Analysis of extracellular vesicles as a potential index for monitoring differentiation of neural lineage cells from induced pluripotent stem cells.

To improve cell production efficacy, it is important to evaluate cell conditions during culture. Extracellular vesicles (EVs) secreted from various cells are involved in stem cell differentiation. As EVs carry information about their source cells, we hypothesized that they may serve as a noninvasive index of cell conditions. We evaluated changes in EV morphology, concentration, and microRNA (miRNA) and protein expression in culture supernatants during the differentiation of induced pluripotent stem cells (iPSCs) into neural lineage cells, for application in regenerative medicine for Parkinson's disease. We observed EVs (50-150 nm) in culture supernatants of iPSCs and differentiated cells. The EVs expressed the exosome markers CD63, CD81, and CD9. Throughout differentiation, the EV concentration in the supernatants decreased, and EV miRNA and protein expression changed substantially. Especially, miR-106b, involved in neural stem cell differentiation and normal brain development, was considerably downregulated. CD63 expression correlated with the CORIN-positive cell rate, which is an index of differentiation. Thus, EV concentration and miRNA and protein expression may reflect the differentiation status of iPSCs. These findings pave the way for the development of novel and sensitive cell culture monitoring methods.

Regulation of Matrix Metalloproteinases-2 and -9 Gene Expression in Cultured Human Fetal Membrane Cells by Influenza Virus Infection.

In order to understand a possible etiology of adverse pregnancy outcomes associated with intrauterine influenza virus infection, we examined the effect of influenza virus infection on gene expression of matrix metalloproteinases (MMPs) in cultured amnion epithelial, amnion mesenchymal and chorion trophoblast cells prepared from human fetal membrane tissues by gelatin zymography, Western blotting and reverse transcriptase-PCR. The cells were infected with influenza A (H1N1) virus. The levels of pro-MMP-9 activity in culture supernatants of three types of cells were increased during the period of 24-48 h after the virus infection as compared to those of mock infection. Chorion trophoblast cells spontaneously released a much greater level of pro-MMP-2 activity than amnion epithelial and amnion mesenchymal cells. The cleavage of pro-MMP-2 into an active intermediate form was enhanced in chorion trophoblast cells by the virus infection. The activity levels of MMP-2 and MMP-9 in culture supernatants were consistent with their protein levels. The virus infection induced the mRNA expression of MMP-9, but not MMP-2, in three types of cells. These results suggest that influenza virus infection induces the gene expression of MMP-9 and the cleavage of pro-MMP-2 into an active intermediate form in human fetal membrane cells, resulting in weakening of the membranes through extracellular matrix degradation. Therefore, it is possible that the regulation of MMPs gene expression in fetal membrane cells by influenza virus infection is implicated in a part of the etiology of adverse pregnancy outcomes associated with intrauterine infection with the virus.

Duplex ultrasound for the prediction of vascular events associated with arteriovenous fistulas in hemodialysis patients.

OBJECTIVE: To determine if duplex ultrasound (US) for arteriovenous fistulas (AVFs) can predict vascular events (VEs; thrombosis and stenosis). METHODS: Duplex US was performed for vascular access evaluation in 2557 maintenance hemodialysis (HD) patients between October 1, 2013 and March 31, 2016. Of these patients, 2184 patients were finally included in this study. AVF dysfunction was assessed using the brachial artery blood flow volume (Qa; mL/min), arterial blood flow resistance index (RI), and residual diameter of the fistula vein (RD; mm). Proximal, midpoint, and distal aspects of the fistulas were measured. The baseline measurements were the US assessments, and the endpoint was VEs requiring vascular access intervention therapy or vascular surgery. Associations of US findings and VEs were assessed with receiver operating characteristic curve analysis, log-rank analysis, and multivariate Cox hazard models. RESULTS: The mean Qa was 772.8 ± 441.4 mL/min; RI, 0.56 ± 0.1; and RD, 2.37 ± 1.0 mm. The optimal Qa cut-off point was calculated as 581.5 mL/min, RI cut-off as 0.56, and RD cut-off as 1.85 mm. VEs were more frequent in patients with a Qa <581.5 mL/min than in those with a Qa >581.5 mL/min (p<0.001). In multivariate analysis, Qa, ferritin, transferrin saturation, and warfarin use were significantly associated with VEs. CONCLUSIONS: US evaluation of AVFs in HD patients is a simple method to predict the risks of thrombosis and fistula dysfunction. Qa, ferritin, transferrin saturation, and warfarin use might be associated with VEs.

