6.6 microM. The enzyme was extremely labile in the absence of ATP. As a result, only a 20-fold purification with 25% recovery of activity was possible. However, this preparation was free of nucleoside phosphorylase, nucleoside phosphotransferase and the distinct uridine kinase. The enzyme had a broad optimum of pH 6.5-8. It had a molecular weight of 15000.     

Immune response to Trichomonas vaginalis. IV. Immunochemical and immunobiological analyses of T. vaginalis antigen

The immune response of patients with trichomoniasis vaginitis to Trichomonas vaginalis was examined by using passive hemagglutination assay and skin reactions. Trichomonas-specific immune responses could hardly be detected in the patients by these assay methods. In order to develop a simple and reliable assay for demonstration of T. vaginalis-specific immune responses in the patients, proliferation responses of peripheral mononuclear cells from the patients were examined. Lymphocytes obtained from the patients were shown to incorporate 3H-methyl-thymidine when stimulated with T. vaginalis antigen in vitro. Furthermore, immunochemical analysis of T. vaginalis antigen has been done by Sephadex chromatography and sodium dodecyl sulfate gel electrophoresis. The data revealed that the murine and human IgM antibodies specific to T. vaginalis recognize various antigens with a wide range of molecular weights (between 13,000 and 100,000 daltons), while human and murine IgG antibodies recognize molecules with a much narrower range of molecular weights (50,000-100,000 daltons).     

Induction of interleukin-8 synthesis from monocytes by human C5a anaphylatoxin.

Interleukin-8 (IL-8) is an important mediator of inflammation and has been shown to be a potent chemotactic/cell activator for polymorphonuclear neutrophils (PMNs). T lymphocytes, and basophils. Cellular sources of IL-8 include monocytes, PMNs, endothelial cells, epithelial cells, and keratinocytes when stimulated by factors such as lipopolysaccharide, IL-1, and tumor necrosis factor-alpha. This report demonstrates that C5a, in addition to being a direct mediator of inflammation, can induce both IL-8 synthesis and high levels of release from monocytes. Natural human C5a and a synthetic C-terminal analogue peptide of C5a each induced IL-8 synthesis and release from CD14+ human peripheral blood mononuclear cells. Antigenic reactivity based on enzyme-linked immunosorbent assay gave evidence that IL-8 was present in the culture supernatants of stimulated peripheral blood mononuclear cells. Proof that supernatant levels of IL-8 attain biologically significant quantities was provided by human PMN chemotaxis assays. The quantity of IL-8 recovered from C5a-activated monocytes in peripheral blood mononuclear cells is up to 1,000-fold greater than that released from comparable numbers of PMNs under similar conditions. Therefore, IL-8 released from C5a-activated monocytes may play a significant role in expanding and prolonging cellular infiltration and activation at sites of infection, inflammation, or tissue injury. This observation suggests an important humoral amplification loop for inflammatory events involving both complement activation and cytokine release.     

Antigenicity of Trichomonas vaginalis heat-shock proteins in human infections

Patients infected with Trichomonas vaginalis mount humoral and cellular immune responses that often do not protect against reinfection. The oxidative stressors produced by leukocytes may trigger a heat-shock-like response in T. vaginalis trophozoites, helping the parasite to survive host immune defenses. The antigenicity of T. vaginalis heat-shock proteins (HSPs) was examined by immunoprecipitation of T. vaginalis heat-induced proteins with sera from infected patients and controls. When T. vaginalis was heat-shocked, HSPs of 169–167 and 140–137 kDa were specifically recognized by sera from infected male and female patients. However, the majority of T. vaginalis HSPs were also immunoprecipitated by control sera; all sera recognized 72- to 71-kDa, 47- to 45-kDa, 38- to 37-kDa, 35-kDa, and 31-kDa heat-induced proteins. At least 15 proteins from non-heat-shocked T. vaginalis were immunoprecipitated by sera from infected patients and controls, indicating that natural or cross-reacting antibodies could participate in host responses to trichomoniasis. Molecules of 158, 135, 89, and 74–72 kDa were immunoprecipitated from some non-heat-shocked parasites only by patients' sera. A 38-kDa T. vaginalis protein was immunoprecipitated only by sera from infected females and may be specific for infection in women. Received: 26 August 1999 / Accepted: 15 September 1999     

Comparison of four methods to detect Trichomonas vaginalis.

