Adhesion development and the expression of endothelial nitric oxide synthase.

OBJECTIVE: This study was conducted to determine whether nitric oxide (NO), a potent vasodilator and inhibitor of thrombus formation, is involved in the formation and maintenance of adhesions. METHODS: Skin, subcutaneous tissues, peritoneum and adhesions were collected from surgical patients and total RNA was isolated. Quantitative reverse transcription polymerase chain reaction (QRT-PCR) was performed to quantitate endothelial nitric oxide synthase (eNOS) and beta-actin mRNA levels. RESULTS: eNOS mRNA levels for skin, subcutaneous tissue, peritoneum and adhesions were < or = 3.12 x 10(-4), < or = 3.12 x 10(-4), 6.24 x 10(-4) and 2.5 x 10(-3) attomoles/microl, respectively. Beta-actin mRNA levels for all tissues were between 1.25 x 10(-1) and 6.25 x 10(-2) attomoles/microl. CONCLUSION: eNOS mRNA can be identified in tissue adhesions, and may therefore play a role in adhesion formation and maintenance.

Detection of cytomegalovirus in the meconium of infected newborns by polymerase chain reaction.

OBJECTIVE: Congenital cytomegalovirus (CMV) infection is a leading cause of hearing loss and mental retardation throughout the world. Detection of the CMV DNA by polymerase chain reaction (PCR) offers a sensitive, rapid, and specific means of identification. Meconium, the stool formed in utero, may be an ideal specimen for CMV detection. The objective of this study was to develop a PCR-based methodology for the detection of CMV in the meconium of neonates. METHODS: Meconium was collected from 10 newborn infants (seven with positive viral cultures and three uninfected infants born to CMV-seropositive mothers). For each, DNA was isolated from meconium by organic extraction and attachment to a DNA-binding matrix, and PCR was performed using amplimers specific for the major intermediate early (MIE) and late antigenic (LA) regions of CMV. RESULTS: Gel electrophoresis demonstrated an anticipated PCR product of 250 base pairs (bp) corresponding to the MIE region of CMV in all infected and positive control meconium samples. Furthermore, a single band of 150 bp corresponding to the LA region of CMV was also amplified in several of the infected infants. Conversely, no amplification of these antigenic regions was noted in either uninfected infants born to CMV-seropositive mothers or negative controls. CONCLUSIONS: CMV is present within the meconium of infected neonates and is readily detectable by PCR.

Expression and regulation of vascular endothelial growth factor in a first trimester trophoblast cell line.

Embryo implantation and development are critically dependent upon the spatial and temporal regulation of angiogenesis and localized vascular permeability. A key mediator of these effects is the endothelial cell mitogen vascular endothelial growth factor (VEGF). VEGF has been shown to promote endometrial vascular permeability, fetal vasculogenesis and placental, fetal and maternal angiogenesis. However, the mechanism through which this regulation occurs in the placenta is poorly understood. This study was conducted to determine if the pro-angiogenic cytokines, TNF-alpha and TGF-beta1, affect VEGF expression in human first trimester trophoblasts. Culture of a first trimester trophoblast cell line (HTR-8/SVneo), in the presence of either TNF-alpha or TGF-beta1, resulted in the expression of significant levels of VEGF in culture. The trophoblast cell line also showed a time-dependent and a dose-dependent increase in VEGF mRNA levels when cultured in the presence of either TNF-alpha or TGF-beta1. These results suggest that both TNF-alpha and TGF-beta1 may regulate the production of VEGF in early gestational trophoblasts and may therefore serve to modulate placental vascular permeability and angiogenesis that are necessary for embryo implantation and placentation.

Effect of imiquimod on cytokine induction in first trimester trophoblasts.