Glomerulopathy with distinctive fibrillar deposits but lacking glomerular deposition of type III collagen.

A 62-year-old woman with nephrotic syndrome underwent a renal biopsy. Under light microscopy, the biopsy findings included lobulation and enlargement of glomeruli, occasional thickening of glomerular capillary walls, and narrowing of the capillary lumen by swollen endothelial cells. Congo red staining was negative for amyloid. No significant intraglomerular fibrin deposition was found by phosphotungstic acid hematoxylin staining. Immunofluorescence microscopy showed no deposition of immunoglobulin G, A, or M; no κ or λ light chains; and no C3 or C1q. Electron microscopy revealed distinctive subendothelial and mesangial fibrillar deposits, mesangial cell interposition, and swelling and vacuolization of endothelial cells resulting in capillary lumen narrowing. Although some curvilinear fibrillar deposits mimicked the bundles of type III collagen fibers seen in collagenofibrotic glomerulopathy, neither glomerular deposition of type III collagen nor elevation of serum procollagen III peptide was noted. This glomerulopathy does not fulfill any known disease entities with non-amyloid non-immunoglobulin-derived organized glomerular deposits.

Involvement of histone H3 phosphorylation via the activation of p38 MAPK pathway and intracellular redox status in cytotoxicity of HL-60 cells induced by Vitex agnus-castus fruit extract.

We have demonstrated that an extract from the ripe fruit of Vitex angus-castus (Vitex), might be a promising anticancer candidate. In order to further provide a molecular rationale for clinical development in anticancer therapy, a detailed mechanism underlying the efficacy of Vitex against HL-60 cells was investigated. Vitex induced a dose- and time-dependent decrease in cell viability associated with induction of apoptosis and G(2)/M cell cycle arrest, both of which were suppressed by the addition of SB203580, an inhibitor for p38 MAPK. Furthermore, SB203580 significantly suppressed Vitex-induced phosphorylation of histone H3, a downstream molecule of p38 MAPK known to be involved in apoptosis induction in tumor cells. Notably, Vitex induced upregulation of intracellular ATP, known to bind its binding pocket inside activated p38 MAPK and to be required for the activation of p38 MAPK pathway. These results, thus, suggest that upregulation of intracellular ATP and phosphorylation of histone H3 are closely associated with the activation of p38 MAPK pathway, consequently contributing to Vitex-mediated cytotoxicity. Intriguingly, a significant decrease of intracellular ROS levels and downregulation of expression level of gp91(phox), an important component of NADPH oxidase, were observed in Vitex-treated cells. A greater decline in ROS levels along with enhanced apoptosis was observed after treatment with Vitex in combination with SnPP, an inhibitor specific for HO-1. Since NADPH oxidase and HO-1 are closely correlated to redox status associated with intracellular ROS levels, the two enzymes are suggested to be implicated in Vitex-mediated cytotoxicity in HL-60 cells by regulating ROS generation. We also suggest that activation of the p38 MAPK pathway may be dependent on the alterations of intracellular ATP levels, rather than that of intracellular ROS levels. These results may have important implications for appropriate clinical uses of Vitex and provide novel insights into the interaction between Vitex and other conventional drugs capable of affecting intracellular redox status.

Differences in permissive cytomegalovirus infection between primary cultured human fetal membrane chorion and amnion cells.

