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.

Involvement of mast cells in inflammation induced by Trichomonas vaginalis via crosstalk with vaginal epithelial cells.

Vaginal epithelial cells (VECs) are thought to function as immune-responsive cells in trichomoniasis, and mast cells have been detected in vaginal smears and the vaginal wall in trichomoniasis. It therefore seemed possible that the VEC-trichomonad reaction might affect the activity of mast cells present in the lamina propria of the vaginal mucosa. In this study, we tested whether culture supernatants of VEC incubated with Trichomonas vaginalis (TCM) could stimulate mast cells. When VECs (MS74) were incubated with live trichomonads, IL-8, IL-6 and MCP-1 expressions increased in the TCM, and mast cells (HMC-1) and human neutrophils migrated more actively towards the TCM. Also, when the TCM was added to mast cells, ß-hexosaminidase and cytokines (IL-8 and TNF-α) expressions were increased. Moreover, the culture supernatant of mast cells incubated with TCM (M-TCM) had more increased chemotactic activity for neutrophils than that of TCM. We conclude that inflammatory mediators made by VECs in response to activation by T. vaginalis activate and attract mast cells and then stimulate them to induce neutrophil migration. Our results indicate, for the first time, that VECs play a role in the infiltration of mast cells and neutrophils early in T. vaginalis infection.

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.

Asp-Tyr-Leu-Lys tetrapeptide inhibits airway inflammation in toluene-2,4-diisocyanate-induced asthma mice.

BACKGROUND: Airway inflammation and remodelling contribute to chronic airway obstruction of asthma. Currently, no medication effectively controls airway remodelling and related vascular changes. Therefore, new strategies need to be developed. The kringle 5 domain has anti-angiogenic activity resulting from the tetrapeptide Lys-Leu-Tyr-Asp (KLYD). OBJECTIVE: To investigate the therapeutic effect of KLYD and its inverse form Asp-Tyr-Leu-Lys (DYLK) on the inflammation and remodelling of toluene-2,4-diisocyanate (TDI)-sensitization/challenged mice. METHODS: Cell numbers were measured in the presence of various concentrations of KLYD and DYLK using in vitro endothelial cell proliferation assay. The changes of cell number and the level of vascular endothelial growth factor (VEGF) in bronchoalveolar lavage (BAL) fluid and response to methacholine (MCh) were measured using the in vivo TDI-sensitized/challenged mice model. Muc5ac, smooth muscle actin (SMA) and proliferating cell nuclear antigen (PCNA) protein expression was analysed on trachea and intrapulmonary bronchi using immunohistochemical stain. RESULTS: Compared with KLYD, DYLK had a greater inhibitory effect on endothelial cell proliferation (P<0.05). Pre-treatment of DYLK showed dose-dependent reduction in the response to MCh (P<0.05) and numbers of inflammatory cells in BAL fluids of TDI-sensitized/challenged mice. TDI induced increases in Muc5ac, SMA and PCNA protein expression and VEGF levels, which were also abolished by DYLK treatment. CONCLUSIONS: Local administration of DYLK effectively inhibits the airway inflammation and airway remodelling of TDI-sensitized/challenged mice via down-regulation of VEGF. These findings suggest that anti-angiogenic peptide therapies, such as local administration of DYLK, are an effective strategy for the treatment of remodelling in asthma.

Trichomonas vaginalis-induced neutrophil apoptosis causes anti-inflammatory cytokine production by human monocyte-derived macrophages.

Neutrophils are the predominant inflammatory cells found in the vaginal discharge of patients with a Trichomonas vaginalis infection. Neutrophils have a shorter life span than other leucocytes. Our previous study indicated that live T. vaginalis alters Mcl-1 expression and caspase-3 activation, thereby inducing apoptosis of human neutrophils. However, it was previously unknown that the apoptotic neutrophils brought about by T. vaginalis can influence vaginal inflammation. Thus, human monocyte-derived macrophages (HMDM) were incubated with T. vaginalis-induced apoptotic neutrophils. Cytokine production and phagocytosis by HMDM were evaluated by ELISA and myeloperoxidase stain, respectively. HMDM showed increased anti-inflammatory cytokine production (IL-10) and decreased levels of pro-inflammatory cytokines, such as TNF-α and IL-6, compared with macrophages alone.

