No abstract

When a virus encounters a susceptible cell, the virus enters it to initiate cytocidal, persistent, latent or abortive cell infection. The influenza virus induces cytocidal infection accompanied by virus production and cell death in several types of cultures such as HeLa and Madin-Darby canine kidney (MDCK) cell lines and human peripheral blood monocytes in vitro, all of which die through the mechanism of apoptosis. 1-4)The human influenza virus usually infects the human respiratory organs. However, influenza virus was isolated from blood, [5][6][7][8] as well as extrapulmonary regions such as lymph node, spleen, liver, kidney, adrenal glands, meninges, 7,[9][10][11] and cerebrospinal fluid.12,13) These findings substantiate the occurrence of viremia in influenza virus infection and the extrapulmonary dissemination of the virus. Furthermore, influenza virus has been isolated from fetal heart, 10) placenta 14) and amniotic fluid, 10,15) suggesting the occurrence of fetal, placental and amniotic fluid infections with this virus.The influenza virus has not so far been isolated from the fetal membranes of a mother who died of influenza virus infection to our knowledge. However, the virus has been isolated from the placenta and amniotic fluid. 14,15) Additionally, organ cultures of human placenta allow influenza virus replication.16) The influenza virus may spread from the placenta to fetal membranes because they are continuous with the placenta. Furthermore, since amniotic fluid fills amniotic cavity formed by fetal membranes, influenza virus in amniotic fluid may influence the membrane cells. However, little is understood about the influence of influenza virus on human fetal membrane cells.We investigated the relationship between the influenza virus and cultured human fetal membrane cells by studying virus production and the apoptotic death of these cells in vitro. We found that the influenza virus induced cytocidal infection in chorion cells, persistent infection accompanied by virus production and cell survival in amnion cells, and that influenza virus-infected chorion cells became degraded through the apoptotic pathway. The present study demonstrates that the host cells make the choice to commit to degradation through this pathway. MATERIALS AND METHODSChemicals Ribavirin (1-b-D-ribofuranosyl-1,2,4-triazole-3-carboxamide) was purchased from Sigma (MO, U.S.A.).Influenza Virus Propagation and Plaque-Forming Assay Influenza virus type A (PR/8/34, H1N1) was propagated in the allantoic cavity of 11-d-old embryonal chicken eggs for 48 to 72 h at 35°C as described.2) Plaque-forming capacity was assayed on confluent monolayers of MDCK (NBL-2) cells (Human Science Research Resources Bank, Japan) as described.4) The chorioallantoic fluid contained 8ϫ10 8 plaque-forming units (PFU)/ml and the hemagglutination titer using human erythrocytes was 1 : 256. Cell Cultures and Virus Infection Human fetal membranes were prepared aseptically from placentas obtained by cesarean section in the month of normal parturition. Primary cult...