Human cytomegalovirus (CMV) is the most common cause of viral intrauterine infection in developed countries. It has been shown that CMV DNA was frequently detected in the fetal membranes when the placenta was infected in utero. However, it is still not clear whether CMV replicates in constituent cells of the fetal membranes. We investigated CMV infection of primary cultured chorion and amnion cells prepared from human fetal membrane tissues. In both types of cell cultures, rounded cells were observed at day 8 and 12 after CMV inoculation, and virus yields in culture supernatants were increased after the inoculation. In both types of cells, viral immediately early 1 (IE1) protein-positive nuclei were scattered at day 4 after the inoculation, and IE1 mRNA was expressed throughout day 1 to 12 after CMV inoculation. In chorion cell cultures, the number of IE1 protein-positive nuclei increased significantly at day 8 and 12 after CMV inoculation as compared to day 4, by which foci were formed. Furthermore, an evident increase in levels of lactate dehydrogenase leakage from chorion cells was observed after CMV inoculation. Contrary, these phenomena were not observed in amnion cell cultures. These results demonstrated that both chorion and amnion cells were permissive to CMV infection, while the velocity of cell-to-cell spread of CMV infection in amnion cells was much lower than that in chorion cells. Therefore, the present study suggests that CMV may replicate rapidly in the chorion cell layer and slowly in the amnion cell layer during intrauterine infection.

Cytotoxicity of Vitex agnus-castus fruit extract and its major component, casticin, correlates with differentiation status in leukemia cell lines.

We have demonstrated that an extract from the ripe fruit of Vitex agnus-castus (Vitex) exhibits cytotoxic activities against various types of solid tumor cells, whereas its effects on leukemia cells has not been evaluated to date. In this study, the effects of Vitex and its major component, casticin, on leukemia cell lines, HL-60 and U-937, were investigated by focusing on proliferation, induction of apoptosis and differentiation. Identification and quantitation by NMR spectroscopy showed that casticin accounted for approximate 1% weight of Vitex. Dose-dependent cytotoxicity of Vitex and casticin was observed in both cell lines, and HL-60 cells were more sensitive to the cytotoxicity of Vitex/casticin compared to U-937 cells. Furthermore, compared to unstimulated HL-60 cells, phorbol 12-myristate 13-acetate (PMA)- and 1,25-dihydroxyvitamin D3 (VD3)-differentiated HL-60 cells acquired resistance to Vitex/casticin based on the results from cell viability and apoptosis induction analysis. Since the HL-60 cell line is more immature than the U-937 cell line, these results suggested that the levels of cytotoxicity of Vitex/casticin were largely attributed to the degree of differentiation of leukemia cells; that is, cell lines with less differentiated phenotype were more susceptible than the differentiated ones. RT-PCR analysis demonstrated that PMA upregulated the expression of intercellular adhesion molecule-1 (ICAM-1) in HL-60 cells, and that anti-ICAM-1 monoclonal antibody not only abrogated PMA-induced aggregation and adhesion of the cells but also restored its sensitivity to Vitex. These results suggested that ICAM-1 plays a crucial role in the acquired resistance in PMA-differentiated HL-60 cells by contributing to cell adhesion. These findings provide fundamental insights into the clinical application of Vitex/casticin for hematopoietic malignancy.

Possible roles of proinflammatory and chemoattractive cytokines produced by human fetal membrane cells in the pathology of adverse pregnancy outcomes associated with influenza virus infection.

Pregnant women are at an increased risk of influenza-associated adverse outcomes, such as premature delivery, based on data from the latest pandemic with a novel influenza A (H1N1) virus in 2009-2010. It has been suggested that the transplacental transmission of influenza viruses is rarely detected in humans. A series of our study has demonstrated that influenza virus infection induced apoptosis in primary cultured human fetal membrane chorion cells, from which a factor with monocyte differentiation-inducing (MDI) activity was secreted. Proinflammatory cytokines, such as interleukin (IL)-6, tumor necrosis factor (TNF)-α, and interferon (IFN)-ß, were identified as a member of the MDI factor. Influenza virus infection induced the mRNA expression of not only the proinflammatory cytokines but also chemoattractive cytokines, such as monocyte chemoattractant protein (MCP)-1, regulated on activation, normal T-cell expressed and secreted (RANTES), macrophage inflammatory protein (MIP)-1ß, IL-8, growth-regulated oncogene (GRO)-α, GRO-ß, epithelial cell-derived neutrophil-activating protein (ENA)-78, and interferon inducible protein (IP)-10 in cultured chorion cells. These cytokines are postulated to associate with human parturition. This paper, therefore, reviews (1) lessons from pandemic H1N1 2009 in pregnancy, (2) production of proinflammatory and chemoattractive cytokines by human fetal membranes and their functions in gestational tissues, and (3) possible roles of cytokines produced by human fetal membranes in the pathology of adverse pregnancy outcomes associated with influenza virus infection.