Effect of iron on the virulence of Trichomonas vaginalis.

The role of iron was evaluated with respect to the virulence of Trichomonas vaginalis in mice. Iron-supplemented and iron-depleted Diamond's trypticase-yeast extract-maltose (TYM) media were prepared by adding 360 microM of ferrous sulfate and 100 microM of 2,2'-dipyridyl. Trophozoites cultivated from normal TYM and iron-supplemented TYM media produced subcutaneous abscesses; however, trichomonads grown in an iron-deficient TYM medium failed to produce any pathology. In addition to the increased virulence of trophozoites in mice, iron affects the level of adherence and the cytotoxicity of trichomonads to HeLa cells, which are significantly reduced in trophozoites grown in iron-deficient medium. In conclusion, it is suggested that under iron-depleted conditions such as that induced by 2,2'-dipyridyl the virulence of T. vaginalis is reduced.

Phylogenetic relationships between the six superoxide dismutase proteins (FeSOD) of Trichomonas vaginalis and FeSOD6 genetic diversity.

The parasitic protozoan Trichomonas vaginalis is known to contain several types of Fe-containing superoxide dismutase proteins (FeSOD). Using three different methods of phylogenetic analysis, maximum parsimony (MP), neighbor joining (NJ), and maximum likelihood (ML) methods, we examined the phylogenetic relationships among the six FeSOD (FeSOD1-FeSOD6) based on their amino acid sequences. All the analyses consistently suggested that the six proteins formed a monophyletic group implying that they probably be originated from an ancestral protein form through repeated duplication events. Although MP tree was totally unresolved, the NJ and ML trees revealed that FeSOD6 placed the most basal position and thus emerged earlier than the other five gene types during the evolution of T. vaginalis. Phylogenetic relationships among the five remaining proteins were (FeSOD2, FeSOD3), (FeSOD4, (FeSOD1, FeSOD5)) although weakly supported in terms of bootstrapping values. In addition to this, we newly designed two PCR primer specifically amplifying full-length FeSOD6 gene and examined its genetic diversity among 12 T. vaginalis isolates from five countries and three continents. They had the same nucleotide sequences except those of three Korean isolates which showed one to three different nucleotides.

Trichomonas vaginalis promotes apoptosis of human neutrophils by activating caspase-3 and reducing Mcl-1 expression.

Neutrophils are the predominant inflammatory cells found in the vaginal discharge of patients with Trichomonas vaginalis infection. However, it is not known whether neutrophil apoptosis is induced by live T. vaginalis. Therefore, we examined whether T. vaginalis can influence neutrophil apoptosis, and also whether caspase-3 and the Bcl-2 family members are involved in the apoptosis. Thus, human neutrophils were incubated with live T. vaginalis and neutrophil apoptosis was evaluated by Giemsa, annexin V-PI, and DiOC6 stainings. The neutrophil apoptosis was significantly higher in those incubated with T. vaginalis than in the control group. When trichomonads were pre-treated with mAb to AP65 (adhesin protein), or when trophozoites were separated from neutrophils using a Transwell chamber, neutrophil apoptosis was significantly reduced. The activation of caspase-3 was evident in neutrophils undergoing spontaneous apoptosis but was markedly enhanced during T. vaginalis-induced apoptosis. Moreover, the inhibition of caspase-3 effectively reduced T. vaginalis-induced apoptosis. Trichomonad-induced apoptosis was also associated with reduced expression of the neutrophil anti-apoptotic protein, Mcl-1. These results indicate that T. vaginalis alters Mcl-1 expression and caspase-3 activation, thereby inducing apoptosis of human neutrophils.

[Resistance to Naegleria fowleri infection passively acquired from immunized splenocyte, serum or milk].

