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1.
Waddlia chondrophila is an obligate intracellular bacterium of the Chlamydiales order. W. chondrophila has been isolated twice from aborted bovine foetuses and a serological study supported the abortigenic role of W. chondrophila in bovine species. Recently, we observed a strong association between the presence of anti-Waddlia antibodies and human miscarriage. To further investigate the pathogenic potential of W. chondrophila in humans, we studied the entry and the multiplication of this Chlamydia-like organism in human macrophages. Confocal and electron microscopy confirmed that W. chondrophila is able to enter human monocyte-derived macrophages. Moreover, W. chondrophila multiplied readily within macrophages. The proportion of infected macrophages increased from 13% at day 0 to 96% at day 4, and the mean number of bacteria per macrophage increased by 3logs in 24h. Intracellular growth of W. chondrophila was associated with a significant cytopathic effect. Thus, W. chondrophila may enter and grow rapidly within human macrophages, inducing lysis of infected cells. Since macrophages are one of the major components of the innate immune response, these findings indirectly suggest the possible human pathogenicity of W. chondrophila.  相似文献   

2.
Epitheliocystis is an infectious disease affecting gills and skin of various freshwater and marine fishes, associated with high mortality and reduced growth of survivors. Candidatus Piscichlamydia salmonis and Clavochlamydia salmonicola have recently been identified as aetiological agents of epitheliocystis in Atlantic Salmon. In addition, several other members of the Chlamydiales order have been identified in other fish species. To clarify the pathogenicity of Chlamydia-like organisms towards fishes, we investigated the permissivity of two fish cell lines, EPC-175 (Fathead Minnow) and RTG-2 (rainbow trout) to three Chlamydia-related bacteria: Waddlia chondrophila, Parachlamydia acanthamoebae and Estrella lausannensis. Quantitative PCR and immunofluorescence demonstrated that W. chondrophila and, to a lesser extent, E. lausannensis were able to replicate in the two cell lines tested. Waddlia chondrophila multiplied rapidly in its host cell and a strong cytopathic effect was observed. During E. lausannensis infection, we observed a limited replication of the bacteria not followed by host cell lysis. Very limited replication of P. acanthamoebae was observed in both cell lines tested. Given its high infectivity and cytopathic effect towards fish cell lines, W. chondrophila represents the most interesting Chlamydia-related bacteria to be used to develop an in vivo model of epitheliocystis disease in fishes.  相似文献   

3.
Bacterial adherence to mammalian cells and their internalization are thought to participate in Pseudomonas aeruginosa pathogenicity. In this study, we explored the role of alpha5beta1 and alphavbeta5 integrins and their natural ligands, fibronectin (Fn) and vitronectin (Vn), in P. aeruginosa interaction with epithelial cells by using the PAK reference bacterial strain, A549 respiratory, and SKOV-3 human ovarian cell lines. The host cell cytoskeleton and cellular tyrosine kinases seem to be solicited during the PAK-respiratory cell interaction: cytochalasin D and genistein decreased the bacterial adherence and internalization. Blocking antibodies to alphavbeta5 integrins were the only antibodies tested to have inhibitory activity against PAK adherence to A549 cells. PAK internalization by A549 and SKOV-3 cells was markedly decreased in the presence of blocking antibodies to Vn and alphavbeta5 integrins. Addition of Vn in excess restored PAK invasion of both A549 and SKOV-3 cells in the presence of anti-Vn antibodies. Immunofluorescence experiments revealed that, in the presence of bacteria, the Vn fibrillar network disappeared, and alphavbeta5 staining was concentrated in sites where adherent bacteria were present. Taken together, these findings suggest that alphavbeta5 integrins, and their natural ligand Vn, are involved in PAK entry into human epithelial cells.  相似文献   

