Primary Human Lung Alveolus-on-a-chip Model of Intravascular Thrombosis for Assessment of Therapeutics.

Pulmonary thrombosis is a significant cause of patient mortality; however, there are no effective in vitro models of thrombi formation in human lung microvessels that could also assess therapeutics and toxicology of antithrombotic drugs. Here, we show that a microfluidic lung alveolus-on-a-chip lined by human primary alveolar epithelium interfaced with endothelium and cultured under flowing whole blood can be used to perform quantitative analysis of organ-level contributions to inflammation-induced thrombosis. This microfluidic chip recapitulates in vivo responses, including platelet-endothelial dynamics and revealed that lipopolysaccharide (LPS) endotoxin indirectly stimulates intravascular thrombosis by activating the alveolar epithelium, rather than acting directly on endothelium. This model is also used to analyze inhibition of endothelial activation and thrombosis due to a protease activated receptor-1 (PAR-1) antagonist, demonstrating its ability to dissect complex responses and identify antithrombotic therapeutics. Thus, this methodology offers a new approach to study human pathophysiology of pulmonary thrombosis and advance drug development.

Concerning the mechanism for transfer of D-glucuronate from myo-inositol oxygenase to D-glucuronate reductase.

The D-glucuronate product of myo-inositol oxygenase (EC 1.13.99.1) is efficiently reduced by NADPH in the presence of either purified D-glucuronate reductase (EC 1.1.1.19), or reductase that is part of a protein aggregate that also contains the oxygenase. This occurs despite the fact that the maximum concentration of D-glucuronate that could be formed by the oxygenase under the conditions used for the coupled enzyme experiments is 7 microM, and 10 microM externally supplied D-glucuronate (Km = 7.6 mM) does not support any detectable NADPH oxidation under the reaction conditions. The most likely explanation for the results is that the uncyclized aldehyde form of D-glucuronate is the product of the oxygenase reaction, and that it diffuses into solution and is captured by the reductase before it cyclizes to the more stable but less reactive hemiacetal form.

The pregnane X receptor: a promiscuous xenobiotic receptor that has diverged during evolution.

Transcription of genes encoding cytochrome P450 3A (CYP3A) monooxygenases is induced by a variety of xenobiotics and natural steroids. There are marked differences in the compounds that induce CYP3A gene expression between species. Recently, the mouse and human pregnane X receptor (PXR) were shown to be activated by compounds that induce CYP3A expression. However, most studies of CYP3A regulation have been performed using rabbit and rat hepatocytes. Here, we report the cloning and characterization of PXR from these two species. PXR is remarkably divergent between species, with the rabbit, rat, and human receptors sharing only approximately 80% amino acid identity in their ligand-binding domains. This sequence divergence is reflected by marked pharmacological differences in PXR activation profiles. For example, the macrolide antibiotic rifampicin, the antidiabetic drug troglitazone, and the hypocholesterolemic drug SR12813 are efficacious activators of the human and rabbit PXR but have little activity on the rat and mouse PXR. Conversely, pregnane 16alpha-carbonitrile is a more potent activator of the rat and mouse PXR than the human and rabbit receptor. The activities of xenobiotics in PXR activation assays correlate well with their ability to induce CYP3A expression in primary hepatocytes. Through the use of a novel scintillation proximity binding assay, we demonstrate that many of the compounds that induce CYP3A expression bind directly to human PXR. These data establish PXR as a promiscuous xenobiotic receptor that has diverged during evolution.

The use of liver spheroids as an in vitro model for studying induction of the stress response as a marker of chemical toxicity.

Stress protein induction has been advocated as a sensitive indicator of compound-induced toxicity. In monolayer cultures of primary hepatocytes, however, the two stress proteins, Hsp25 and Hsp72/3 are up-regulated, probably due to the effect of the isolation procedure and adaptation of the cells to the culture conditions. The aim of the current studies was to determine whether liver spheroids would provide an improved experimental model for the study of heat shock protein induction in vitro. Primary rat hepatocytes were cultured as liver spheroids and the expression of Hsp25 and Hsp72/3 measured along with the levels of ATP, GSH and albumin secretion. Hsp72/3 was initially increased in spheroid culture but returned to in vivo levels after 3 days of culture. Hsp25 was maintained at in vivo levels until day 6 of culture, after which levels increased slightly. The effects of the two hepatotoxins, hydrazine and cadmium chloride (CdCl(2)), were therefore measured on day 6 of spheroid culture. CdCl(2) had no effect on Hsp25 but increased Hsp72/3 at concentrations that affected other biochemical parameters. Hydrazine caused a rapid reduction in ATP levels and albumin secretion, but did not affect Hsp72/3. Hsp25 was slightly induced by hydrazine at later sampling times at concentrations, however, that affected other biochemical parameters. It can be concluded that liver spheroids provide a model for studying stress protein expression. However, the increase in stress proteins appears to be a relatively insensitive parameter compared to other more conventionally used toxicity endpoints and the response appears to vary with individual toxins under study.

Effects of medium composition on the morphology and function of rat hepatocytes cultured as spheroids and monolayers.

