Biomedical subjects
Publications and source records attributed to W R Hansen.
Giardia lamblia cell populations show 90% detachment from glass under normal forces of 2.43+/-0.33 nN applied by centrifugation. Detachment forces were not significantly different for cells attached to positively charged, hydrophobic, or inert surfaces than for cells attached to plain glass. The insensitivity of attachment force to surface treatment is consistent with a suction-based mechanism of attachment.
A tokay gecko can cling to virtually any surface and support its body mass with a single toe by using the millions of keratinous setae on its toe pads. Each seta branches into hundreds of 200-nm spatulae that make intimate contact with a variety of surface profiles. We showed previously that the combined surface area of billions of spatulae maximizes van der Waals interactions to generate large adhesive and shear forces. Geckos are not known to groom their feet yet retain their stickiness for months between molts. How geckos manage to keep their feet clean while walking about with sticky toes has remained a puzzle until now. Although self-cleaning by water droplets occurs in plant and animal surfaces, no adhesive has been shown to self-clean. In the present study, we demonstrate that gecko setae are a self-cleaning adhesive. Geckos with dirty feet recovered their ability to cling to vertical surfaces after only a few steps. Self-cleaning occurred in arrays of setae isolated from the gecko. Contact mechanical models suggest that self-cleaning occurs by an energetic disequilibrium between the adhesive forces attracting a dirt particle to the substrate and those attracting the same particle to one or more spatulae. We propose that the property of self-cleaning is intrinsic to the setal nanostructure and therefore should be replicable in synthetic adhesive materials in the future.
Virus surveillance in free-flying, nonmigratory ducks living on the eastern shore of Maryland indicated that influenza A viruses were introduced into the area or that the prevalence of endemic infections increased between July 15 and August 27, 1998. Cloacal swabs collected between May 28 and July 15, 1998, were negative for influenza A virus recovery (0/233), whereas 13.9% (29/209) of swabs collected between August 27 and September 2, 1998, were positive for influenza A virus recovery. Five hemagglutinin subtypes (H2, H3, H6, H9, and H12), six neuraminidase subtypes (N1, N2, N4, N5, N6, and N8), and nine HA-NA combinations were identified among 29 influenza A isolates. Interestingly, 18 of the 29 isolates initially appeared to contain two or more HA and/or NA subtypes. The free-flying, nonmigratory ducks served as excellent sentinels for the early detection of type A influenza viruses in the southern half of the Atlantic Migratory Waterfowl Flyway during the earliest phase of the yearly southern migration.
The ecology of the strain of West Nile virus (WNV) introduced into the United States in 1999 has similarities to the native flavivirus, St. Louis encephalitis (SLE) virus, but has unique features not observed with SLE virus or with WNV in the old world. The primary route of transmission for most of the arboviruses in North America is by mosquito, and infected native birds usually do not suffer morbidity or mortality. An exception to this pattern is eastern equine encephalitis virus, which has an alternate direct route of transmission among nonnative birds, and some mortality of native bird species occurs. The strain of WNV circulating in the northeastern United States is unique in that it causes significant mortality in exotic and native bird species, especially in the American crow (Corvus brachyrhynchos). Because of the lack of information on the susceptibility and pathogenesis of WNV for this species, experimental studies were conducted at the USGS National Wildlife Health Center. In two separate studies, crows were inoculated with a 1999 New York strain of WNV, and all experimentally infected crows died. In one of the studies, control crows in regular contact with experimentally inoculated crows in the same room but not inoculated with WNV succumbed to infection. The direct transmission between crows was most likely by the oral route. Inoculated crows were viremic before death, and high titers of virus were isolated from a variety of tissues. The significance of the experimental direct transmission among captive crows is unknown.
