Comparison of LPS-stimulated release of cytokines in punch versus transwell tissue culture systems of human gestational membranes

Abstract Background Cytokine signaling within the amnionic, chorionic and decidual extraplacental gestational membranes plays an important role in membrane rupture and the timing of birth. The predominant in vitro explant culture system for evaluating cytokine induction in human gestational membranes has been the free-floating biopsy punch culture. Punch systems have been used to investigate the impact of various toxicants, pharmaceuticals and genetic variation on expression of pro-inflammatory cytokines. More recently, a dual compartment transwell culture system has been developed that more closely mimics the intrauterine compartment. The current study compares these two systems with respect to release of pro- and anti-inflammatory cytokines in response to lipopolysaccharide (LPS), a model stimulant. Methods Tissue samples were exposed to 100 ng/ml LPS for 12 h and cytokines were measured by ELISA. Data are expressed as increase relative to non-treated controls. Results Levels of interleukin-6 increased in punch culture medium samples to a significantly greater extent (34.2 fold) compared with medium from transwell cultures in the amnion (6.6 fold) or choriodecidual (7.1 fold) compartments. Interleukin-8 also showed a significantly greater induction in punch (4.8 fold) than transwell amnion (1.6 fold) or choriodecidual (1.7 fold) samples. The anti-inflammatory interleukin-10 showed a significant difference between punch (36.5 fold) and transwell amnion (15.4 fold) samples, but no difference was observed between punch and transwell choriodecidual (28.5 fold) samples. Neither interleukin-1beta nor tumor necrosis factor-alpha (TNF-alpha) showed a significant difference between the punch and transwell samples. Conclusions These results indicate that the pattern of LPS-stimulated cytokine release from gestational membranes in vitro depends on the culture system used, confounding comparisons of studies that use different gestational membrane culture systems to study inflammatory responses.http://deepblue.lib.umich.edu/bitstream/2027.42/78253/1/1477-7827-8-121.xmlhttp://deepblue.lib.umich.edu/bitstream/2027.42/78253/2/1477-7827-8-121.pdfPeer Reviewe

\u3cem\u3eRoberts v. Johnson\u3c/em\u3e—A Welcome Change Tainted by an Outmoded Approach to Statutory Interpretation

In 1974, the Washington State Legislature repealed its automobile guest statute, intending to establish ordinary negligence as the proper standard of liability in host-guest automobile tort actions. Nevertheless, in March 1978, in Lau v. Nelson, the Washington Supreme Court, ignoring clear indicia of legislative intent, held that the repeal of the guest statute revived the common law of this state, which, like the guest statute, predicated a guest\u27s recovery on proving the host grossly negligent. Having effectively reinstated the very law the legislature repealed, the Lau court declined to decide whether the majority rule of ordinary negligence should replace Washington\u27s common law rule of gross negligence. In December 1978, in Roberts v. Johnson, the court resolved that issue by overruling Lau and abandoning gross negligence in favor of ordinary negligence as the applicable standard of liability. This laudatory and long overdue result was tainted, however, by the court\u27s specific reaffirmation of Lau\u27s holding that the repeal of a statute reinstates the common law regardless of the legislative intent behind the repeal. The court\u27s failure to ascertain and implement the legislative intent is the unfortunate manifestation of a rigid, insensitive, and rule-oriented approach to statutory interpretation

Historical Reconstruction of Population Density of the Echinoid Diadema antillarum on Florida Keys Shallow Bank-Barrier Reefs