Vitex agnus-castus is a shrub belonging to the Verbenaceae family, which grows naturally and is common in the Middle East and southern Europe. Its ripened fruits have been used as a folk medicine for a long time for alleviation and/or improvement of symptoms from obstetric and gynecological diseases. Hobbs, in "Vitex: The Women's Herb", described Vitex as an effective remedy for emmeniopathy, including amenorrhea, oligomenorrhea, and menorrhagia, and that due to estrogen hypersecretion before menstruation in premenstrual tension, long-term use of V. agnus-castus reduced those symptoms and prevented relapse of the disease. 1)It also appears that the function of the corpus luteum in corpus luteum deficiency syndrome improved with V. agnus-castus due to the crinogenic effect of luteinizing hormones.Hirobe et al. reported that an antitumor effect results from the extract of ripened Israeli-grown V. agnus-castus fruits (Vitex extract) on the Chinese hamster lung carcinoma cells line V-79, and that Vitex extract has high antitumor activity. 2)Furthermore, analysis of the components of Vitex extract showed four types of new flavonoid compound and four known flavonoids.3) We investigated the cytotoxicity of Vitex extract against a normal human cell line, human uterine cervical fibroblasts (HCF), and the cancer cell lines breast carcinoma (MCF-7) and ovarian cancer (SKOV 3). Using these three cell lines, we found that the cytotoxic activity of Vitex extract may be attributed to the growth activity of the respective cell, and demonstrated the possibility that the cytotoxicity is related to the cell cycle stage. 4)To investigate the antitumor effects of Vitex extract in more detail, we examined the cytotoxicity of Vitex extract against two noncancerous and six cancerous human cell lines. The cytotoxic activity of Vitex extract was confirmed to depend on the growth rate of the cells, the cytotoxic cell death of cells with relatively low growth activity occurs through apoptosis, and apoptotic cell death results from an increase in intracellular oxidation. MATERIALS AND METHODS Cells and Media The breast carcinoma (MCF-7)5) and gastric signet ring carcinoma (KATO III) 6) cell lines were described previously. Cervical carcinoma (SKG-3a), colon carcinoma (COLO 201), and small cell lung carcinoma (Lu-134-A-H) cell lines were provided by the JCRB Cell Bank (Tokyo, Japan). The ovarian cancer (SK-OV-3) cell line was provided by the American Type Culture Collection (Rockville, MD, U.S.A.). Fetal fibroblasts (HE-21) were obtained from the Research Institute for Functional Peptides (Yamagata, Japan). HCF was prepared as previously described.7) Pyrrolidine dithiocarbamate (PDTC) and N-acetyl-L-cysteine (NAC) were purchased from Dojindo Laboratories and Wako Pure Chemical Industries, respectively. Preparing Ethanol Extract from Dried Ripened V. agnus-castus Fruits Dried, ripened, Israeli-grown V. agnuscastus fruits were triturated, and a crude extract was prepared from the triturate (1 g) with ethanol (10 ml) under reflux for 2 h. After...

When a virus encounters a susceptible cell, the virus enters and initiates cytocidal, persistent, latent or abortive infection. We recently reported that influenza virus (IV) induces persistent infection accompanied by cell survival and virus production in cultured amnion cells. In addition, cytocidal infection accompanied by apoptotic cell death and virus production occurred in cultured chorion cells. Both cell cultures were prepared from human fetal membranes. 1)IV induces cytocidal infection in a variety of cultures, such as HeLa and Madin-Darby canine kidney (MDCK) cell lines and human peripheral blood monocytes in vitro, all of which die through the mechanism of apoptosis.2-5) Furthermore, IV infection induces gene expression of inflammatory cytokines such as interleukin (IL)-1, IL-6, tumor necrosis factor (TNF)-a and interferon (IFN)-a/b, and C-C and C-X-C chemokines such as monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)-1a, RANTES (regulated on activation, normal T cell expressed and secreted) and IFN-inducible protein (IP)-10, 6,7) and proapoptotic factors such as Fas and Fas ligand,8) which occur in infected host cells undergoing apoptosis. Apoptosis and inducible gene expression of inflammatory mediators therefore seem to be correlated. However, whether IV induces gene expression of inflammatory mediators during persistent infection remains unknown.We therefore extensively examined expression of mRNAs for inflammatory cytokines such as IL-1b, IL-6, TNF-a, IFN-a, IFN-b, IFN-g, macrophage colony-stimulating factor (M-CSF) and granulocyte macrophage colony-stimulating factor (GM-CSF), and Fas during cytocidal and persistent infections by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Of note is the fact that IV infection induced the gene expression of a set of inflammatory cytokines during cytocidal infection accompanied by apoptotic cell death, but this induction was not present during persistent infection in the absence of apoptosis. In this paper we report a noticeable difference in gene expression of a set of inflammatory cytokines between cytocidal and persistent infections. MATERIALS AND METHODSMaterials PCR primers for IFN-a, IFN-b, IFN-g, M-CSF and GM-CSF were originally designed according to published cDNA sequences. 9-13) PCR primers for Fas were based on the published DNA sequences for the primers. 14)PCR primers for IL-1b, IL-6, TNF-a and glycerol-3-phosphate dehydrogenase (G3PDH) were purchased from CLON-TECH Labs. Inc. (CA, U.S.A.). DNA sequences for the PCR primers are shown in Table 1. Neutralizing antibodies against Fas (Medical & Biological Labs. Co., Ltd., Nagoya, Japan), IFN-b (Yamasa Shoyu Co., Ltd., Tokyo, Japan), TNF-a and IFN-g (Genzyme Diagnostic, MA, U.S.A.) were purchased from the listed suppliers.Cell Culture and Virus Infection Primary cultured chorion and amnion cells were prepared from human fetal membranes obtained by cesarean section during the month of normal parturition as described.15) HeLa cell line was obtained from...