4.
We have examined the effects of medroxyprogesterone acetate (MPA) and 4-hydroxytamoxifen (OH-TAM) on the cell proliferation and the expression of TGF- and TGF-β genes in Ishikawa cells and HEC-50 human endometrial adenocarcinoma cells. The effects of exogenous TGF-, TGF-β and anti-EGF receptor monoclonal antibody on cell proliferation were also determined. Antisense oligonucleotides were used to determine the effects of endogenous expression of TGF- and TGF-β. In both cell lines, MPA resulted in a time and dose-dependent inhibition of cell proliferation whereas OH-TAM had no effect on HEC-50 cell proliferation. The relative abundance of TGF- mRNA was significantly reduced by MPA in Ishikawa cells but not in HEC-50 cells. In Ishikawa cells, a reduction in TGF- mRNA abundance was observed with OH-TAM under conditions where both inhibition and stimulation of cell proliferation were demonstrated. Anti-EGF receptor monoclonal antibody inhibited Ishikawa cell growth but had little effect on HEC-50 cell proliferation. Exogenous TGF- stimulated proliferation of both cell lines whereas exogenous TGF-β inhibited proliferation of Ishikawa cells but stimulated proliferation of HEC-50 cells. Antisense oligonucleotides to TGF-β inhibited proliferation of HEC-50 cells. From these data we conclude that the antiproliferative effects of progestins and OH-TAM on endometrial cancer cells appear to be mediated by different mechanisms.  相似文献   

5.
Human embryo implantation is a critical multistep process consisting of embryo apposition/adhesion, followed by penetration and invasion. Through embryo penetration, the endometrial epithelial cell barrier is disrupted and remodeled by an unknown mechanism. We have previously developed an in vitro model for human embryo implantation employing the human choriocarcinoma cell line JAR and the human endometrial adenocarcinoma cell line Ishikawa. Using this model we have shown that stimulation with ovarian steroid hormones (17β-estradiol and progesterone, E2P4) and suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, enhances the attachment and adhesion of JAR spheroids to Ishikawa. In the present study we showed that the attachment and adhesion of JAR spheroids and treatment with E2P4 or SAHA individually induce the epithelial-mesenchymal transition (EMT) in Ishikawa cells. This was evident by up-regulation of N-cadherin and vimentin, a mesenchymal cell marker, and concomitant down-regulation of E-cadherin in Ishikawa cells. Stimulation with E2P4 or SAHA accelerated Ishikawa cell motility, increased JAR spheroid outgrowth, and enhanced the unique redistribution of N-cadherin, which was most prominent in proximity to the adhered spheroids. Moreover, an N-cadherin functional blocking antibody attenuated all events but not JAR spheroid adhesion. These results collectively provide evidence suggesting that E2P4- and implanting embryo-induced EMT of endometrial epithelial cells may play a pivotal role in the subsequent processes of human embryo implantation with functional control of N-cadherin.  相似文献   

6.
Extracellular lysophosphatidic acid (LPA) and the G-protein-coupled LPA receptors (LPAR) are involved in cell migration and invasion and found in the human endometrium. However, underlying mechanisms resulting in cellular invasion have been rarely investigated. We used stromal endometrial T-HESC, epithelial endometriotic 12Z, 49Z and Ishikawa cells. Interestingly, proliferation of T-HESC cells was strongly increased after LPA treatment, whereas the epithelial cell lines only showed a moderate increase. LPA increased invasion of 12Z and 49Z strongly and significantly. The LPAR inhibitor Ki16425 (LPAR1/3) attenuated significantly LPA-induced invasiveness of 12Z, which was confirmed by LPAR1 and LPAR3 siRNAs, showing that both LPA receptors contribute to invasiveness of 12Z cells. Investigation of cell invasion with an antibody-based protease array revealed mainly differences in cathepsins and especially cathepsin B between 12Z compared to the less invasive Ishikawa. Stimulation with LPA showed a time- and dose-dependent increased secretion of cathepsin B which was inhibited by the Gq inhibitor YM-254890 and Gi/o inhibitor pertussis toxin in the 12Z cells, again highlighting the importance of LPAR1/3. The activity of intracellular and secreted cathepsin B was significantly upregulated in LPA-treated samples. Inhibition of cathepsin B with the specific inhibitor CA074 significantly reduced LPA-increased invasion of 12Z. Our results reveal a novel role of LPA-mediated secretion of cathepsin B which stimulated invasion of endometriotic epithelial cells mainly via LPAR1 and LPAR3. These findings may deepen our understanding how endometriotic cells invade into ectopic sites, and provide new insights into the role of LPA and cathepsin B in cellular invasion.  相似文献   