The 1983–1984 caribbean-wide mass mortality of the once ubiquitous long-spined sea urchin Diadema antillarum Philippi, 1845, is one of several factors considered responsible for coral reef change throughout the region. Unfortunately, there is a paucity of pre-mortality event density data for D. antillarum in the Florida Keys, making it difficult to determine pre-1983 population density levels. results from surveys conducted during 1970–1973 in the lower Florida Keys, in shallow (\u3c12 m) fore reef habitats, yielded relatively abundant and widespread D. antillarum densities in qualitative transects at five reefs prior to the 1983–1984 die-off. In quantitative surveys at one reef, Middle sambo reef in 1972, up to 7.9 individuals m–2 were recorded using quadrats in high-relief spur and groove habitat. A second mortality event in the Florida Keys, beginning in April 1991, again depressed urchin densities that had begun to recover from the 1983–1984 mass mortality. By 1992, D. antillarum densities (\u3c0.01 m–2) were two orders of magnitude lower than pre-die-off estimates (range of 0.07–0.57 m–2 from several spur and groove reefs in the lower Florida Keys) and remained so through 2009. The pre-mortality echinoid density estimates detailed in the Florida Keys provide a baseline to compare with their current population status and should help inform managers about realistic recovery or restoration targets for D. antillarum

Accelerated return to sport after osteochondral autograft plug transfer

Background:Previous studies have reported varying return-to-sport protocols after knee cartilage restoration procedures.Purpose:To (1) evaluate the time for return to sport in athletes with an isolated chondral injury who underwent an accelerated return-to-sport protocol after osteochondral autograft plug transfer (OAT) and (2) evaluate clinical outcomes to assess for any consequences from the accelerated return to sport.Study Design:Case series; Level of evidence, 4.Methods:An institutional cohort of 152 OAT procedures was reviewed, of which 20 competitive athletes met inclusion and exclusion criteria. All patients underwent a physician-directed accelerated rehabilitation program after their procedure. Return to sport was determined for all athletes. Clinical outcomes were assessed using International Knee Documentation Committee (IKDC) and Tegner scores as well as assessment of level of participation on return to sport.Results:Return-to-sport data were available for all 20 athletes; 13 of 20 athletes (65%) were available for clinical evaluation at a mean 4.4-year follow-up. The mean time for return to sport for all 20 athletes was 82.9 ± 25 days (range, 38-134 days). All athletes were able to return to sport at their previous level and reported that they were satisfied or very satisfied with their surgical outcome and ability to return to sport. The mean postoperative IKDC score was 84.5 ± 9.5. The mean Tegner score prior to injury was 8.9 ± 1.7; it was 7.7 ± 1.9 at final follow-up.Conclusion:Competitive athletes with traumatic chondral defects treated with OAT managed using this protocol had reduced time to preinjury activity levels compared with what is currently reported, with excellent clinical outcomes and no serious long-term sequelae.</jats:sec

Florida Keys Population Abundance Estimates for Nine Coral Species Proposed for Listing Under the U.S. Endangered Species Act

This report presents abundance and size-class distribution estimates for nine coral species in the Florida Keys and Dry Tortugas, all of which are proposed for listing or reclassification under the U.S. Endangered Species Act (ESA). The lack of population data for these species was highlighted as a deficiency in the ESA Review Process by the Biological Review Team (BRT) in their Status Review (Brainard et al. 2011) and also by the National Oceanic and Atmospheric Administration (NOAA) (Federal Register 2012). Field sampling protocols were adapted from Aronson et al. (1994) and the Atlantic and Gulf Rapid Reef Assessment program (Kramer and Lang 2003) to measure population-level metrics of scleractinian corals, with population data analyses following Smith et al. (2011). The data in this report are based on focused surveys for Acropora corals in the Florida Keys during 2005, 2007, and 2012; and for all scleractinian coral species during 2005, 2009, and 2012; and for all coral species in the Dry Tortugas for 2006 and 2008. Colony density within belt transects and size measurements were obtained for each species present. Statistical estimation procedures for population abundance metrics – means (e.g. coral density) and totals (e.g. coral abundance) – for a two-stage stratified random sampling design were adapted from Cochran (1977), and computations were carried out using SAS statistical software. Domain-wide estimates are presented in this report

The Information Content of Pore Fluid δ^(18)O and [CI^-]