A pathogenic free-living amoeba, Naegleria fowleri, causes primary amoebic meningoencephalitis to human and experimental animals. This infection is rare, but the mortality is very high. Nowadays, drug treatment or active immunization of human or mice are being tried with partial effectiveness. This study shows passive immunization effect by transfer of immunized spleen cells, serum, or milk from immunized mother in mouse experimental model. Young BALB/c mice were immunized intraperitoneally with 2-3 X 10(6) trophozoites of N. fowleri, and spleen cells and sera were collected for injection to recipient mice. There were seven transfer groups, i.e., immunized mouse serum, spleen cells, serum and spleen cells, normal mouse serum, spleen cells, serum and spleen cells, and control group. Three days later, BALB/c mice were inoculated with 1 x 10(4) trophozoites of N. fowleri intranasally. After infection, decreased mortality and prolonged survival time of mice were noted in immunized groups compared with non-immunized control group. The groups injected with immunized spleen cells or normal serum showed lower mortality than that of controls but showed no changes of serum IgG level. The groups injected with immunized serum or normal spleen cells showed increased serum IgG level after immunization but hundred percent mortality was observed. Mother mice were immunized intraperitoneally with 2-3 X 10(6) trophozoites of N. fowleri at the end of pregnancy and weaning period. Soon after the delivery, litters born of non-immunized mother were matched with immunized mother for feeding immune milk. After three weeks, the litters were infected with 1 X 10(4) trophozoites of N. fowleri or sacrificed for serum collection to measure the IgG levels.(ABSTRACT TRUNCATED AT 250 WORDS)

[Cytotoxicity of resident and lymphokine-activated mouse peritoneal macrophage against Trichomonas vaginalis].

This study was aimed to observe the direct and lymphokine-activated cell mediated cytotoxic effects against Trichomonas vaginalis by mouse peritoneal macrophages. Cytotoxicity was measured as release of 3H-thymidine from prelabeled protozoa, and tested in U-bottom microtiter plates. A 0.1 ml suspension of labeled protozoa (2 x 10(5)/ml) was placed in each well, followed by 0.1 ml of a suspension containing increasing numbers of peritoneal cells. After a 24 hr incubation at 37 degrees C, 0.1 ml of the supernatant was collected and counted in liquid scintillation counter. Mouse peritoneal macrophages had appreciable level of spontaneous cytotoxicity against T. vaginalis at the effector to target cell ratios from 5:1 to 50:1. Treatment of macrophages with lymphokine, produced by PHA-stimulated spleen cells, increased the cytotoxicity in comparison with resident macrophages against T. vaginalis. The degree of macrophage activation for the killing was not dependent upon the lymphokine concentration. Peritoneal cells adherent to plastic displayed significant levels of cytotoxicity against T. vaginalis. This study indicates that mouse peritoneal macrophages are spontaneously cytotoxic for T. vaginalis and lymphokine increases the cytotoxicity by activating macrophages to kill T. vaginalis.

Identification of surface antigen of Trichomonas vaginalis.

Plasma membrane proteins of a Korean isolate of Trichomonas vaginalis HY-1 were fractionated for antigen analysis. Homogenates of T. vaginalis were fractionated by the differential centrifugation using sucrose step-gradient method. The interface layer from the 25%/45% sucrose was collected as a plasma membrane fraction and its purity was examined by transmission electron microscopy. The antigenicity of plasma membrane fraction was analysed by enzyme-linked immunoelectrotransfer blot technique with immune rabbit serum and compared with surface antigen labelled with N-hydroxysuccinimide-biotin. The fluffy fraction of 25%/45% sucrose interface was homogeneous and membrane particles were present as extended sheet and concentric vesicles showing typical trilamellar appearance under transmission electron microscope. Seven fractions at 40, 50, 60, 110, 130, 140 and 150 kDa were identified as the antigenic membrane proteins in EITB with anti HY-1 rabbit serum. The common band at 60 kDa was detected both in antigenic fractions of plasma membrane and surface protein labelled with NHS-biotin. This result indicates that this protein is considered as a major surface antigen of T. vaginalis. The role of this surface antigen at 60 kDa should be studied further.

[Antigen analysis of Toxoplasma gondii lysate and excretory-secretory materials by enzyme-linked immunoelectrotransfer blot (EITB)].