Primary hepatocytes cultured as monolayers or as spheroids were studied to compare the effects of four different culture media (Williams' E, Chee's, Sigma Hepatocyte, and HepatoZYME medium). Rat hepatocytes were cultured as conventional monolayers for 3 d or as spheroids for 2 wk. For spheroid formation a method was emplOyed that combined the use of a nonadherent substratum with rotation of cultures. Hepatocyte integrity and morphology were assessed by light and electron microscopy and by reduced glutathione content. Hepatocyte function was measured by albumin secretion and 7-ethoxycoumarin metabolism. Chee's medium was found to be optimal for maintenance of hepatocyte viability and function in monolayers, but it failed to support spheroid formation. For spheroid formation and for the maintenance of spheroid morphology and function, Sigma HM was found to be optimal. These results demonstrate that the medium requirements of hepatocytes differ markedly depending on the culture model employed. Spheroid culture allowed better preservation of morphology and function of hepatocytes compared with conventional monolayer culture. Hepatocytes in spheroids formed bile canaliculi. and expressed an actin distribution resembling that found in hepatocytes in vivo. Albumin secretion was maintained at the same level as that found during the first d in primary culture, and 7-ethoxycoumarin metabolism was maintained over 2 wk in culture at approximately 30% of the levels found in freshly isolated hepatocytes. The improved morphology and function of hepatocyte cultures as spheroids may provide a more appropriate in vitro model for certain applications where the maintenance of liver-specific functions in long-term culture is crucial.

A comparison of the recovery period for women and men after an acute myocardial infarction.

OBJECTIVES: To compare return to work, participation in cardiac rehabilitation, and sexual activity in women and men recovering from acute myocardial infarction (AMI). DESIGN: A descriptive survey design was used. Descriptive statistics and chi square analysis were used to compare differences between women and men after an AMI. SETTING: The survey was mailed to the subject's home. SUBJECTS: A purposive sample of 20 women and 42 men. RESULTS: Comparing women with men, there were significant differences in the following activities with women evidencing higher percentages in responsibility for household duties before AMI, and cooking, washing dishes, reading, bed making, laundry, dusting and sweeping within 4 weeks after AMI. For those subjects who were sexually active before AMI, all resumed sexual activities after an average of 8 weeks. Women reported a decrease in frequency, less satisfactory relationship, and more reports of chest pain during sexual activity. Subjects reported that nurses gave little or no counseling concerning resumption of household activities, return to work issues, and sexual activity. Women received less counseling than men after AMI. CONCLUSIONS: The findings are not generalizable to the population at large; however, the study indicates a need to investigate further the recovery period for women who experience AMI.

Changes in health status and quality of life and the impact of uncertainty in patients who survive life-threatening arrhythmias.

OBJECTIVE: The purpose of this study was to describe the changes in perception of health status and quality of life from before treatment to 6 months after and the impact of uncertainty on these variables in survivors of life-threatening arrhythmia. DESIGN AND SETTING: A descriptive correlational design at a large urban teaching hospital. MEASURES: We measured health status, quality of life, and uncertainty before treatment and 6 months after a life-threatening arrhythmia. RESULTS: Survivors included 66 men and 15 women, 41 of whom received pharmacologic therapy and 36 of whom received an implantable cardioverter defibrillator (ICD), completed the Medical Outcomes Survey (SF-36), Ferrans and Powers Quality of Life Index (QLI), and the Mishel Uncertainty in Illness Scale (MUIS-C) before treatment and 6 months after. There were significant improvements in the mental and physical health composite summaries as measured by the SF36 (P <.01). Conversely, there were significant reductions in the overall score and specifically in socioeconomic and psychological/spiritual quality of life domains as measured by the QLI (P <.05). An increased perception of uncertainty was related to decreased perception of health status and quality of life at both measurement times, with higher correlations 6 months later. CONCLUSIONS: Survivors demonstrated improvements in perceived health status, although this did not appear to translate into improvements in the subjective domains of quality of life. The overall quality of life and the domains of psychological/spiritual state and socioeconomic status were lower 6 months after a life-threatening arrhythmia. Uncertainty had a significant impact on these perceptions, identifying an area for nursing interventions.

An overview of evaluation research methods with implications for nursing staff development.

This article presents an overview of evaluation research methods. A clear distinction is made between evaluation research and research methods, and evaluation research and quality assurance. Examples from nursing staff development are used to illustrate selected evaluation research methods.

The importance of systematic inquiry in nursing research: An example; women and recovery from acute myocardial infarction.

A review of the literature related to recovery from acute myocardial after discharge from the hospital revealed that very little is known about the recovery period in women. The recovery period has been studied in men with the major focus on cardiac rehabilitation, return to work, and sexual activity. In this descriptive exploratory study women were compared to men in these three areas of recovery within 3 to 9 months after discharge from the hospital. There were statistically significant differences in the following areas: marital status, more women were widowed or single; women had more responsibility for household duties after acute myocardial infarction, a decrease in frequency of sexual activity, and more reports of chest pain during sexual activity. Subjects reported little or no counselling by nurses with regard to cardiac rehabilitation, return to work or household duties, and resumption of sexual activity.