Tumour necrosis factor (TNF)-alpha-stimulated prostaglandin (PG) E(2)biosynthesis by amnion-derived AV3 cells is accompanied by increased prostaglandin H synthase (PGHS)-2 mRNA expression. PGHS-1 mRNA expression is unchanged. PGHS-2 promoter-reporter constructs (-891/+9 and 5' deletions thereof) were prepared. The regions containing concensus nuclear factor kappaB (NF-kappaB) elements (-447/-438 and -222/-213) did not enhance promoter activity. Elements associated with both basal and TNF-alpha-stimulated expression lie between bases -52 and -203. Site-directed mutagenesis of nuclear factor of interleukin-6 (NF-IL6) and cyclic AMP response elements (CREs) in this region reduced both basal and induced transcriptional activity of the -203/+9 construct by over 95 per cent. Electrophoretic mobility-shift assays using oligonucleotides derived from these sites demonstrated formation of specific DNA-protein complexes. Both NF-IL6 and CRE unlabelled oligonucleotides inhibited complex formation with the NF-IL6 oligonucleotide probe. Unlabelled CRE oligonucleotide also effectively inhibited formation of the complex with the CRE probe, but reduced effectiveness was observed when the NF-IL6 oligonucleotide was the competitor. Finally, unlabelled, consensus NF-kappaB oligonucleotide failed to compete for either probe. TNF-alpha treatment did not increase levels of these complexes. Thus NF-kappaB does not enhance basal or TNF-alpha-responsive PGHS-2 transcription in amnion-derived AV-3 cells. A permissive role for NF-IL6/CRE binding proteins in regulating PGHS-2 expression in these cells is indicated, but requires further clarification.
Prostaglandin H synthase (PGHS)-2 promoter fragments (-528 to +9 bp and 5' unidirectional deletions thereof) were cloned upstream of the chloramphenicol acetyl-transferase (CAT) reporter gene. These were transfected into amnion-derived AV3 cells. The region, -528 to -203, which includes NF-kappa B sites, had little influence on CAT expression. The region, -203 and -52, however, was responsible for most of the basal promoter activity and also conferred responsiveness to interleukin (IL)-1 beta (>3-times basal). Point mutations of NF-IL6 and cAMP response element (CRE) in this region reduced both basal and IL-1 beta-stimulated production of CAT; dual mutation eliminated IL-1 beta responsiveness. Factors in nuclear extracts from control or IL-1 beta-stimulated AV3 cells specifically complexed the NF-IL6 and CRE sequences. However, the NF-IL6 and CRE oligonucleotides cross-competed, suggesting a common factor. C/EBP beta was identified by supershift assay as interacting with both sequences. To a lesser extent C/EBP alpha and delta also interacted with the NF-IL6 site. However, CRE binding protein (CREB), was absent from the complex with the CRE. In conclusion, NF-IL6 and CRE elements principally account in AV3 amnion cells for basal and IL-1 beta-inducible transcriptional activity of the proximal 528 bp of the PGHS-2 promoter, while NF-kappa B elements play no substantial role. C/EBPs, particularly C/EBPbeta, are implicated in control of PGHS-2 transcription through the NF-IL6 and CRE sites.
Preterm labor is frequently associated with ascending intrauterine infection, accompanied by leukocytes infiltration and enhanced local production of cytokines and other inflammatory mediators. The resulting amplification of the inflammatory response, and of prostanoid production in particular, is postulated to be a principal mechanism of infection-driven preterm labor. In this review the effects of pro- and anti-inflammatory cytokines are discussed with respect to the expression of enzymes involved in three key steps of prostanoid biosynthesis and metabolism: liberation of arachidonic acid (AA), conversion of AA to bioactive prostanoids, and prostanoid catabolism. We suggest that by exerting coordinate actions on all three key steps, through multiple molecular mechanisms, inflammatory cytokines acutely up-regulate prostanoid production in intrauterine tissues.
The metabolism of arachidonic acid results in the production of prostaglandins (PGs), which are involved in the initiation of labour at term and preterm. The fetal membranes are a source of pro-inflammatory cytokines which promote increased PG biosynthesis via increased release of arachidonic acid and its conversion to biologically active metabolites such as PGE2 and PGF2alpha. In the amnion, the liberation of arachidonic acid from membrane glycerophospholipid stores can be catalysed by cytosolic phospholipase A2 (cPLA2). In amnion-derived WISH cells, the addition of tumour-necrosis factor alpha (TNF-alpha) (50 ng/ml) provoked a time-dependent increase in the expression of the cPLA2 mRNA which was greatest at 8 and 16 h post-treatment (3.62+/-0.52 and 3.15+/-0.45-fold of control, n=3). The increase in cPLA2 mRNA expression by TNF-alpha was unaffected by the prior addition of interleukin-4 (IL-4) (10 ng/ml), a known inhibitor of prostaglandin endoperoxide H synthase (PGHS)-2 mRNA and protein expression in WISH cells. TNF-alpha also increased the level of immunoreactive cPLA2 protein in a time-dependent manner with the highest levels evident after 8 and 16 h. As with the mRNA, cPLA2 protein levels were unaffected by pre-incubation with IL-4. The inclusion of the cPLA2-specific inhibitor arachidonyl trifluoromethyl ketone (AACOCF3) resulted in a concentration-dependent inhibition of PGE2 biosynthesis in WISH cells treated with TNF-alpha (>95 per cent at 2 microM). We conclude that TNF-alpha increases the abundance of the cPLA2 mRNA and protein in amnion epithelial cells, an effect which plays an important role in amnion PG biosynthesis in the presence of intrauterine infection.