Four methods for the detection of Trichomonas vaginalis in vaginal secretions from 88 symptomatic patients were compared: wet-mount examination, Kupferberg liquid medium, Hirsch charcoal agar, and the Papanicolaou smear. Hirsch diphasic slants and Kupferberg medium did not significantly differ in sensitivity from direct examination of wet mounts. The Papanicolaou smear identification of trichomonads was found to be the least sensitive method evaluated.     

Inorganic pyrophosphatase of Trichomonas vaginalis.

Trichomonas vaginalis homogenates were found to have an acid inorganic pyrophosphatase activity with a specific activity at pH 4.8 of about 7 nmol min-1 (mg protein)-1. This activity was localized predominantly in hydrolase containing particles, showed structure-bound latency and was tightly membrane-bound. The activity showed no magnesium dependence, a Km of about 2 mM inorganic pyrophosphate, a pH optimum of 5.2 and was inhibited by fluoride at millimolar levels. No evidence was obtained for the existence of a cytosolic magnesium-dependent activity but the existence of a low level of magnesium-independent cytosolic activity cannot be excluded. These observations correlate with the importance of cytosolic inorganic pyrophosphate in the carbohydrate catabolism in T. vaginalis.     

Contact-dependent cytopathogenic mechanisms of Trichomonas vaginalis.

The cytopathogenic mechanisms of Trichomonas vaginalis have been debated since the 1940s. We examined the following three proposed pathogenic mechanisms: contact-dependent extracellular killing, cytophagocytosis, and extracellular cytotoxins. Serial observations of Chinese hamster ovary (CHO) cell monolayers exposed to trichomonads revealed that (i) trichomonads form clumps, (ii) the clumps adhere to cells in culture, and (iii) monolayer destruction occurs only in areas of contact with T. vaginalis. Kinetic analysis of target cell killing by trichomonads revealed that the probability of CHO cell death was related to the probability of contact with T. vaginalis, supporting the observation by microscopy that trichomonads kill cells only by direct contact. Simultaneous studies of 111indium oxine label release from CHO cells and trypan blue dye exclusion demonstrated that T. vaginalis kills target cells without phagocytosis. Filtrates of trichomonad cultures or from media in which trichomonads were killing CHO cells had no effect on CHO cell monolayers, indicating that trichomonads do not kill cells by a cell-free or secreted cytotoxin. The microfilament inhibitor cytochalasin D (10 micrograms/ml) inhibited trichomonad killing of CHO cell monolayers by 80% (P less than 0.0001). In contrast, the microtubule inhibitor vinblastine (10(-6) M) caused only 17% inhibition of trichomonad destruction of CHO cell monolayers (P less than 0.020), whereas colchicine (10(-6) M) had no effect. T. vaginalis kills target cells by direct contact without phagocytosis. This event requires intact trichomonad microfilament function; microtubule function appears not to be essential.     

Pseudocyst forms of Trichomonas vaginalis from cervical neoplasia

Trichomonas vaginalis, a flagellated protozoan parasite causes a variety of adverse health consequences in both men and women. The parasite exists in the trophozoite and the pseudocystic stage. The study reports for the first time that pseudocyst forms of T. vaginalis isolated from cervical neoplasia (CN) patients demonstrated distinct, different and significant in vitro growth profiles when grown in vitro cultures from day 1 up to day 5 (p<0.05, Mann-Whitney test) when compared with the same life cycle stages isolated from non-cervical neoplasia but symptomatic patients (NCN). Pseudocysts from CN and NCN isolates remained viable in distilled water until 3 h 10 min and 2 h 10 min, respectively. The nucleus of pseudocysts in CN isolates using acridine orange and DAPI showed more intense staining revealing higher nuclear content. The FITC-labeled Concanavalin A stained stronger green fluorescence with surface of pseudocysts in CN isolates showing more rough and creased surface with higher numbers of deep micropores with larger numbers of chromatin masses, vacuoles, and hydrogenosomes. The study confirms that pseudocystic stage from CN, despite the uniformity in appearance of being rounded and showing no motility without a true cyst wall under light microscopy, demonstrated different biochemical, surface, and ultrastructural properties. The study provides evidence that phenotypic variant forms of pseudocysts does exist and possibly does play a role in exacerbating cervical cancer.     