A case of anemia with schistocytosis, thrombocytopenia, and acute renal failure caused by adenomyosis.

A 51-year-old woman with adenomyosis was admitted because of anemia with schistocytosis, thrombocytopenia, and acute renal failure (ARF). Thrombotic microangiopathy (TMA) was considered. Plasma exchange and steroid therapies improved laboratory results. However, renal biopsy specimen revealed acute tubular necrosis (ATN), but not TMA, and thrombocytopenia, diagnosed it as disseminated intravascular coagulation (DIC) but not TMA. Few cases of DIC associated with benign tumors of the uterus and, especially, adenomyosis have been reported. In adenomyosis patients, ARF is usually caused by obstructive uropathy. However, the rare case suggests that hemolytic anemia, DIC, and ARF due to ATN can occur in adenomyosis patients.

A case of hypokalemia-induced fatal arrhythmia caused by indapamide in an anorexic elderly patient.

An 84-year-old man was referred to our hospital for atrioventricular block and severe hypokalemia. He had been treated for hypertension since 2007 with indapamide, a thiazide-like diuretic. His laboratory data had not been tested for a long time. One week before his first visit, he suffered from a common cold and anorexia. He was admitted to our hospital because his electrocardiogram showed ventricular flutter, and pulmonary arrest occurred at the time of his visit. Cardiopulmonary resuscitation was successfully performed. Hypokalemia (K, 1.7 mEq/L) was considered as the cause of acute cardiopulmonary failure. His oral intake of potassium decreased, but potassium loss from the kidney persisted (urinary potassium, 14.0 mEq/L; transtubular potassium gradient, 5.00). These results suggested that although hypokalemia was suspected to have been present for a long time due to indapamide, severe hypokalemia was induced during the period of anorexia. After discontinuation of indapamide and intravenous administration of potassium L: -aspartate for potassium supplementation, the patient's serum potassium levels increased and his general condition improved. Although it is well known that hypokalemia is caused by indapamide, the incidence is not frequent and if observed is not severe. However, we experienced an unusual case of hypokalemia-induced fatal arrhythmia caused by indapamide. Hence, the serum potassium concentration of patients under the drug, especially anorexic elderly patients, should be monitored.

A case presenting with rapid renal damage caused by immunoglobulin D lambda-type multiple myeloma accompanied by granular lymphocyte proliferative disorder.

A 69-year-old man was referred to our hospital for severe anemia. The atypical lymphocyte count, including granular lymphocytes, was 2,750/µL. Lymphocyte surface marker analysis showed CD3+, CD5+, CD16+, and CD56+ cells. Mixed T cell- and natural killer cell-type granular lymphocyte proliferative disorder (GLPD) was diagnosed. Because his serum creatinine levels deteriorated rapidly over the next 3 months, from 0.96 to 3.27 mg/dL, he was admitted to our hospital. The serum levels of immunoglobulins (Ig) other than IgD had decreased, and monoclonal protein was detected in the gamma-globulin region. Immunoelectrophoresis revealed IgD and lambda (λ) proteins in the serum and λ-type Bence-Jones protein in the urine. Renal biopsy examination revealed widespread tubular atrophy and interstitial fibrosis, and cast formation with λ protein deposits in tubular lumens, indicating cast nephropathy. These results indicated that the rapid renal damage was caused by IgD λ-type multiple myeloma accompanied by GLPD. The clinical course of GLPD is not usually aggressive and the findings of physical examinations are not significant. GLPD is usually associated with cytopenia (neutropenia or anemia), viral infections, collagen diseases, neoplasms such as malignant lymphoma, or chronic infections. To date, there are only 2 case reports of GLPD accompanied by multiple myeloma but without renal function or renal histological findings. When the clinical course of GLPD is aggressive and is accompanied with rapid renal damage, multiple myeloma should be considered as a complication.