Cytokines and growth factors have been proposed to act as in vivo modulators of amnion prostaglandin production at parturition. To characterize the effects of the 'anti-inflammatory' cytokine interleukin (IL)-4 on amnion prostaglandin production, amnion epithelium-derived WISH cells were treated with IL-4 in the presence/absence of IL-1beta, tumour necrosis factor-alpha (TNF-alpha) or epidermal growth factor (EGF). IL-4 (0.08-10 ng/ml) potently inhibited cytokine-stimulated PGE2 production over 16 h (maximal inhibition approximately 66% at 2.0 ng/ml IL-4). Delaying addition of IL-4 (1 ng/ml) by up to 8 h after IL-1beta addition only slightly attenuated its inhibitory effects, from approximately 65% to approximately 50%. EGF-stimulated PGE2 production was either not inhibited or slightly stimulated by IL-4. Immunoblotting studies revealed that IL-4 (10 ng/ml) significantly suppressed prostaglandin-H synthase-2 (PGHS-2) levels in cells stimulated with IL-1beta and TNF-alpha over 16 h, but had no consistent effects on cytosolic phospholipase A2 (cPLA2) levels under any condition. In the presence of arachidonic acid (10 microM), IL-4 again inhibited cytokine-stimulated, but not EGF-stimulated, PGE2 production. The presence of IL-4 also failed to alter the amount of arachidonic acid released in response to EGF. These findings suggest a role and potential therapeutic application for IL-4 in inhibiting amnion PGHS-2 expression and hence prostaglandin production in infection-driven preterm labour, but not labour in the absence of inflammatory initiators.
We have isolated the prostaglandin H synthase-1 (PGHS-1) promoter from the human amnion cell line WISH by long template PCR. The fragment was 1124 base pairs in length and shared a 96% sequence identity with the sequence in GenBank. The putative transcription start site is located 18 bp upstream of the start codon. The sequence is TATA-less, but contains multiple Sp-1 sites and a GC box at -132. The fragment was subcloned into the promoterless reporter construct pBLCAT3 to produce the promoter reporter construct pPGHS1CAT. pPGHS1CAT expression in amnion-derived AV3 cells was inhibited by the pro-inflammatory cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin 1-beta (IL-1beta). PGHS-1 mRNA levels however, were unchanged over a 16-h time course with either treatment. These results suggest that PGHS-1 transcription is regulated in a negative manner by cytokines in human amnion-derived cells.
Increased production of prostaglandins and cytokines by amnion, particularly prostaglandin (PG) E2, interleukin (IL)-6 and IL-8, is thought to be an important event in infection-associated preterm labour. We characterized the amnion-derived AV3 cell line to determine its appropriateness as a model for investigation of the regulation of amnion cytokine and PG production. Amnion-derived AV3 cells were treated with tumour necrosis factor-alpha (TNF-alpha, interleukin-1beta (IL-1beta), epidermal growth factor (EGF) and phorbol 12-myristate 13-acetate (PMA) and IL-6, IL-8 and prostaglandin production was determined by immunoassay. Production of IL-6 and IL-8 rose dramatically with all treatments. PGE2, but not PGF2alpha or 6-keto-PGF1alpha, biosynthesis was also increased in a concentration-dependent manner with all treatments. A rapid increase in PGHS-2 (but not PGHS-1) mRNA expression was observed in response to TNF-alpha and IL-1beta. We conclude that the AV3 cell line inflammatory response profile is similar to those observed in primary amnion and other amnion-derived cell lines, and is an appropriate model for human amnion.