7.
The purpose of this study was to identify an endometrial cell line that maintained the E2 up-regulation of estrogen receptor (ER) mRNA by enhanced message stability and to assess its dependence on ER protein. Estradiol (E2) effects on gene expression were measured in three cell lines: one immortalized from sheep endometrial stroma (ST) and two from human endometrial adenocarcinomas (Ishikawa and ECC-1). E2 up-regulated ER mRNA levels in ST and Ishikawa cells, but down-regulated ER mRNA levels in ECC-1 cells. E2 up-regulated progesterone receptor (PR), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and transforming growth factor-alpha (TGF-alpha) in both Ishikawa and ECC-1 cells. The selective estrogen receptor modulator ICI 182,780 antagonized the E2-induced up-regulation of ER and/or PR mRNA levels in all three cells, while another, GW 5638, antagonized the up-regulation of PR mRNA in Ishikawa and ECC-1 cells. In mechanistic studies, E2 had no effect on ER mRNA stability in ST cells and it destabilized ER mRNA in ECC-1 cells. Thus, Ishikawa cells appear to be the most physiologically relevant cell line in which to study the up-regulation of ER mRNA levels by enhanced mRNA stability. Its antagonism by ICI 182,780 reveals that ER protein is involved in this E2 response.  相似文献   

8.
The effects of the transforming growth factor-beta 1 (TGF-beta 1) and epidermal growth factor (EGF) on the growth of cells from 2 endometrial cancer lines, Ishikawa and HEC-50 were evaluated by measuring rates of DNA synthesis and changes in cell numbers during culture. EGF at 17 and 1.7 nM concentrations consistently enhanced HEC-50 cell proliferation. TGF-beta 1 inhibited Ishikawa cell proliferation but, unexpectedly for epithelium-derived cells, stimulated HEC-50 cell growth. This effect is of interest as it indicates that endometrial cells can acquire an altered responsiveness to a growth inhibitor during the process of malignant transformation. Northern blot analyses showed expression of TGF-alpha, TGF-beta 1 and EGF receptors mRNA in both cell lines. Neither estradiol (E2) nor 4-hydroxytamoxifen (OHTam) affected mRNA levels for either TGF-alpha or TGF-beta in HEC-50 cells, a line unresponsive to E2 for proliferation. In Ishikawa cells, previously shown to respond to both E2 and OHTam by increasing proliferation rates, E2 increased TGF-alpha mRNA and reduced TGF-beta mRNA levels. OHTam lowered the levels of both mRNA species, although the effect was greater on TGF-beta than TGF-alpha mRNA. These data are consistent with, but do not prove, the existence of a possible autocrine regulation by TGF-alpha and TGF-beta of human cancer cell proliferation, which might be under E2 influence in Ishikawa cells.  相似文献   

9.
The etiology of endometrial cancers remains poorly understood, particularly with respect to signal transduction pathways underlying the development and progression of the more aggressive, type II steroid‐independent tumors. Protein kinase C alpha (PKCα) regulates cellular processes critical to malignancy and has been implicated in the pathogenesis of endometrial cancers. The objective of these studies was to determine the functional role of PKCα in endometrial cancer cell proliferation, anchorage‐independent growth, and invasion. PKCα expression in endometrial cancer cell lines was examined by Western blotting. PKCα levels were increased in type II HEC‐50, HEC‐1‐A and HEC‐1‐B cell lines relative to the type I Ishikawa and RL‐95‐2 lines. Retroviral constructs were used to either overexpress PKCα or selectively knockdown levels by shRNA in Ishikawa and HEC 50 cells, respectively. Knockdown of PKCα expression in HEC‐50 cells resulted in a diminished growth rate and attenuation of anchorage‐independent growth. Correspondingly, Ishikawa cells overexpressing PKCα protein exhibited increased proliferation, resistance to growth factor deprivation and enhanced anchorage‐independent growth. Consistent with the observed changes in cell proliferation, PKCα also modulated cyclin D1 promoter activity in both cell lines. A reduction in PKCα levels rendered HEC‐50 cells significantly less invasive, whereas PKCα overexpression enhanced invasion of Ishikawa cells. These data indicate that PKCα promotes growth and invasion of endometrial cancer cells, suggesting that PKCα dependent signaling pathways could provide novel prognostic indicators or therapeutic targets, particularly in clinically aggressive type II endometrial tumors. J. Cell. Physiol. 220: 112–118, 2009. © 2009 Wiley‐Liss, Inc.  相似文献   