Two faces of nurse faculty: teacher and researcher.

The purpose of this paper is to examine three topics related to nurse educators and research productivity. The first section is an analysis and synthesis of what is known about nurse educator involvement in research activities from published studies. The second section is an examination of factors that may enhance or deter nurse educators from engaging in research activities. The focus of the last section is on the relationship between research and teaching quality. Empirical data are examined and recommendations made for nurse faculty.

Recovery from acute myocardial infarction in women.

This paper is a review of the literature on recovery from acute myocardial infarction in women. The topic has been subdivided into three areas for presentation: cardiac rehabilitation, return to work and sexual activity. The exploration of the literature revealed the paucity of research on women, but some comparisons could be made between men and women. Compared to men, women appear to utilize cardiac rehabilitation programs less frequently than men and have higher dropout rates, they return to work less frequently and after a longer period of time and resume sexual activity after a longer period of time reporting more symptoms during and after the activity. Investigation of the literature showed that the recovery period for women is incompletely explored and that there is a critical need for research.

Inhibition of the catalytic subunit of phosphorylase phosphatase by oxalyl thioesters and its possible relevance to the mechanism of insulin action.

Oxalyl thioesters, especially S-oxalylglutathione, are shown to be effective inhibitors of the catalytic subunit of phosphorylase phosphatase. The amount of inhibition was found to be time dependent and partially reversed by thiols, thus suggesting that at least part of the inhibition is due to oxalylation of an enzymic thiol group. The possibility that the inhibition of the phosphatase by oxalyl thioesters may be important in vivo and that oxalyl thioesters may be functioning as negative intracellular messengers for insulin is discussed.

Oxalyl thiolesters and N-oxalylcysteine are normal mammalian metabolites.

A method for the quantitative analysis of N-oxalylcysteine and oxalyl thiolesters (RSCOCOO-) in biological samples is described. These compounds were found in all rat tissues examined (kidney, liver, brain, heart, muscle and fat), with the amount of N-oxalylcysteine ranging up to 18 nmoles/g wet weight and that of oxalyl thiolesters up to 65 nmoles/g wet weight. The identification of such compounds in animal tissues adds further credence to the hypothesis that they may be important metabolic effectors.

Inhibition of malic enzyme by S-oxalylglutathione, a probable in vivo effector.

Various oxalyl thiol esters (RSCOCOO-), especially S-oxalylglutathione (GS-Ox), were found to be very effective inhibitors of chicken liver malic enzyme. When the conditions are similar to those encountered physiologically [high reduced nicotinamide adenine dinucleotide phosphate (NADPH) concentrations], inhibition is detectable with less than 1 microM concentrations of GS-Ox. The amount of inhibition is not reversed by excess glutathione, thus indicating that it is not due to oxalyl transfer to some enzymic thiol group with release of glutathione. Detailed kinetic studies show that the inhibition by GS-Ox can be treated as a simple reversible binding to the enzyme; the double reciprocal plot patterns indicate that the inhibition is linear noncompetitive (mixed type), vs. both L-malate in the oxidative decarboxylation reaction and pyruvate in the reverse reaction. At pH 7.4 and 25 degrees C in the presence of 100-200 microM NADPH, the Kis and Kii values for GS-Ox are 0.7 and 5 microM, respectively, and are the same for reactions run in either direction. The high specificity for GS-Ox is indicated by the observation that, under similar conditions, the Kis values for S-oxalyl coenzyme A and S-oxalyl-N-acetylcysteamine are 40 and 150 microM, respectively. Such high specificity indicates that the enzyme has evolved a specific binding site for the glutathione part of GS-Ox. The current results, when considered in conjunction with recent evidence that oxalyl thiol esters are present in animal tissues at concentrations up to 50 microM, imply that GS-Ox is an important in vivo regulator of malic enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Determination of oxalyl thiolesters, N-oxalylcysteine and N-oxalylcysteamine in biological materials.

A convenient method is described for the quantitative analysis of oxalyl thiolesters (OTEs), a newly discovered class of mammalian metabolites, in biological samples. By this particular technique the total concentration of all OTEs in the sample is determined. The method involves first reacting the biological material with cysteamine (2-aminoethanethiol) or cysteine under conditions that convert OTEs quantitatively to N-oxalylcysteamine (or N-oxalylcysteine), followed by reaction with monobromobimane to give a highly fluorescent derivative that is analyzed by reversed-phase ion-pair chromatography, with tetrabutylammonium ion as the counterion and N-(2-mercaptopropionyl)glycine as an internal standard. The method is capable of detecting as little as 0.6 pmol of the bimane derivative of the N-oxalyl compound in a single HPLC injection. The application of this method has led to the discovery that not only OTEs but also N-oxalylcysteine and N-oxalylcysteamine are normal mammalian metabolites. In various rat tissues the OTE concentration ranges up to 65 nmol/g (wet wt), the N-oxalylcysteine concentration is approximately 10 nmol/g, and the N-oxalylcysteamine concentration is 0-3 nmol/g.