Recently, the importance of toxoplasmosis is raised as a complication in immunosuppressed or AIDS patients. Our study focused on the identification of a variety of Toxoplasma antigens by immunoblotting. Rabbits and BALB/c mice were immunized with Toxoplasma lysate (RH strain), frozen tachyzoites (RH strain) or cysts (Beverly and Fukaya strain). Blood were collected from ear vein, heart or orbital plexus for detecting the serum antibody levels. For excretory-secretory (E.S) antigens, T. gondii (RH) tachyzoite were cultured in CHL (Chinese hamster lung) cells with MEM containing of 5% FCS. After 72 hrs, culture supernatant was collected. BALB/c mice were inoculated with RH tachyzoite intraperitoneally and peritoneal fluids were extracted three days later. E.S antigens were detected in culture supernatant and infected mouse peritoneal fluid by EITB. Serum IgG levels in rabbit were 1:512 of 10 days after primary immunization, 1:2,048 of 10 days after secondary immunization, 1:1,024 of 20 days after secondary immunization by IFAT, respectively. Serum IgG levels of immunized mice were 1:128 after 7 weeks. Tachyzoite antigens of the RH strain were detected 25 protein bands ranging 10 kDa-220 kDa of molecular weights with Coomassie blue stain. Toxoplasma major antigens corresponding to MW of 24 kDa, 27 kDa, 30 kDa, 35 kDa, 38 kDa were recognized by IgG and IgM antibodies. Excretory-secretory antigens present in culture supernatant with M. W. of 20, 30 kDa and in infected mouse peritoneal fluid with M.W. of 33 (P30), 45 kDa. When RH tachyzoite antigen was probed with different mice sera immunized with 2 strains of T. gondii, the IgG antibody band of Fukaya and Beverly strain (8 week-serum) is identical to those of RH strain. It is considered that the 30 kDa polypeptide detected in excretory-secretory materials and lysate was important major antigen of T. gondii (RH).

Partially purified Toxoplasma gondii antigens by immunoaffinity chromatography.

Tachyzoite antigens of Toxoplasma gondii (RH) were partially purified by immunoaffinity chromatography. The cultivated Toxoplasma in vivo (mouse) and in vitro (Hep-2 cell) and peritoneal fluid of T. gondii infected mice were collected for antigen analysis. Tachyzoite antigens collected from infected mouse showed positive bands of 76 kDa, 70 kDa, 64 kDa, 53 kDa, 46 kDa, 44 kDa, 41 kDa, 35 kDa, 25 kDa, 18 kDa, and 13 kDa on immunoblot with anti-Toxoplasma rabbit sera, and those from infected Hep-2 cells revealed reactive bands of 70 kDa, 64 kDa, 53 kDa, 35 kDa 28 kDa, and 13-10 kDa. After applying to an IgG-Sepharose column, two elution peaks, E-1 and E-2 fractions, were obtained from both soluble antigen of T. gondii and the peritoneal fluid of infected mice, respectively. Immunoblots of soluble antigen with immunized rabbit sera revealed positive bands of 97 kDa, 63 kDa, 53 kDa, and 35 kDa from E-1 fraction and 53 kDa and 35 kDa from E-2. In the case of the eluted peaks from mice peritoneal fluid, E-1 showed protein bands of 84 kDa, 76 kDa, 53 kDa, and 29 kDa bands and 53 kDa and 45 kDa from E-2 on immunoblots. Serum IgG antibody titer of mice immunized with T. gondii tachyzoites was increased on 1 week after booster immunization when analysed by ELISA using crude antigen, while it was elevated on 3 weeks after booster immunization by ELISA using purified antigen.

Toxoplasma gondii: ultrastructural localization of specific antigens and inhibition of intracellular multiplication by monoclonal antibodies.