10.
InlB is one of the two Listeria monocytogenes invasion proteins required for bacterial entry into mammalian cells. Entry into human epithelial cells such as Caco-2 requires InlA, whereas InlB is needed for entry into cultured hepatocytes and some epithelial or fibroblast cell lines such as Vero, HEp-2 and HeLa cells. InlB-mediated entry requires tyrosine phosphorylation, cytoskeletal rearrangements and activation of the host protein phosphoinositide (PI) 3-kinase, probably in response to engagement of a receptor. In this study, we demonstrate for the first time that InlB is sufficient to promote internalization. Indeed, coating of normally non-invasive bacteria or inert latex beads with InlB leads to internalization into mammalian cells. In addition, a soluble form of InlB also appears to promote uptake of non-invasive bacteria, albeit at a very low level. Similar to entry of L. monocytogenes , uptake of InlB-coated beads required tyrosine phosphorylation in the host cell, PI 3-kinase activity and cytoskeletal reorganization. Taken together, these data indicate that InlB is sufficient for entry of L. monocytogenes into host cells and suggest that this protein is an effector of host cell signalling pathways.  相似文献   

11.
Few data exist on the interaction of Campylobacter upsaliensis with host cells, and the potential for this emerging enteropathogen to invade epithelial cells has not been explored. We have characterized the ability of C. upsaliensis to invade both cultured epithelial cell lines and primary human small intestinal cells. Epithelial cell lines of intestinal origin appeared to be more susceptible to invasion than non-intestinal-derived cells. Of three bacterial isolates studied, a human clinical isolate, CU1887, entered cells most efficiently. Although there was a trend towards more efficient invasion of Caco-2 cells by C. upsaliensis CU1887 at lower initial inocula, actual numbers of intracellular organisms increased with increasing multiplicity of infection and with prolonged incubation period. Confocal microscopy revealed C. upsaliensis within primary human small intestinal cells. Both Caco-2 and primary cells in non-confluent areas of the infected monolayers were substantially more susceptible to infection than confluent cells. The specific cytoskeletal inhibitors cytochalasin B, cytochalasin D and vinblastine attenuated invasion of Caco-2 cells in a concentration-dependent manner, providing evidence for both microtubule- and microfilament-dependent uptake of C. upsaliensis. Electron microscopy revealed the presence of organisms within Caco-2 cell cytoplasmic vacuoles. C. upsaliensis is capable of invading epithelial cells and appears to interact with host cell cytoskeletal structures in order to gain entry to the intracellular environment. Entry into cultured primary intestinal cells ex vivo provides strong support for the role of host cell invasion during human enteric C. upsaliensis infection.  相似文献   

12.
Infection by Cryptococcus neoformans begins with inhalation of infectious propagules. Fungi reach the lung tissue and interact with epithelial cells in a crucial but poorly understood process. In this study, the interaction of C. neoformans with the human alveolar epithelial cell lineage A549 was investigated, focusing on the relevance of the capsular polysaccharide in this process. The association of encapsulated strains with A549 cells was significantly inhibited by a monoclonal antibody to glucuronoxylomannan (GXM), a major component of the cryptococcal capsule. A purified preparation of GXM produced similar results, suggesting the occurrence of surface receptors for this polysaccharide on the surface of alveolar cells. A549 cells were in fact able to bind soluble GXM, as confirmed by indirect immunofluorescence analysis using the anti-polysaccharide antibody. C. neoformans is internalized after GXM-mediated interaction with A549 cells in a process that culminates with death of host cells. Our results suggest that C. neoformans can use GXM for attachment to alveolar epithelia, allowing the fungus to reach the intracellular environment and damage host cells through still uncharacterized mechanisms.  相似文献   