OBJECTIVES: Imiquimod (IQ) is used clinically for the topical treatment of external genital warts. IQ is an immune response modifier and induces the expression of interferon-alpha and other cytokines in human Peripheral Blood Monocytes (PBMC). Trophoblasts have been previously shown to express inflammatory cytokines upon lipopolysaccharide (LPS) stimulation. The objective of this study was to evaluate the ability of IQ to induce transcription of cytokines in trophoblasts. METHODS: A transformed human first trimester trophoblast cell line, HTR-8/SVneo, was cultured in DMEM containing IQ at concentrations of 0 to 5.0 microg/ml. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) viability assays were conducted to control for any drug-induced cell death. Total RNA was isolated from trophoblasts at 0, 8 and 24 hours of culture and Reverse Transcription-Polymerase Chain Reaction (RT-PCR) was conducted using specific amplimers for the inflammatory cytokines interleukin (IL)-1alpha, IL-1beta, IL-6 and IL-8. RT-PCR of beta-actin was performed to control for equal RNA loading. RESULTS: RT-PCR was unable detect an increase in either IL-1alpha, IL-1beta, IL-6 or IL-8 mRNA in first trimester trophoblasts cultured in the presence of 0 to 5.0 microg/mL of IQ for up to 24 hours. RT-PCR confirmed equal RNA loading and MTT viability assays did not show loss of cell viability at concentrations of IQ up to 5.0 microg/ml. CONCLUSIONS: IQ, at the concentrations tested, did not induce the transcriptional expression of inflammatory cytokines in human first trimester trophoblasts. These data suggest that IQ would not induce the expression of inflammatory cytokines in placental trophoblasts.

Ethanol-induced expression of cytokines in a first-trimester trophoblast cell line.

OBJECTIVES: Altered cytokine expression at the fetoplacental interface may be a potential mechanism for the development of fetal immune dysfunction in children with fetal alcohol syndrome. This study was conducted to determine whether first-trimester trophoblasts respond to ethanol exposure by the induction of specific cytokines. STUDY DESIGN: HTR-8/SVneo trophoblast cells were cultured in vitro in the presence of either ethanol (0.5% [vol/vol]), lipopolysaccharide (1 microg/mL), or ethanol and lipopolysaccharide. Expression of granulocyte colony-stimulating factor, regulated on activation normal T cell expressed and secreted, and interleukin-6 was examined by Northern analysis and enzyme-linked immunosorbent assay. RESULTS: Culture in the presence of ethanol, lipopolysaccharide, or lipopolysaccharide and ethanol resulted in the increased transcription and secretion of granulocyte colony-stimulating factor, regulated on activation normal T cell expressed and secreted, and interleukin-6 at significantly greater levels (P < .01) than control cultures. CONCLUSIONS: Human first-trimester trophoblasts express high levels of cytokines when cultured in the presence of ethanol. Trophoblasts may therefore be an important exogenous source of cytokines for the fetus, and altered cytokine levels during early gestation may have an adverse effect on the development of the fetal immune system.

Decreased gene expression of endothelial nitric oxide synthase in newborns with persistent pulmonary hypertension.

Previous studies in adults have shown that chronic pulmonary hypertension is associated with decreased endothelial nitric oxide synthase (eNOS) expression in pulmonary arteries. However, the role of decreased eNOS expression in persistent pulmonary hypertension of the newborn (PPHN) is unknown. We investigated the hypothesis that umbilical vein endothelial cells cultured from infants with PPHN will have decreased eNOS expression. Umbilical cords were collected from meconium-stained infants at birth, and endothelial cells were isolated if the infants developed PPHN. Endothelial cells were grown in primary culture, and total RNA was isolated. cDNA was reverse transcribed from mRNA and amplified by PCR. An expected product of approximately 550 bp was found in all control infants but only in two of the six infants with PPHN. Identity of the PCR product was confirmed by Southern hybridization to a separate internal eNOS-specific probe. Amplification of beta-actin cDNA, an internal control, was detected in all controls and in all infants with PPHN, including the four infants without the eNOS band. There was no difference in the course and outcome of patients with presence or absence of the eNOS band. However, there was an acidotic arterial blood pH (7.19-7.29) and intrapartum fetal heart rate decelerations in all four infants without eNOS expression. In conclusion, eNOS mRNA was detected in all normal term infants but was notably absent in the majority of infants with PPHN in this pilot study. The development of PPHN is multifactorial, and a decrease in eNOS gene expression may occur in some infants. Whether the decreased eNOS transcript is a cause of PPHN or a result of intrapartum stress remains to be determined.