This experiment was focused on the characterization of anti-Toxoplasma monoclonal antibodies (mAbs) and the effect of mAbs on the parasite invasion of mouse peritoneal macrophages. Twenty eight mAbs including M110, M556, R7A6 and M621 were characterized by Ab titer, immunoglobulin isotyping and western blot pattern. Antibody titer (optical density) of 4 mAbs, M110, M556, R7A6 and M621, were 0.53, 0.67, 0.45 and 0.39 (normal mouse serum; 0.19) with the same IgG1 isotypes shown by Enzyme-linked immunosorbent assay (ELISA). Western blot analysis showed that M110, M556, R7A6 and M621 reacted with the 33 kDa (p30), 31 kDa (p28), 43 kDa and 36 kDa protein. Immunogold labelling of mAbs M110, M556, R7A6 and M621 reacted with the surface membrane, dense granules and parasitophorous vacuolar membrane (PVM), rhoptries and cytoplasm of tachyzoite, respectively. For in vitro assay, preincubation of tachyzoites with four mAbs, M110, M556, R7A6 and M621 resulted in the decrease of the number of infected macrophages (p < 0.05) and the suppression of parasite multiplication at 18 h post-infection. Four monoclonal antibodies including M110 (SAG1) were found to have an important role in the inhibition of macrophage invasion and T. gondii multiplication in vitro, and these mAbs may be suitable for vaccine candidates, diagnostic kit and for chemotherapy.

Three dimensional shape reconstruction and finite element analysis of femur before and after the cementless type of total hip replacement.

Computerized tomography was used to reconstruct a shape, and stresses in three-dimensional objects were analysed. The human femur, which has a very irregular shape, was chosen as an object. CT image data of a cadaver femur were transferred to a computer, and an edge extraction program generated the cross-section of bone by specifying a range of CT values for each slice. Pixel data from the CT scan are converted into a vector of points (x, y, z) which can specify the boundaries of bone. Lateral surfaces are defined by stacking up the slices and making use of the vectorized data. Intermediate and oblique cross-sections can be obtained by an interpolation technique. The constructed model was used as input data for the finite element analysis. To understand the stress distributions before and after the cementless type of total hip replacement, a three-dimensional finite element stress analysis of the bone-implant system was carried out, assuming micromotions between the stem and the femur. The analysis was done for both frictionless and friction cases, modelling the contact point with a gap element having isotropic friction. The analysis shows that the stress is not concentrated on the femoral calcar when the friction coefficient is large.

Human syngamosis: the first case in Korea.

Parasites of the genus Mammomonogamus affect the respiratory tract of domestic mammals but have only rarely been reported in humans. In this case report the diagnosis of human syngamosis is described following bronchoscopic examination of a patient whose initial symptoms were simply of community acquired pneumonia. The patient had a persistent and productive cough with intermittent fever during 10 days of observation. After bronchoscopic extraction of the parasites and treatment with albendazole he recovered fully. This is one of the first recognised cases of human syngamosis in Korea.

Degradations of human immunoglobulins and hemoglobin by a 60 kDa cysteine proteinase of Trichomonas vaginalis.

The present study was undertaken to investigate the role of cysteine proteinase of Trichomonas vaginalis in escaping from host defense mechanism. A cysteine proteinase of T. vaginalis was purified by affinity chromatography and gel filtration. Optimum pH for the purified proteinase activity was 6.0. The proteinase was inhibited by cysteine and serine proteinase inhibitors such as E-64, NEM, IAA, leupeptin, TPCK and TLCK, and also by Hg2+, but not affected by serine-, metallo-, and aspartic proteinase inhibitors such as PMSF, EDTA and pepstatin A. However, it was activated by the cysteine proteinase activator, DTT. The molecular weight of a purified proteinase was 62 kDa on gel filtration and 60 kDa on SDS-PAGE. Interestingly, the purified proteinase was able to degrade serum IgA, secretory IgA, and serum IgG in time- and dose-dependent manners. In addition, the enzyme also degraded hemoglobin in a dose-dependent manner. These results suggest that the acidic cysteine proteinase of T. vaginalis may play a dual role for parasite survival in conferring escape from host humoral defense by degradation of immunoglobulins, and in supplying nutrients to parasites by degradation of hemoglobin.

A human case of tick bite by Ixodes nipponensis.

A human case of the tick bite on the back of 36-year-old man was found in September 1995. On admission he complained of itching sensation and pain at the site. The removed tick was identified morphologically as Ixodes nipponensis.