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.

Experimental infection of early pregnant cows with bovine viral diarrhea virus: transmission of virus to the reproductive tract and conceptus.

The objective of this study was to chronologically investigate the effect of bovine viral diarrhea virus (BVDV) infection on early pregnant cows before placenta formation. Six cows were intravenously inoculated with either BVDV (treated, n=4) or growth medium (control, n=2) (day 0) at day 26 of pregnancy. Two treated cows and one control cow were euthanized on day 3 post-infection and the remaining animals were euthanized on day 6. BVDV was isolated from maternal tissues such as lymphoid or reproductive tissues of treated animals on days 3 and 6 post-infection. Additionally, one treated cow autopsied on day 6 post-infection had evidence of infectious BVDV in the allantoic membranes, allantoic fluid and embryos. In three treated cows, a significant decline in progesterone concentration was also observed post-infection while in control cows they remained constant. Therefore, BVDV can infect bovine embryos before placenta formation and may affect progesterone profiles in cows during early pregnancy.

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.

Induction of pro-inflammatory cytokine gene expression and apoptosis in human chorion cells of fetal membranes by influenza virus infection: possible implications for maintenance and interruption of pregnancy during infection.

Human fetal membranes are composed of amnion, chorion and decidua tissues, which play a critical role in defense barriers as well as maintenance of pregnancy and parturition. Pro-inflammatory cytokines, such as interleukin (IL)-1beta, IL-6 and tumor necrosis factor (TNF)-alpha, produced by the tissues are postulated to facilitate parturition. Influenza virus infection is one of causes of pregnancy-associated complications, such as premature delivery, abortion and stillbirth. Recent studies have demonstrated that influenza virus infection induced the gene expression of a set of pro-inflammatory cytokines, such as IL-1beta, IL-6, TNF-alpha, interferon (IFN)-beta, IFN-gamma and granulocyte macrophage colony-stimulating factor (GM-CSF), and the secretion of unidentified monocyte differentiation-inducing factor(s) from primary cultured chorion cells undergoing apoptosis. These phenomena were not observed in primary cultured amnion cells infected with the virus. This article reviews, (1) the production of cytokines in fetal membrane tissues and their functions; (2) the differential induction of pro-inflammatory cytokine gene expression and apoptosis in fetal membrane chorion and amnion cells by influenza virus infection. An accumulating number of evidence suggests that interactive reactions between fetal membrane chorion cells and maternal monocytes/macrophages may play a critical role in defense barriers against the virus infection. Understanding the interactions would make important contributions to the elucidation of the pathogenesis of influenza virus infection during pregnancy.

Detection of equine herpesvirus-1 in the fetal membranes of aborted equine fetuses by immunohistochemical and in-situ hybridization techniques.

Formalin-fixed, paraffin wax-embedded fetal membranes from 76 cases of equine abortion were examined immunohistochemically for equine herpesvirus (EHV)-1 antigen. Of the 76 cases, 11 had been proved EHV-1-positive by diagnostic methods applied to the aborted fetuses (viral isolation in tissue culture, or immunohistochemical examination, or both). Of the 11 fetal membranes from the virus-positive animals, five gave positive results on immunohistochemical examination, and three on in-situ hybridization; the positive signals were detected in trophoblastic cells and occasionally in monocytes and endothelial cells. The distribution of virus appeared to be related to areas of (1) vacuolar degeneration and desquamation of chorionic epithelium, (2) mild lympho-histiocytic vasculitis and placentitis, and (3) increased metabolic activity of mesenchymal cells in the villi of the fetal membranes. This is the first report of EHV-1 antigen and nucleic acid detection in the trophoblasts of fetal membranes from spontaneous cases of equine abortion.

Apoptosis in cultured human fetal membrane cells infected with influenza virus.

We investigated the induction of apoptosis in cultured human fetal membrane cells infected with influenza virus type A. We found that influenza virus yield in supernatants of primary cultured chorion and amnion cells prepared from human fetal membranes increased 6 h after infection. Chromosomal DNA was fragmented into oligonucleosomes at 48 h after influenza virus infection in chorion cells but not in mock-infected chorion cells, mock-infected amnion cells or influenza virus-infected amnion cells. The DNA fragmentation in influenza virus-infected chorion cells was evident at 24 h after infection and depended on the multiplicity of infection at 48 h. Incubating influenza virus-infected chorion cells with ribavirin, an inhibitor of viral RNA synthesis, reduced the increase in virus yield and simultaneously blocked DNA fragmentation. These results suggest that both chorion and amnion cells become infected with influenza virus, but that influenza virus infection induces apoptosis in chorion, but not typical apoptosis characterized by DNA ladder formation in amnion cells. We further observed that influenza virus replication is associated with the induction of apoptosis.

Differential mRNA expression of inflammatory cytokines in cultured human fetal membrane cells responding to influenza virus infection.