13.
Polarized epithelial cells represent the primary barrier to virus infection of the host, which must also be traversed prior to virus dissemination from the infected organism. Although there is considerable information available concerning the release of enveloped viruses from such cells, relatively little is known about the processes involved in the dissemination of nonenveloped viruses. We have used two polarized epithelial cell lines, Vero C1008 (African green monkey kidney epithelial cells) and Caco-2 (human intestinal epithelial cells), infected with poliovirus and investigated the process of virus release. Release of poliovirus was observed to occur almost exclusively from the apical cell surface in Caco-2 cells, whereas infected Vero C1008 cells exhibited nondirectional release. Structures consistent with the vectorial transport of virus contained within vesicles or viral aggregates were observed by electron microscopy. Treatment with monensin or ammonium chloride partially inhibited virus release from Caco-2 cells. No significant cell lysis was observed at the times postinfection when extracellular virus was initially detected, and transepithelial resistance and vital dye uptake measurements showed only a moderate decrease. Brefeldin A was found to significantly and specifically inhibit poliovirus biosynthetic processes by an as yet uncharacterized mechanism. The vectorial release of poliovirus from the apical (or luminal) surface of human intestinal epithelial cells has significant implications for viral pathogenesis in the human gut.  相似文献   

14.
We have investigated the interrelationship between two anti-apoptotic factors, XIAP and Akt, and their role in chemoresistance of uterine cancer cells. We used one cervical cancer cell line (HeLa) and two endometrial cancer cell lines (KLE and Ishikawa) as a model. The three drugs decreased Akt and XIAP content and induced apoptosis in P-Akt-negative HeLa cells. In P-Akt1/3-positive Ishikawa cells apoptosis induction correlated with XIAP decrease. P-Akt1/2/3-positive KLE cells showed maximum chemoresistance as XIAP and Akt levels/phosphorylation remained stable in response to the three drugs, and only cisplatin could significantly induce apoptosis. We found that XIAP and Akt were functionally linked in uterine cancer cells, as downregulation of XIAP with RNAi decreased P-Akt levels, and inhibition of PI3-K/Akt activity using LY294002 decreased XIAP content. Overexpression of constitutively active Akt isoforms in HeLa cells induced isoform-specific sensitivity to doxorubicin and taxol but not cisplatin. XIAP RNAi increased the cell-specific sensitivity to cisplatin and doxorubicin but not taxol. Finally, we found P-Akt immunoreactivity in epithelial cells from multiple human endometrial carcinoma tumors, suggesting that Akt may also regulate chemosensitivity in uterine cancers in vivo. Altogether these results highlight an intertwined role for specific Akt isoforms and XIAP in chemoresistance of uterine cancer cells.  相似文献   

15.
Endometrial cancer cell lines have provided a valuable model to study endometrial epithelial cells in vitro. Since the first development of HEC1B over 35 yr ago, many different cell lines have been isolated and described. One valuable cell line that maintains hormone responsiveness and unique stability over time is the ECC-1 cell line, developed originally by the late P.G. Satyaswaroop. In this study, we investigated some of the properties of these cells and present their salient characteristics. Like Ishikawa cells, ECC-1 cells maintain both estrogen receptors (ESR1 [ER alpha] and ESR2 [ER beta]), progesterone receptors (PR A and B; PGRs), and androgen receptors (ARs), along with the p160 steroid receptor coactivators NCOA1 (formerly SRC1), NCOA2 (formerly TIF2), and NCOA3 (formerly AIB1). The karyotype of these cells is abnormal, with multiple structural rearrangements in all cells analyzed. Unlike Ishikawa cells that express glandular epithelial antigens, ECC-1 cells maintain a luminal phenotype, with expression of KRT13 (cytokeratin 13) and KRT18 (cytokeratin 18). Apparent differences in the regulation of ESR2 also were evident in ECC-1 cells compared to Ishikawa cells. Like other endometrial cell lines, ECC-1 cells express the steroid receptor coactivators and exhibit epidermal growth factor-stimulated expression of known luminal proteins thought to be involved in implantation, including the hyaluronate receptor CD44 and SPP1 (formerly osteopontin) and CD55 (decay-accelerating factor). These characteristics appear to be stable and persistent over multiple cell passages, making this well-differentiated cell line an excellent choice to study endocrine and paracrine regulation of endometrial epithelium in vitro.  相似文献   