Isolation and characterization of flagella from Trichomonas vaginalis

As a first step in the biochemical and biomedical analyses of flagella from Trichomonas vaginalis the flagella were isolated, purified, and analyzed. The flagella were detached by mechanical shearing and a crude flagellar preparation was obtained by low-speed differential centrifugation. The crude flagellar preparation was subjected to further purification by discontinuous sucrose density-gradient centrifugation. Electron micrographs (EM) of the purified flagella showed the typical 9 + 2 axonemal arrangement. The structural integrity and the flagellar membrane were not destroyed by the deflagellation method or the purification scheme employed. The flagellar preparations were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified preparation contained many flagellar enriched proteins ranging from 20 to 120 kDa. Three major proteins of 65 kDa and a doublet of about 50–58 kDa were observed. The protein patterns and EM appearance of the fractions were highly reproducible. Received: 12 December 1997 / Accepted: 5 June 1998     

Characterization of Trichomonas vaginalis virus proteins in the pathogenic protozoan T. vaginalis

Summary.  The 4.6-kb double-stranded (ds) RNA of Trichomonas vaginalis virus (TVV)-T1 has been shown to encode two overlapping genes, cap and pol. In this study, a serum for specifically detecting viral cap gene product was raised against a recombinant protein, and sera for specifically detecting pol gene product were raised against synthetic oligopeptides. A 75-kDa major protein and a 160-kDa minor protein were detected by anti-CAP serum in a TVV-T1 sample, indicating that the 75-kDa protein is the viral capsid protein. The 160-kDa protein alone was also detected by two distinct anti-POL sera, indicating that the pol gene is expressed as a CAP-POL fusion protein. These results suggest that the TVV-T1 genome is arranged into a cap-pol organization in a manner similar to that of viruses in family Totiviridae. Accepted December 12, 1997 Received September 8, 1997     

Lipoteichoic acid induces secretion of interleukin-8 from human blood monocytes: a cellular and molecular analysis.

Invasion by gram-positive and gram-negative bacterial organisms is characterized immunopathologically by the activation of mononuclear phagocytic cells, leading to the elaboration of macrophage-derived regulatory and chemotactic factors, and the resultant influx of inflammatory leukocytes. Little is known regarding the mechanisms by which gram-positive organisms initiate macrophage activation and subsequent inflammation. In this investigation, we postulated that lipoteichoic acid (LTA) purified from two different gram-positive bacterial species was an important signal for the expression of chemotactic cytokines from human peripheral blood monocytes (PBM). In initial experiments, we demonstrated that cell-associated interleukin-8 (IL-8) was expressed by mononuclear phagocytes present in inflamed areas of endocardium in cases of acute Staphylococcus aureus endocarditis. We next demonstrated that LTA purified from either Staphylococcus aureus or Streptococcus pyogenes induced the time- and dose-dependent expression of IL-8 mRNA and protein from human PBM. The expression of IL-8 mRNA from LTA- but not lipopolysaccharide (LPS)-treated PBM was superinduced by concomitant treatment with cycloheximide, indicating that the expression of IL-8 mRNA from LTA-treated PBM was negatively controlled by repressor proteins. Furthermore, mRNA stability studies indicated that IL-8 mRNA was less stable in the presence of LTA than in the presence of LPS. Our findings indicate that LTA can induce the secretion of the polymorphonuclear leukocyte chemotactic factor IL-8 and that LTA may be an important cellular mediator of inflammatory cell recruitment that characterizes immune responses to gram-positive bacterial infections.     