Detection of human defensins in the placenta.

PROBLEM: The placenta is a highly selective barrier against the hematogenous dissemination of infectious agents. Despite the presence of seemingly intact physical and immunologic barriers, infections nonetheless occur. These observations prompted the examination of placental tissue, amnion, and chorion for previously unrecognized protective mechanisms. METHOD OF STUDY: Messenger RNA from term placenta, amnion, and chorion were reverse transcribed using a 3' RACE adapter. 3' rapid amplification of cDNA ends (RACE)-polymerase chain reaction (PCR) was conducted on cDNA from these tissues to detect the presence of human defensins. Southern analysis and partial sequence analysis were subsequently performed to confirm identity. RESULTS: PCR amplification of placental, amnion, and chorion cDNA yielded a 468-bp product and a weakly detectable band of 300 bp. Southern analysis demonstrated two corresponding hybridizing bands in the placenta, amnion, and chorion but not from a negative cDNA control. Partial sequence analysis of the 468-bp product from placenta confirmed the presence of either defensin 1 or 3 in human placenta. CONCLUSIONS: The human placenta, amnion, and chorion express defensins at the level of transcription. These findings suggest that a novel and previously unrecognized mechanism of protecting the fetus against infection may be present within these tissues.

Detection of human defensin 5 in reproductive tissues.

OBJECTIVES: This study was undertaken to determine whether tissue-specific defensins are expressed within female reproductive tissues. STUDY DESIGN: Messenger ribonucleic acid from amnion, chorion, endometrium, endocervix, myometrium, placenta, small intestine, peripheral blood lymphocytes, and cervical, endometrial, and trophoblast cell lines was reverse transcribed with a 3'-RACE adapter. 3'-RACE polymerase chain reaction was conducted with an upstream human defensin 5 primer and 3'-RACE adapter primer. Polymerase chain reaction products hybridizing to a human defensin 5 probe were cloned for sequence analysis. Sequence data were compared against a nucleotide sequence database, and secondary structure predictions were made. RESULTS: Chorionic tissue, endocervical tissue, endometrial tissue, and an endometrial cell line all demonstrated a single hybridizing 362 bp polymerase chain reaction product. Sequence analysis of all clones demonstrated near-perfect identity with human defensin 5. CONCLUSIONS: Human endocervix, endometrium, and chorion express defensin 5 at the level of transcription. These findings suggest that a previously unrecognized mechanism of protecting female reproductive tissues against infection, by means of a natural antimicrobial system (defensins), may be present.

Induction and postranslational expression of G-CSF and RANTES in a first trimester trophoblast cell line by lipopolysaccharide.

PROBLEM: Comparatively little is known about the capacity of first trimester trophoblasts to respond to an infection and coordinate an immune response. This study characterizes the LPS induction of G-CSF and RANTES in a first trimester trophoblast cell line. METHODS: HTR-8/SV neo cells were exposed to LPS (1 micrograms/ml) for 0, 2, 4, 6, 8, and 24 hours in DMEM-Ham's F-12 media supplemented with 10% fetal bovine serum. Cytokine levels in culture supernatants were measured by ELISA. Northern analysis of total RNA was conducted using antisense cytokine probes. RESULTS: Levels of immunoreactive G-CSF and RANTES from LPS induced cultures at 24 hours were 10-fold and 8.5-fold greater than cytokine levels from non-induced cells at 24 hours, respectively (P < 0.01). Under LPS induction, maximal rates of G-CSF and RANTES transcription occurred at 24 hours and 8 hours, respectively. CONCLUSION: The LPS induction of proinflammatory cytokines in a first trimester trophoblast cell line supports the contention that first trimester trophoblasts participate in cytokine based immune signaling in response to infection.

Induction and posttranslational expression of cytokines in a first-trimester trophoblast cell line by lipopolysaccharide.