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We have examined the expression of the intercellular adhesion molecule-1 (ICAM-1) mRNA in primary and established amnion-derived cell cultures and regulation of this expression by tumour necrosis factor-alpha (TNF-alpha) and interleukin (IL)-1beta. TNF-alpha (50 ng/ml) and IL-1beta (1.0 ng/ml) induced 18- and 11-fold increases respectively in expression of the ICAM-1 mRNA in WISH cells (an amnion epithelium-derived cell line). The increase was detectable within one hour of treatment and peaked by two hours. The protein synthesis inhibitor, cycloheximide (10 microg/ml) did not inhibit this induction. Increased levels of ICAM-1 protein were detected in the cells within 4 h after initiation of treatment with either cytokine. By 16 h of treatment with IL-1beta or TNF-alpha ICAM-1 reached 40 and 73 pg/microg cellular protein, representing 6- and 11-fold stimulations respectively. In primary amnion cells, basal expression of ICAM-1 mRNA was undetectable. However, TNF-alpha (50 ng/ml) induced ICAM-1 mRNA within two hours, peak expression being reached between four and eight hours after initiation of treatment. The present report demonstrates for the first time that amnion derived cells can express ICAM-1 and, further, that this expression is regulated by pro-inflammatory cytokines. This has implications for the amnion as a possible source for soluble ICAM-1, for this gene product as a marker for preterm labour, and for participation of the amnion, additional to its reported secretory role, in inflammatory processes of the fetal membranes.
We have evaluated the mechanism by which tumour necrosis factor-alpha (TNF-alpha) induces increased prostaglandin (PG) biosynthesis in amnion-derived WISH cells. WISH cells were treated with 50 ng/ml TNF-alpha or vehicle for 0-24 h. PGE2 production was stimulated by TNF-alpha within 2 h and continued to accumulate for at least 24 h. Increased prostaglandin endoperoxide H synthase (PGHS)-2 mRNA expression was evident within 30 min and was highest by 1 h, returning to unstimulated levels by 2 h. The PGHS-2 mRNA was re-induced at 8 h and was also elevated at 16 h. Immunoreactive PGHS-2 protein was nearly undetectable in control cells. However, within 30 min of TNF-alpha treatment, PGHS-2 protein was elevated and was induced for at least 16 h suggesting rapid production of both the PGHS-2 mRNA and protein. Transcription run-on assays indicated that the initial increase in the PGHS-2 mRNA was due to a 20-fold increase in the rate of transcription. The PGHS-2 mRNA decayed with an apparent half-life of 1 h in TNF-alpha-stimulated WISH cells. Induction of PGHS-2 expression proceeded in the presence of 10 microg/ml cycloheximide which agrees with the classification of PGHS-2 as an immediate early gene. These results indicate that a bi-phasic induction of the PGHS-2 mRNA is due, in part, to an initial transcriptional activation which results in rapid and continued synthesis of the PGHS-2 protein. This may be a unique characteristic of amnion cells which may be partially responsible for increased PG concentrations in the amniotic fluid during infection-associated preterm labour.
Increased prostaglandin biosynthesis during intrauterine infection may be a possible mechanism by which preterm labour is initiated. Inflammatory cytokines and growth factors are known to stimulate prostaglandin production through an increase in prostaglandin endoperoxide H synthase (PGHS)-2 synthesis and activity. Interleukin-4 (IL-4), an anti-inflammatory cytokine, can downregulate PGHS-2 expression and inhibit prostaglandin production. Therefore, the aims of the current study were to determine the effects of IL-4 on PGHS-1 and PGHS-2 expression in amion-derived WISH cells treated with inflammatory cytokines and growth factors. In WISH cells, near-maximal production of the PGHS-2 mRNA occurred using 5 ng/ml EGF, 1 ng/ml IL-1beta or 50 ng/ml TNF-alpha. Time-course experiments determined that the PGHS-2 mRNA was induced maximally by these stimuli by 1 h. Pretreatment of WISH cells with IL-4 reduced PGHS-2 mRNA levels at 1 h by 67% in cells treated with EGF, 62% in cells treated with IL-1beta and 54% in cells treated with TNF-alpha. Pretreatment with IL-4 more effectively inhibited PGHS-2 expression than simultaneous addition with EGF or IL-1beta but not TNF-alpha. Immunoblot analysis showed a correlation between inhibition of mRNA levels and levels of PGHS-2 protein, although stimulation of PGHS-2 protein production by EGF was undetectable. Levels of PGHS-1 protein and mRNA remained unchanged in all experiments. Increased production of prostaglandin E2 (PGE2) in response to TNF-alpha and IL-1beta treatment was attenuated by IL-4 pretreatment, by 52% and 72%, respectively. No attenuation of EGF-stimulated PGE2 levels was seen. We conclude that IL-4 inhibits PGHS-2 mRNA and protein production in cytokine-stimulated WISH cells, but does not affect EGF-stimulated PGE2 production, suggesting that EGF can induce prostaglandin biosynthesis by a mechanism other than through increased PGHS-2 expression.