We examined the expression of mRNAs for inflammatory cytokines and Fas in cultured human fetal membrane cells responding to influenza virus (IV) infection using the reverse transcriptase-polymerase chain reaction (RT-PCR). Primary cultured chorion and amnion cells prepared from human fetal membranes were infected with IV. Chorion cells expressed significant amounts of interleukin (IL)-1beta, IL-6, tumor necrosis factor (TNF)-alpha, interferon (IFN)-beta, IFN-gamma and granulocyte macrophage colony-stimulating factor (GM-CSF) mRNAs and small amounts of Fas mRNA in response to IV infection. Amnion cells expressed TNF-alpha and IFN-beta mRNAs in response to IV infection, while expression of the other mRNAs was not altered. We also examined whether or not TNF-alpha, IFN-beta, IFN-gamma and Fas participated in IV infection-induced apoptotic DNA fragmentation in chorion cells. Neutralizing antibodies against them did not inhibit DNA fragmentation. These results suggested that chorion cells expressed significant amounts of mRNAs for inflammatory cytokines in response to IV infection, and that, in contrast, mRNA expression was quiescent in amnion cells. Moreover, TNF-alpha, IFN-beta, IFN-gamma and Fas do not appear to be directly involved in the apoptosis induction of IV-infected chorion cells. The results indicated that chorion cells may play a role in defense against IV through an antiviral immune response and apoptosis to eliminate own cells and viral pathogens in infected organs, whereas amnion cells do not play such a role.

Vertical transmission of human immunodeficiency virus.

Sensitive detection methods, such as DNA PCR and RNA PCR suggest that vertical transmission of human immunodeficiency virus (HIV) occurs at three major time periods; in utero, around the time of birth, and postpartum as a result of breastfeeding (Fig. 1). Detection of proviral DNA in infant's blood at birth suggests that transmission occurred prior to delivery. A working definition for time of infection is that HIV detection by DNA PCR in the first 48 h of life indicates in utero transmission, while peripartum transmission is considered if DNA PCR is negative the first 48 h, but then it is positive 7 or more days later [1]. Generally, in the breastfeeding population, breast milk transmission is thought to occur if virus is not detected by PCR at 3-5 months of life but is detected thereafter within the breastfeeding period [2]. Using these definitions and guidelines, studies has suggested that in developed countries the majority, or two thirds of vertical transmission occur peripartum, and one-third in utero [3-6]. The low rate of breastfeeding transmission is due to the practice of advising known HIV-positive mothers not to feed breast milk. However, since the implementation of antiretroviral treatment in prophylaxis of HIV-positive mothers, some studies have suggested that in utero infection accounts for a larger percentage of vertical transmissions [7]. In developing countries, although the majority of infections occurs also peripartum, a significant percentage, 10-17%, is thought to be due to breastfeeding [2, 8, 9].

Distribution of viral antigen in uterus, placenta and foetus of cattle persistently infected with bovine virus diarrhoea virus.

The tissue distribution and cellular localisation of bovine virus diarrhoea virus (BVDV) was investigated in the uterus, placentomes, intercotyledonary foetal membranes and foetal organs of three persistently infected (PI) pregnant heifers. The uterus and ovaries of a non-pregnant PI heifer were also included in the study. Cryostat sections were examined using immunohistochemical techniques and monoclonal antibodies against BVDV. A double immunofluorescence technique was used to identify BVDV positive cells that also showed staining for either the leukocyte common antigen CD45 or the cytoskeletal filament vimentin. BVDV antigen was detected in all the organs examined, and was present in both epithelial and non-epithelial cells. In all organs many of the virus-positive cells also showed reactivity for vimentin. In the foetal liver and spleen a small, scattered population of virus-positive cells showed reactivity for CD45. A few cells showed reactivity both for BVDV antigen and for CD45 in the placentomes and intercotyledonary foetal membranes. In contrast to earlier reports, only scattered cells in the foetal part of the placentomes, the cotyledons, showed reactivity for BVDV antigen. However, in the chorion of the intercotyledonary foetal membranes, a larger proportion of the trophoblast cells showed reactivity for BVDV, especially the binuclear trophoblast cells. In the uterus, pregnancy appeared to favour virus replication, as the section from the pregnant heifers showed much stronger staining and a higher proportion of viral antigen-positive cells than sections from the non-pregnant PI heifer.

Transport of viruses through fetal membranes: an in vitro model of perinatal transmission.

A model system for perinatal transmission of viral infections was developed and transport of infectious virus particles through fetal membranes was investigated. Viruses of different families known to cause serious intrauterine infections were selected, including relevant and model viruses: the DNA-viruses HSV-1 and -2 as well as the animal herpes viruses BHV-1 and SHV-1, the RNA-virus BVDV as a model for hepatitis C virus, HIV-1 and -2, and PPV as a model for parvovirus B19. Migration of infectious virus from the maternal to the fetal side of the membrane could be detected as early as 20 min after the start of incubation. A peak of virus migration was observed after 1-2 hr. 0.02-1% of HSV-1 and 0.03-0.2% of HSV-2 were transported from the maternal side of the membrane to the fetal side. Only 0.01% of PPV migrated to the fetal side, whereas no transport of BVDV was observed. HIV-1 (1.4%) and HIV-2 (0.8%) seemed to be transported at higher rates. The concept of an active transport of infectious virus is compatible with the kinetics of penetration of the fetal membrane. The question of whether different receptors for the individual viruses on the cellular surface account for differences in virus transport will require further investigation. The fetal membrane acts as a protective barrier for the fetus, reducing greatly infectious titers or even preventing completely penetration of virus.