OBJECTIVES: The response to infection by human first-trimester trophoblasts is a poorly understood event. This study was undertaken to determine whether first-trimester trophoblasts are capable of responding to an infection stimulus and mediating an immune response. STUDY DESIGN: HTR-8/SVneo cells were exposed to lipopolysaccharide (1 microgram/ml) or media alone for either 0, 2, 4, 6, 8, or 24 hours. Northern analysis was conducted by use of a panel of antisense cytokine probes. Enzyme-linked immunosorbent assays specific for either interleukin-1 alpha, interleukin-6, interleukin-8, or transforming growth factor-beta 1 were conducted on corresponding cell culture supernatants, and the kinetics of expression were determined. RESULTS: Interleukin-1 alpha, interleukin-6, interleukin-8, and transforming growth factor-beta 1 transcription occurred maximally between 2 and 8 hours of culture in media containing lipopolysaccharide, with a subsequent diminution of response. Enzyme-linked immunosorbent assay analysis corroborated lipopolysaccharide induction seen at the level of transcription, with significant posttranslational expression of these cytokines being detected between 2 and 24 hours in culture (p < 0.01). CONCLUSIONS: Expression of the proinflammatory cytokines interleukin-1 alpha, interleukin-6, interleukin-8 and transforming growth factor-beta 1 strongly support the contention that human first-trimester trophoblasts are capable of responding to an infection stimulus and eliciting an immune response through cytokine-based immune signaling.

Human trophoblast cell adhesion to extracellular matrix protein, entactin.

PROBLEM: Trophoblast interaction with endometrial extracellular matrix (ECM) is crucial during human embryo implantation and placentation. Entactin, a ubiquitous basement membrane glycoprotein, plays a central role in ECM assembly, cell attachment, and chemotaxis. The present study was conducted to examine the possible role of entactin in promoting human trophoblast adhesion. METHODS: Using an extended life span first trimester trophoblast cell line HTR-8/SVneo (HTR) and a cell adhesion assay, we measured the adherence of human first trimester trophoblasts to recombinant entactin and its domains. Also, we used flow cytometry and indirect immunofluorescence to detect the presence of integrins that may be involved in human trophoblast-entactin interaction; these methods were used to analyze HTR cells, as well as tissue sections and freshly isolated human trophoblasts from first trimester and term placenta. RESULTS: We found that first trimester trophoblast cells were highly adherent to entactin and its E and G2 domains but not to G1 or G3 domains. Using indirect immunofluorescence and flow cytometry, we found that both beta 1 and beta 3 integrin subunits were expressed on the surface of HTR trophoblast cells adhering to entactin; in contrast, beta 2 and beta 4 integrin subunits were not detected. In addition, we found that alpha v beta 3 was expressed on freshly isolated villous cytotrophoblasts and cytotrophoblast and syncytiotrophoblasts in tissue sections from term placenta. The beta 3 integrin subunit was expressed in cytotrophoblasts and syncytiotrophoblasts in villi of first trimester placental tissue sections. CONCLUSION: Recombinant entactin promotes human trophoblast cell adhesion through both its E and G2 domains and these specific adhesive interactions may be mediated by beta 1 and/or beta 3 class integrins.

The mouse lysyl oxidase gene (Lox) resides on chromosome 18.

Lysyl oxidase initiates crosslink formation of the connective tissue matrix. This enzyme can also revert the ras phenotype in mouse NIH 3T3-transformed cells. Even though lysyl oxidase may participate in many different biological processes, its chromosomal assignment in the mouse genome remains to be addressed. Southern analysis of a panel of Chinese hamster x mouse somatic cell hybrids was utilized to assign the lysyl oxidase gene (Lox) to mouse Chromosome 18.

Gene assignment by quantitative hybridization analysis of somatic cell hybrids.

Since the inception of somatic cell hybridization technology, the number of genes mapped to a particular chromosome or region of a chromosome has increased exponentially. Conventional assignment relies on the interpretation and designation of concordance to the various panel members. Assignment of genes to individual chromosomes may be ambiguous if the representation of the individual chromosomes within the hybrid panel is not considered. To overcome this inherent limitation, we have developed a computer-assisted method to assign genes to chromosomes. This assignment utilizes an integrative statistical analysis procedure to reconcile chromosomal representation of each member of the somatic cell hybrid panel. In this manner, the intensity of the corresponding bands appearing on the autoradiographic image reflects the prevalence of that specific gene-containing chromosome within the somatic cell hybrid. The statistical method described above provides the foundation for an independent means to assign genes to a specific chromosome. We have utilized this method to assign the human lysyl oxidase gene to chromosome 5.