Various segments of the 3'-nontranslated region of the renal glutaminase (GA) mRNA were tested for their ability to enhance turnover and pH responsiveness. The combined effects were retained in the 340-base R-2 segment. However, the combined R-1 and R-3 fragments also imparted a partial destabilization and pH responsiveness to a chimeric beta-globin mRNA. RNA electrophoretic mobility shift assays indicated that cytosolic extracts of rat renal cortex contain a protein that binds to the R-2 and R-3 RNAs. The binding observed with the R-2 RNA was mapped to a direct repeat of an 8-base AU sequence. This binding was effectively competed with an excess of the same RNA, but not by adjacent or unrelated RNAs. UV cross-linking experiments identified a 48-kDa protein that binds to the AU repeats of the R-2 RNA. The apparent binding of this protein was greatly reduced in renal cytosolic extracts prepared from acutely acidotic rats. Two related RNA sequences in the R-3 segment also exhibited specific binding. However, the latter binding was more effectively competed by R-2 RNA than by itself, indicating that the homologous sites may be weaker binding sites for the same 48-kDa protein. Thus, a single protein may bind specifically to multiple instability elements within the 3'-nontranslated region of the GA mRNA and mediate its pH-responsive stabilization.
Rat kidney expresses two forms of glutaminase (GA) mRNA which probably result from the use of alternative polyadenylation signals. The two mRNAs are increased coordinately in response to metabolic acidosis via a mechanism that apparently does not involve transcriptional or translational regulation. A 956-bp fragment that contains the 3'-nontranslated sequence of the smaller GA cDNA was cloned into an expression vector (p beta G) that encodes a chimeric beta-globin growth hormone mRNA. Both the parent and the derived construct (p beta G-GA) were transfected into LLC-PK1-F+ cells. Stable transfectants express sixfold lower levels of beta G-GA mRNA than that of the parent beta G mRNA. However, only the beta G-GA mRNA is increased 2.5-fold by growth in acidic medium (pH 6.9, 10 mM HCO3-). The apparent half-life of the beta G mRNA (> 24 h) is unaffected by the pH of the growth media. In contrast, the apparent half-life of the beta G-GA mRNA is increased from 4.5 h to approximately 24 h when cells are transferred to acidic medium for 8 h. The observed pH response is not reproduced when the beta G-GA construct is stably transfected into COS-7 cells or when a beta-globin-phosphoenolpyruvate carboxykinase chimeric gene is expressed in LLC-PK1-F+ cells. Thus the 3'-nontranslated region of the GA mRNA contains a pH-responsive stability element.
LLC-PK1-F+ cells are porcine proximal tubule-like cells that have been used to model the renal ammoniagenic response to metabolic acidosis. A 3.2-kb porcine glutaminase (GA) cDNA (pGA201) containing 528 bp of coding sequence and 2.7 kb of 3'-untranslated region was cloned and sequenced. Probes derived from both porcine and rat GA cDNAs were used to characterize the expression of putative GA mRNAs in LLC-PK1-F+ cells. Two larger putative GA mRNAs (approximately 5.0 and 4.5 kb in length) were resolved and a smaller 2.5-kb species was also observed. The level of the 5.0-kb mRNA is detectable in freshly split LLC-PK1-F+ cells and increases as the cells reach confluence. In contrast, the amount of the 4.5-kb GA mRNA is greatest in freshly split cells and decreases gradually as the cells approach confluence. The levels of the 5.0- and 2.5-kb mRNAs are also affected by refeeding the cells, and the 2.5-kb mRNA accumulates to high levels if cells are retained in the same media for 4 days. Exposure to acidic media had little or no effect on the levels of GA mRNAs expressed in confluent or postconfluent cells, whereas, in growing and undifferentiated cells, this treatment did affect the level of the 4.5-kb mRNA. Thus the putative GA mRNA species are differentially expressed. Given this complexity, a careful assessment of GA mRNA species, of basal expression, and of growth conditions are essential for a meaningful analysis of GA mRNA levels in cultured cells.