Complement-mediated regulation of Trichomonas vaginalis infection in mice.

Trichomonas vaginalis is a flagellated protozoan which causes trichomoniasis, a sexually transmitted disease of the human genitourinary tract. The importance of the alternative complement pathway in host defence against T. vaginalis was investigated in vitro. Kinetic studies utilising immunofixation following electrophoresis showed that both a strongly and weakly virulent strain of T. vaginalis activated murine serum C3. In vivo studies with congenic-resistant, C5-deficient, B10.D2/OSn- and C5-sufficient, B10.D2/nSn mice showed that the presence of C5 is a significant factor in the innate host resistance to primary infection with a strongly virulent, but not a weakly virulent trichomonad strain.     

Growth of Trichomonas vaginalis in commercial culture media.

There are only two commercially available, ready-to-use culture media which are approved by the Food and Drug Administration for clinical diagnosis of vaginal trichomoniasis: Kupferberg's STS and Diamond's medium (modified). Diamond's medium (Klaas modification), recommended by the Centers for Disease Control for the isolation of Trichomonas vaginalis, was compared in vitro to Kupferberg's (STS) medium. Growth studies using six fresh clinical isolates, all from different patients, showed that while generation time was about 6 h in both STS and Diamond's, the period of exponential growth was longer in Diamond's. More important, in STS there was a 4-h lag period during which the population significantly decreased prior to exponential growth. This did not occur in Diamond's medium. Three hundred organisms inoculated into Diamond's reached a population of over 10(5) organisms in 72 h. In STS, the same inoculum could multiply to only 6 x 10(3) organisms. The fact that there is a lag phase in STS which is not seen in Diamond's could explain why low numbers of T. vaginalis do not multiply in STS but do multiply and can be detected in Diamond's. We conclude that because Diamond's medium (modified) allows more prolific growth over a shorter period of time, it is more suitable than Kupferberg's (STS) for detecting T. vaginalis in patients with vaginitis.     

Characterization of an ecto-phosphatase activity in the human parasite Trichomonas vaginalis

We have characterized phosphatase activity present on the external surface of Trichomonas vaginalis, using intact living parasites. This enzyme hydrolyzes the substrate p-nitrophenylphosphate (p-NPP) at a rate of 134.3+/-14.8 nmol Pi/h per 10(7) cells. This phosphatase activity decreased by increasing the pH from 6.8 to 8.4, a pH range in which cell viability was maintained for at least 1 h. Experiments using classical inhibitors of acid phosphatases, such as ammonium molybdate and sodium fluoride, as well as inhibitors of phosphotyrosine phosphatase, such as sodium orthovanadate, [monoperoxo(picolinato)oxovanadate(V)] (mpV-PIC) and [potassiumbisperoxo(1,10-phenanthroline)oxovanadate(V)] (bpV-PHEN), showed a decrease in this phosphatase activity, with different patterns of inhibition. Cytochemical analysis showed the localization of this enzyme on the parasite surface (cell body and flagellum) and in intracellular vacuoles. Phosphatase reaction products were also observed in exocytosed membrane-bound material.     

Antigen-specific proliferation responses of peripheral blood lymphocytes to Trichomonas vaginalis antigen in patients with Trichomonas vaginitis.

This report describes the development of an assay system which overcomes the difficulty of detecting immune responses of patients with Trichomonas vaginitis by making use of peripheral blood leukocytes obtained from such patients. When peripheral blood leukocytes from the patient were stimulated in microcultures with the soluble antigen extracted from Trichomonas vaginalis, significant degrees of proliferation ensued, as measured by the incorporation of [methyl-3H]thymidine 4 to 5 days after initiation. The antigen-induced proliferation response of peripheral blood leukocytes is specific for T. vaginalis antigen. The T. vaginalis-specific [methyl-3H]thymidine incorporation is mediated by Leu-1-positive cells, namely, T lymphocytes, in the peripheral blood leukocyte population. This assay system should prove useful for the analysis of the immune response to the protozoa in patients with Trichomonas vaginitis.