Paleoceanographic proxies indicate that the ocean state during the Last Glacial Maximum (LGM) differed from the modern ocean state. Depth profiles of ocean sediment pore fluid δ^(18)O and [Cl^−] have been used to reconstruct the δ^(18)O and salinity at the LGM. Here, it is investigated whether pore fluid profiles can constrain ocean δ^(18)O and salinity at other times and, simultaneously, their ability to constrain the LGM δ^(18)O and salinity. An inverse framework is developed that relies on Bayesian parameter estimation, thus allowing formal separation of prior assumptions from the information in observations. Synthetic problems are used to explore the information about past ocean tracers that can be recovered from pore fluid profiles. It is concluded that prior knowledge of deep ocean mixing time scales is essential to an accurate inverse estimate of LGM ocean salinity and δ^(18)O from modern pore fluid profiles. The most recent 10 000 years of ocean salinity and δ^(18)O and the error in their estimates are better constrained by the pore fluid profiles than are the LGM values. The inverse estimate of salinity and δ^(18)O is strongly correlated with the estimate of diffusivity of oxygen isotopes and [Cl^−] in sediment pore fluids. Uncertainty on the diffusivity of oxygen isotopes and chloride in sediments is reduced through inversion of the pore fluid profiles, but simultaneous estimation of δ^(18)O or salinity and diffusivity increases the total uncertainty. The error reported in previous work may underestimate the true uncertainty of LGM deep ocean salinity and δ^(18)O

The Human Gestational Membranes as a Site of Polybrominated Diphenyl Ether Toxicity.

Preterm birth is a major public health concern impacting one in eight babies born in the U.S.A. Although a precise cause for preterm birth cannot be established in most cases, one of the most promising predictors of preterm birth is inflammation of the extra-placental gestational membranes. Gestational membrane production of pro-inflammatory cytokines has been shown to initiate term parturition pathways and is thought to play a role in preterm parturition as well. Pregnant woman are exposed to a wide array of environmental pollutants, but few have been investigated epidemiologically for association with preterm birth and none have been satisfactorily investigated mechanistically. Polybrominated diphenyl ethers (PBDEs) are emerging toxicants of concern that have received limited attention regarding potential risks to human health and birth outcomes. Preliminary research suggests that PBDEs may stimulate the gestational membranes to secrete pro-inflammatory cytokines in vitro. This dissertation tests the hypothesis that polybrominated diphenyl ethers bioaccumulate in the human gestational compartment and stimulate cytokine secretion from human gestational membranes. Total PBDE levels were measured in the human gestational membranes and found at levels of 17.4±3.9 pg/g tissue (5.6±1.3 ng/g lipid). Human amniotic fluid levels were 3795±1592 pg/ml fluid (404±126 ng/g lipid). Congener-specific profiles for the 21 congeners measured identified only tri- through hexa-BDEs in gestational membranes. In contrast, tri- through deca-BDE congeners were found in amniotic fluid. To assess stimulation of inflammatory responses in human gestational membranes, two tissue culture systems were compared for cytokine release into the culture medium: a biopsy punch explant culture system and a transwell mounted explant culture system. Although lipopolysacharide stimulated a robust increase of interleukins-1β, 6, 8, 10 and tumor necrosis factor-α in both systems, with an amplified release of interleukins-6, 8 and 10 in the punch culture system, no increase in cytokine release was observed in response to PBDE stimulation. This is the first report of PBDE accumulation in the human gestational membranes, providing a basis for future investigations of toxic action in this tissue and the first to suggest amniotic fluid as a significant route of PBDE exposure for both the gestational membranes and the developing fetus.Ph.D.ToxicologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/64669/1/markmil_1.pd

Nonclassical Degrees of Freedom in the Riemann Hamiltonian

The Hilbert-Polya conjecture states that the imaginary parts of the zeros of the Riemann zeta function are eigenvalues of a quantum hamiltonian. If so, conjectures by Katz and Sarnak put this hamiltonian in Altland and Zirnbauer's universality class C. This implies that the system must have a nonclassical two-valued degree of freedom. In such a system, the dominant primitive periodic orbits contribute to the density of states with a phase factor of -1. This resolves a previously mysterious sign problem with the oscillatory contributions to the density of the Riemann zeros.Comment: 4 pages, no figures; v3-6 have minor corrections to v2, v2 has a more complete solution of the sign problem than v

Oral dosing for antenatal corticosteroids in the Rhesus macaque.