16.
Legionella oakridgensis occasionally causes pneumonia in humans. We report here the characteristic morphology of intracellular microcolonies of L. oakridgensis OR-10 in infected epithelial cells. By light microscopy after Gimenez staining, the bacteria showed serpentine-like chain, disk-like conglomerate, and granular forms when they grew intracellularly in Vero cells, HeLa cells, and A549 cells. In a time-lapse study, we observed the progressive change from a serpentine-like chain form to a conglomerate form in Vero cells. Transmission electron microscopy showed that L. oakridgensis OR-10 proliferated both inside membrane structures and in the cytoplasm. Such highly serpentine chain growth has not been reported in any intracellular bacteria. Furthermore, these results imply that L. oakridgensis OR-10 may be proliferating inside the endoplasmic reticulum.  相似文献   

17.
Tremella mesenterica (TM) is a common food and folk medicine widely used in several Asian countries as a tonic for the lungs. In the present study, we compared the effects of extracellular polysaccharides (EPS), intracellular polysaccharides (IPS), and ethanol extract (EE) of Tremella mesenterica on the induction of apoptosis into human lung carcinoma A549 epithelial cells. The EE, but not the EPS or the IPS, almost completely inhibited the growth of A549 cells. The results of Annexin V-FITC/PI staining and flow cytometric analysis indicated that the percentage of Annexin V(+)/PI(-) cells in EE-treated cells increased to 32.8%. The results of further investigation showed a disruption of mitochondrial transmembrane potential (DeltaPsi(m)), the production of reactive oxygen species (ROS), and the activation of caspase-3 protein in EE-treated cells. These findings suggest that EE can decrease cell viability and induce apoptosis in A549 cell lines by activating a mitochondrial pathway.  相似文献   

18.
The oestrogen receptor (ER) α-β+ HEC-1B and the ERα+β+ Ishikawa (IK) cell lines were investigated to dissect the effects of oestrogen exposure on several parameters of Chlamydia trachomatis infection. Antibody blockage of ERα or ERβ alone or simultaneously significantly decreased C. trachomatis infectivity (45-68%). Addition of the ERβ antagonist, tamoxifen, to IK or HEC-1B prior to or after chlamydial infection caused a 30-90% decrease in infectivity, the latter due to disrupted eukaryotic organelles. In vivo, endometrial glandular epithelial cells are stimulated by hormonally influenced stromal signals. Accordingly, chlamydial infectivity was significantly increased by 27% and 21% in IK and HEC-1B cells co-cultured with SHT-290 stromal cells exposed to oestrogen. Endometrial stromal cell/epithelial cell co-culture revealed indirect effects of oestrogen on phosphorylation of extracellular signal-regulated kinase and calcium-dependant phospholipase A2 and significantly increased production of interleukin (IL)-8 and IL-6 in both uninfected and chlamydiae-infected epithelial cells. These results indicate that oestrogen and its receptors play multiple roles in chlamydial infection: (i) membrane oestrogen receptors (mERs) aid in chlamydial entry into host cells, and (ii) mER signalling may contribute to inclusion development during infection. Additionally, enhancement of chlamydial infection is affected by hormonally influenced stromal signals in conjunction with direct oestrogen stimulation of the human epithelia.  相似文献   

19.
Plasminogen activator activity was demonstrated in two carcinoma cell lines: A549 cells derived from a human alveolar epithelial carcinoma; and ZHC cells derived from a rat hepatoma. Both cells had intracellular plasminogen activator activity throughout their cell cycles and in each case this activity reached a maximum. For A549 cells the maximal activity took place either during the G2 phase or in the course of the S to G2 transition, suggesting that plasminogen activator might play a role in cell division. For ZHC cells, the maximal activity occurred at the start of the S phase, suggesting that in these cells plasminogen activator might be involved in DNA replication.  相似文献   

20.
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