Regulation of the SLT-1A toxin operon by a ferric uptake regulatory protein in toxinogenic strains of Shigella dysenteriae type 1.

The regulation of Shiga toxin expression in a clinical isolate of S. dysenteriae 1 by the Fe-Fur (Iron-ferric uptake regulatory protein) repressor complex was investigated. The presence of an endogenous Fur repressor protein capable of binding to either a Fur binding consensus sequence or the regulatory region of SLT-1A was determined in toxinogenic strains of S. dysenteriae. Plasmid constructs bearing Fur binding sites fused to readily assayable reporter genes were used. Plasmid pSC27.1 contains a 21 bp synthetic oligonucleotide Fur protein binding consensus sequence located upstream to the gene for beta-galactosidase. Plasmid pSC105 contains the regulatory sequences of Shiga-like toxin-1A located upstream to the gene for alkaline phosphatase. In an analogous fashion to Shiga toxin regulation in S. dysenteriae 1, transformants bearing either pSC27.1 or pSC105 plasmid DNA were repressed in gene product expression when grown in minimal medium supplemented with iron. Conversely, transformants were de-repressed when grown under iron limiting conditions. These data suggest the presence of Fe-Fur mediated regulation of toxinogenesis in clinical isolates of S. dysenteriae.

Characterization of the human lysyl oxidase gene locus.

Lysyl oxidase (EC 1.4.3.13) is a copper-dependent enzyme acting principally on collagen and elastin catalyzing the formation of aldehyde cross-links. It is also believed to possess a tumor suppressor activity as the anti-oncogene of ras. While rat, human, and mouse lysyl oxidase cDNAs have been cloned, little is known about the structure of the gene, its organization, or regulation. This paper describes the cloning of an intronic segment of the human lysyl oxidase gene. Sequence analysis defined the location of an intron that separates the prepro-coding segments from the segment encoding the catalytic domain. Genomic restriction mapping and gene copy number data established that multiple lysyl oxidase mRNA transcripts originate from a single gene and thus are products of alternative splicing. Northern analysis of adult and fetal fibroblast RNA showed a dominant approximately 4.3-kilobase lysyl oxidase mRNA transcript that varied in abundance as a function of cell line. These data are consistent with a complex mechanism regulating the expression of the lysyl oxidase gene.

Association of adhesive, invasive, and virulent phenotypes of Salmonella typhimurium with autonomous 60-megadalton plasmids.

The plasmid DNA content of six invasive and adhesive strains of Salmonella typhimurium was determined, and all six strains (CR8500 [S850], CR6600 [TML], W118, NY, PR, and S2204) were found to harbor at least one plasmid equivalent in size to the 60-megadalton plasmid ("cryptic" plasmid), pSLT, which is normally resident in S. typhimurium strain LT2. The role of such 60-megadalton plasmids in the adhesive and invasive properties of strain CR6600, a commonly encountered salmonella pathogen that produces type 1 fimbriae, and strain CR8500, a representative FIRN biotype which does not produce type 1 fimbriae, was studied further by obtaining derivatives of these strains that no longer harbored an autonomous 60-megadalton plasmid. Strains CR6260 and CR6190 and strains CR8100 and CR8353, which were "cured" derivatives of strains CR6600 and CR8500, respectively, were significantly less adhesive and invasive in the HeLa cell test. A 53.5-megadalton colicin plasmid harbored by strain CR6600 did not detectably influence these properties. Additionally, strain CR6260 was avirulent, and strain CR8100 was 1,000 to 10,000-fold less virulent for orally infected mice as compared with their respective parental strains. Significantly, the virulence of strain CR8100 correlated with tissue colonization by bacteria that exhibited autonomous copies of a 60-megadalton plasmid. We propose that this plasmid exists in both autonomous and integrated states and that the in vivo environment selects for bacteria with autonomous plasmid copies which can express the virulent phenotype, thus enabling such strains to survive the defense mechanisms of the host.