Antenatal corticosteroids (ACS) are standard of care for women at risk of preterm delivery, although choice of drug, dose or route have not been systematically evaluated. Further, ACS are infrequently used in low resource environments where most of the mortality from prematurity occurs. We report proof of principle experiments to test betamethasone-phosphate (Beta-P) or dexamethasone-phosphate (Dex-P) given orally in comparison to the clinical treatment with the intramuscular combination drug beta-phosphate plus beta-acetate in a Rhesus Macaque model. First, we performed pharmacokinetic studies in non-pregnant monkeys to compare blood levels of the steroids using oral dosing with Beta-P, Dex-P and an effective maternal intramuscular dose of the beta-acetate component of the clinical treatment. We then evaluated maternal and fetal blood steroid levels with limited fetal sampling under ultrasound guidance in pregnant macaques. We found that oral Beta is more slowly cleared from plasma than oral Dex. The blood levels of both drugs were lower in maternal plasma of pregnant than in non-pregnant macaques. Using the pharmacokinetic data, we treated groups of 6-8 pregnant monkeys with oral Beta-P, oral Dex-P, or the maternal intramuscular clinical treatment and saline controls and measured pressure-volume curves to assess corticosteroid effects on lung maturation at 5d. Oral Beta-P improved the pressure-volume curves similarly to the clinical treatment. Oral Dex-P gave more variable and nonsignificant responses. We then compared gene expression in the fetal lung, liver and hippocampus between oral Beta-P and the clinical treatment by RNA-sequencing. The transcriptomes were largely similar with small gene expression differences in the lung and liver, and no differences in the hippocampus between the groups. As proof of principle, ACS therapy can be effective using inexpensive and widely available oral drugs. Clinical dosing strategies must carefully consider the pharmacokinetics of oral Beta-P or Dex-P to minimize fetal exposure while achieving the desired treatment responses

A new approach to generating research-quality data through citizen science: The USA National Phenology Monitoring System

Phenology is one of the most sensitive biological responses to climate change, and recent changes in phenology have the potential to shake up ecosystems. In some cases, it appears they already are. Thus, for ecological reasons it is critical that we improve our understanding of species&#x2019; phenologies and how these phenologies are responding to recent, rapid climate change. Phenological events like flowering and bird migrations are easy to observe, culturally important, and, at a fundamental level, naturally inspire human curiosity&#x2014; thus providing an excellent opportunity to engage citizen scientists. The USA National Phenology Network has recently initiated a national effort to encourage people at different levels of expertise&#x2014;from backyard naturalists to professional scientists&#x2014;to observe phenological events and contribute to a national database that will be used to greatly improve our understanding of spatio-temporal variation in phenology and associated phenological responses to climate change.&#xd;&#xa;&#xd;&#xa;Traditional phenological observation protocols identify specific dates at which individual phenological events are observed. The scientific usefulness of long-term phenological observations could be improved with a more carefully structured protocol. At the USA-NPN we have developed a new approach that directs observers to record each day that they observe an individual plant, and to assess and report the state of specific life stages (or phenophases) as occurring or not occurring on that plant for each observation date. Evaluation is phrased in terms of simple, easy-to-understand, questions (e.g. &#x201c;Do you see open flowers?&#x201d;), which makes it very appropriate for a citizen science audience. From this method, a rich dataset of phenological metrics can be extracted, including the duration of a phenophase (e.g. open flowers), the beginning and end points of a phenophase (e.g. traditional phenological events such as first flower and last flower), multiple distinct occurrences of phenophases within a single growing season (e.g multiple flowering events, common in drought-prone regions), as well as quantification of sampling frequency and observational uncertainties. These features greatly enhance the utility of the resulting data for statistical analyses addressing questions such as how phenological events vary in time and space, and in response to global change. This new protocol is an important step forward, and its widespread adoption will increase the scientific value of data collected by citizen scientists.&#xd;&#xa