Lactate dehydrogenase leakage as a marker for apoptotic cell degradation induced by influenza virus infection in human fetal membrane cells.

OBJECTIVE: In order to elucidate the implication of apoptosis in lactate dehydrogenase (LDH) leakage from influenza virus-infected cells, the effects of a general caspase inhibitor, N-t-Boc-Asp(OMe)-fluoromethylketone (Boc-D-fmk), on LDH leakage, apoptosis induction and virus proliferation were examined. METHODS: Cultured human fetal membrane chorion and amnion cells were incubated with or without Boc-D-fmk after influenza virus infection. LDH leakage was estimated by measuring LDH activities in the culture supernatants and cell lysates. The extent of apoptosis was determined by caspase-3 protein cleavage and DNA fragmentation. Virus proliferation was determined by a plaque-forming assay. RESULTS: While virus proliferation was observed in both chorion and amnion cells, the virus infection resulted in LDH leakage, caspase-3 protein cleavage, and oligonucleosomal DNA fragmentation, all of which were observed only in the chorion cells and inhibited by the presence of Boc-D-fmk except for the virus proliferation. CONCLUSION: LDH level in amniotic fluid is known to be one of markers for predicting fetal membrane damage. Therefore, this study provides a possible diagnostic application of LDH level to predict the extent of tissue damage of fetal membranes via apoptosis induced by influenza virus infection.

Direct contribution of inducible nitric oxide synthase expression to apoptosis induction in primary smooth chorion trophoblast cells of human fetal membrane tissues.

We have previously demonstrated that apoptosis induction is observed only in smooth chorion laeve trophoblast cells, and not in amnion epithelial cells of human fetal membrane tissues prepared at the term. Apoptosis induction was suppressed by the presence of an inhibitor specific for inducible nitric oxide synthase (iNOS), suggesting that intracellular oxidative stress plays a critical role in this process. In this study, we transfected the iNOS gene into primary cultured chorion and amnion cells to examine the direct contribution of iNOS gene expression to the apoptosis induction in these cells. We identified a significant increase in the levels of iNOS protein expression and nitrite accumulation in both chorion and amnion cells after the iNOS gene transfection. However, the induction of apoptosis was observed in an approximately 70% of chorion cells transfected with iNOS gene. Transfection of the iNOS gene into chorion cells resulted in the activation of p38 mitogen-activated protein kinase (MAPK) and downregulation of hemeoxygenase-1 protein expression, whereas no such events were observed in the transfected amnion cells. These results suggest that apoptosis induced in the chorion trophoblast cells by the iNOS gene expression is closely linked to a physiological consequence, such as the rupture of fetal membranes.

Cytotoxic effects of flavonoids against a human colon cancer derived cell line, COLO 201: a potential natural anti-cancer substance.

The proliferation of a human colon carcinoma cell line, COLO 201, was effectively suppressed through apoptosis in the presence of flavonoids, an ethanol extract from Vitex agnus-castus fruits. The induction of apoptosis was not inhibited by the presence of an anti-oxidant, N-acetyl-L-cysteine, whereas only HO-1 gene expression levels increased among other typical oxidative stress-associated genes examined after Vitex treatment. These results suggest that Vitex treatment activates a pathway associated with HO-1 gene activation, resulting in the induction of apoptosis in COLO 201. Results also implicate a potential clinical chemotherapeutic application of Vitex for the treatment of colon cancer patients.

Sucutiniranes A and B, new cassane-type diterpenes from Bowdichia nitida.