No abstract

Previous study showed that mice lacking modulator recognition factor-2 (Mrf-2) were lean, with significant decreases in white adipose tissue. One postulated mechanism for the lean phenotype in Mrf-2 knockout mice is a defect in adipogenesis. In order to investigate this further, we examined the effects of Mrf-2 deficiency on adipogenesis in vitro. In mouse fibroblasts (MEFs) derived from Mrf-2(-/-) embryos, and in 3T3-L1 cells after knockdown of Mrf-2 by small interference RNA (siRNA) there was a potent inhibition of hormone-induced lipid accumulation, and significant decreases in the expression of the adipogenic transcription factors CCAAT/enhancer-binding protein (C/EBP) alpha and peroxisome proliferator-activated receptor-gamma and the mature adipocyte genes they control. Transduction of Mrf-2(-/-) MEFs with a retroviral vector expressing the longer Mrf-2 splice variant (Mrf-2B) stimulated both gene expression and lipid accumulation. Because 3T3-L1 cells are committed to the adipocyte lineage, we used this simpler model system to examine the effects of Mrf-2 deficiency on adipocyte maturation. Analyses of both mRNA and protein revealed that knockdown of Mrf-2 in 3T3-L1 cells prolonged the expression of C/EBP homologous protein-10, a dominant-negative form of C/EBP. Consistent with these findings, suppression of Mrf-2 also inhibited the DNA-binding activity of C/EBPbeta. These data suggest that Mrf-2 facilitates the induction of the two key adipogenic transcription factors C/EBPalpha and peroxisome proliferator-activated receptor-gamma indirectly by permitting hormone-mediated repression of the adipogenic repressor C/EBP homologous protein-10.

In the trophoblast layer of the chorion laeve of human fetal membranes obtained by cesarean section at the month of normal parturition, cells with condensed nuclei could be observed by histochemical examination. Incubating fetal membranes at 37 degrees C in vitro in cultivation medium, the frequency of cells with condensed nuclei increased in the chorion laeve, associating with an increase in DNA fragmentation and the population of in situ TUNEL (terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-X nick end labeling) staining-positive cells. The progressed apoptotic cell death in the chorion laeve in vitro was suppressed by incubation of the tissue in the presence of glucocorticoids, cortisone or hydrocortisone, which was also demonstrated by DNA fragmentation analysis and in situ TUNEL staining. These results reveal that a substantial proportion of trophoblast cells in the chorion laeve of human fetal membranes are induced to undergo apoptosis at the end of pregnancy, and that the apoptosis progresses rapidly in vitro as the incubation period increases. It is suggested that certain hormones such as glucocorticoid, may be related to the regulation of the apoptosis in human fetal membranes.

The ability of heated scallop-shell powder HSSP to work against Listeria sp. biofilm formed at a low temperature was investigated. A biofilm of L. innocua ATCC 33090 was grown on a glass plate at 15 for 15 days, then immersed in HSSP slurry. Following treatment, the disinfection ability of the HSSP against the biofilm was non-destructively quantified by conductimetric assay. The biofilm grown at 15 was less sensitive than that grown at 37 to HSSP treatment and alkaline treatment. The biofilm grown at 15 was completely deactivated by 30 min of HSSP treatment 10 mg/mL, pH 12.5 . In contrast, after 30 min treatment with alkaline solution at pH 12.5 or sodium hypochlorite 100 ppm , the activity was reduced by only one order of magnitude. The disinfection efficacy of HSSP 10 mg/mL against L. innocua is similar to or higher than that of sodium hypochlorite 200 ppm . Fluorescence microscopy validated the results of the conductimetric assay. Therefore, HSSP treatment is a potentially powerful alternative control agent against Listeria sp. biofilms that present hazards in the food industry.