Two new cassane-type diterpenes, sucutiniranes A (1) and B (2), have been isolated from the seeds of Bowdichia nitida together with 6alpha-acetoxyvouacapane (3) and 6alpha,7beta-diacetoxyvouacapane (4), and the structures of 1 and 2 were elucidated by using 2D NMR data and chemical correlations. Sucutinirane A (1) and 3 showed a moderate cytotoxicity against human colon carcinoma COLO201 cells, and 6alpha,7beta-diacetoxyvouacapane (4) showed in vitro antiplasmodial activity against parasite Plasmodium falciparum 3D7.

Imbalance between ROS production and elimination results in apoptosis induction in primary smooth chorion trophoblast cells prepared from human fetal membrane tissues.

We have previously demonstrated that induction of apoptosis was observed in the smooth chorion trophoblast cells of human fetal membranes prepared at term, and that apoptosis progressed rapidly during in vitro incubation of the tissues. Furthermore, we identified the contribution of ROS production system (e.g., oxidant enzymes, such as iNOS and Cox-2) to the apoptosis induction in the chorion cells, suggesting an important role of the two inducible enzymes in the induction process. In this study, we examined the role of ROS elimination system (e.g., antioxidant enzymes, such as glutathione peroxidase (GPx) and catalase) in the apoptosis induction of the chorion cells, since the apoptosis induction by oxidative stress is a result of imbalance between production and elimination of ROS. Treatment of chorion and amnion cells with mercaptosuccinic acid (MS, GPx inhibitor) and 3-amino-1,2,4-triazole (ATZ, catalase inhibitor) resulted in an inhibition of GPx and catalase activity, respectively. Furthermore, incubation with MS alone induced apoptosis in the chorion cells and apoptosis level was enhanced by the addition of ATZ, while ATZ alone hardly induced apoptosis in the chorion cells. However, none of these reagents induced apoptosis in the amnion cells. Moreover, an increase of the level of hemeoxygenase-1 gene expression was observed only in the amnion cells when both antioxidant enzyme activities were suppressed. Therefore, we concluded that GPx played a more critical role than catalase in the control of the apoptosis induction of the chorion cells, suggesting that the threshold levels of stress tolerance in the chorion cells are much lower than those in the amnion cells.

Effects of mitogen-activated protein kinase inhibitors on tumor necrosis factor-alpha gene expression and apoptosis induction in cultured human fetal membrane chorion cells infected with influenza virus.

OBJECTIVES: We investigated the involvement of p38 mitogen-activated protein (MAP) kinase in tumor necrosis factor (TNF)-alpha gene expression, apoptosis induction and virus replication in cultured human fetal membrane chorion cells infected with influenza virus. METHODS: Influenza virus-infected chorion cells were incubated in the absence or presence of inhibitors of p38 MAP kinase, SB203580 and SB202190. TNF-alpha mRNA and hemagglutinin viral RNA (HA vRNA) were amplified with reverse transcriptase-polymerase chain reaction techniques. TNF-alpha protein concentrations were determined by enzyme-liked immunosorbent assay. The extent of apoptosis induction was estimated by DNA agarose gel electrophoresis. Pyrrolidine dithiocarbamate (PDTC) and ribavirin, which have been shown to inhibit apoptosis induction via the inhibition of viral gene replication, were used as positive control reagents. RESULTS: PDTC and ribavirin inhibited the accumulation of TNF-alpha mRNA and HA vRNA in the virus-infected chorion cells, resulting in the suppression of TNF-alpha protein secretion. Both SB203580 and SB202190 suppressed TNF-alpha protein secretion, but not the accumulation of TNF-alpha mRNA as well as HA vRNA and the induction of apoptosis. CONCLUSIONS: These results suggest that p38 MAP kinase pathway is critical in TNF-alpha gene expression at a post-transcriptional level but not in the apoptosis induction and influenza virus replication in cultured chorion cells.

Characterization of monocyte differentiation-inducing (MDI) factors derived from human fetal membrane chorion cells undergoing apoptosis after influenza virus infection.

Influenza virus infection during pregnancy has been implicated as one of cause of premature delivery, abortion and stillbirth. We have reported that cultured human fetal membrane chorion cells undergoing apoptosis by influenza virus infection secrete unidentified heat-stable monocyte differentiation-inducing (MDI) factors. In this study, cellular, biological and immunochemical characteristics of MDI factors were investigated using human monocytic leukemia THP-1 cells by nitroblue tetrazolium reduction and cell adhesion assays. The treatment of THP-1 cells with culture supernatants from the influenza virus-infected chorion cells induced the nitroblue tetrazolium reduction ability, which was inhibited by the addition of superoxide dismutase and diphenyleneiodonium chloride, an inhibitor for reduced nicotinamide adenine dinucleotide phosphate oxidase. The phenomenon was also observed in human peripheral blood monocytes and histiocytic leukemia U937 cells, but not in promyelocytic leukemia HL-60 cells. The induction of nitroblue tetrazolium reduction and adhesion abilities in THP-1 cells was closely correlated with the concentrations of interleukin-6 protein in the culture supernatants. These abilities were inhibited to approximately 60% by the addition of antibodies against interleukin-6, or alpha-chain (gp80) or beta-chain (gp130) of IL-6 receptor. The induction of nitroblue tetrazolium reduction was increased by the addition of supernatants from amniochorion tissue cultures after influenza virus infection. These results indicate that chorion cell-derived interleukin-6 is partly responsible for monocyte differentiation to macrophages capable of generating superoxide anion. It is possible that these pathways represent part of the mechanism for birth complications associated with intrauterine influenza infection in pregnancy.

Contribution of inducible nitric oxide synthase and cyclooxygenase-2 to apoptosis induction in smooth chorion trophoblast cells of human fetal membrane tissues.

We examined the contribution of apoptosis- and oxidative stress-associated genes to apoptosis induction in trophoblast cells of human fetal membrane tissues undergoing apoptosis during in vitro incubation. RT-PCR analyses demonstrated an increased level of HO-1, Mn-SOD, Cox-2, iNOS, TNFalpha, TNFR1, IL-1beta, IL-6, Bax, Bak, and Bad gene expression, while Bcl-2 mRNA expression level decreased. Western blot analyses demonstrated an increase in iNOS, Cox-2, and HO-1 protein levels; a decrease in pro-caspase-3 and 9, proform-PARP, and Apaf-1 protein levels; a leakage of cytochrome c from the mitochondria. An antioxidative reagent, general and selective Cox-2 inhibitors, and an iNOS inhibitor suppressed in vitro progression of the apoptosis. Furthermore, an NO donor reagent induced apoptosis in primary cultured trophoblast cells. Therefore, we concluded that the induction of apoptosis in the smooth chorion trophoblasts is mediated through oxidative stress induction followed by mitochondria damage, suggesting that iNOS and Cox-2 play an important role in the apoptosis induction in trophoblasts of human fetal membrane tissues.

Inhibition of influenza-virus-induced apoptosis in chorion cells of human fetal membranes by nordihydroguaiaretic Acid.

OBJECTIVES: It has been postulated that the pathogenesis of influenza virus infection involves not only the virus-proliferation-mediated apoptotic cell death in infected cells, but also a direct reactive oxygen species (ROS)-induced cellular injury in the infected organs. We examined effects of an antioxidant, nordihydroguaiaretic acid (NDGA), on apoptosis induction and viral proliferation. Subsequently, the results were compared with those of pyrrolidine dithiocarbamate (PDTC), another antioxidant. METHODS: The levels of ROS production were measured with 2',7'-dichlorofluorescein diacetate; apoptosis induction and viral proliferation were analyzed by DNA fragmentation and plaque-forming assays, respectively. RESULTS: The treatment of infected cells with NDGA inhibited ROS overproduction, apoptotic DNA fragmentation and virus proliferation. The maximum inhibition against DNA fragmentation (76%) was observed with 500 microM NDGA. The antiviral activity of NDGA against influenza virus was more potent than that of PDTC. CONCLUSIONS: The present study, therefore, suggests for the first time that NDGA, a known antioxidant reagent, inhibits the induction of apoptosis in human fetal membrane chorion cells infected with influenza virus through the more potent antiviral activity than that of PDTC.