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.

Regulation of cell morphology and cytochrome P450 expression in human hepatocytes by extracellular matrix and cell-cell interactions.

The influence of extracellular matrix conditions and plating density on cell cytoarchitecture and the constitutive and chemically induced expression of cytochrome P450 3A4 (CYP3A4) was examined in primary cultures of human hepatocytes. Constitutive and drug-induced microsomal CYP3A4 expression occurred equally well in human hepatocyte cultures maintained on either a complex or simple substratum (Matrigel vs collagen, type I), or in a sandwich configuration (i.e., between two layers of extracellular matrix), despite the markedly different morphological properties exhibited by each condition. However, a density-dependent decrease in both the constitutive and induced levels of CYP3A4 was observed in hepatocytes maintained on a simple collagen substratum as plating density was reduced from 100% to 25%. Marked alterations in cell shape and cytoarchitecture were noted concomitant with decreases in the expression and localization of intercellular gap junctions and E-cadherin-mediated cell adhesions. In addition, the intracellular distribution of microtubules and microfilaments was altered substantially and the expression of immunoreactive actin and beta-tubulin increased as cell density was decreased. These effects were reversed to some extent by overlaying monolayers with extracellular matrix or by co-culturing with another cell type. Efforts to maintain normal cell shape and cytoskeletal distribution in hepatocytes at low cell density with a Matrigel substratum failed to restore normal basal levels of CYP3A4 expression or responsiveness to rifampicin (RIF). Likewise, E-cadherin and Cx-32 expression was again reduced, even though the distribution and expression of cytoskeletal elements returned to normal levels. These results suggest that cell-cell contacts, but not the extracellular matrix configuration or composition, play a critical role in determining normal responsiveness to chemical modulators in human hepatocytes.

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.

Safety and efficacy of fast track in patients undergoing coronary artery bypass surgery.

BACKGROUND: The incidence of coronary artery bypass surgery has been increasing annually with increasing pressure on the health care system. Fast track has been proposed as a means to increase efficiency and volume, without an increase in hospital resources. To date this approach has not been critically assessed in Canada. METHODS: We examined 617 consecutive patients undergoing isolated CABG surgery. The patients were divided into (1) fast track (FT) recovery (n = 219), without admission to an ICU, and (2) non-fast track (NFT) recovery (n = 398) with direct admission to the ICU. There were no differences in age, gender, timing of surgery, left main stenosis, preoperative myocardial infarction, renal failure, diabetes, peripheral vascular disease, or in the incidence of chronic obstructive pulmonary disease between the two groups. The NFT group had a higher proportion of patients with NYHA Class III/IV symptoms preoperatively (65.7% vs. 57.3%, p = 0.048), in patients with an ejection fraction < 40% (42.5% vs. 30.6%, p = 0.004), or in the number of individuals with an IABP inserted before surgery (13 vs. 1, p < 0.001). RESULTS: In the FT group the average period of aortic occlusion (40.7 +/- 15.2 min vs. 71.8 +/- 26.5 min, p < 0.001) and perfusion time (67.8 +/- 24.5 min vs. 117.5 +/- 40.2 min, p < 0.001) were significantly less than in the NFT group. The number of grafts per patient was 3.3 +/- 1.0 vs. 3.2 +/- 1.0, respectively (p = 0.38). Operative mortality was 0.9% in the FT group and 1.3% in the NFT group (p = 1.0). Significant differences were seen in the proportion of patients that suffered from postoperative ventilatory failure (3.2% in FT vs. 12.1% in NFT, p < 0.001), and the proportion of patients that suffered any postoperative complication was significantly higher in the NFT group (21.4%) than in the FT group (9.1%, p < 0.001). The differences in postoperative complications resulted in a shorter length of stay (LOS) in FT patients (5.6 +/- 4.1 days vs. 9.7 +/- 9.4 days NFT, p < 0.001). Only 4.1% of patients that entered the FT group failed and required admission to the ICU. Multivariate stepwise logistic regression analysis identified non-fast track recovery as an independent predictor of morbidity in CABG surgery patients. CONCLUSIONS: The data indicate it is possible to perform isolated CABG surgery, in a large proportion of the population, without the need for admission to an ICU for postoperative care.

Comparative toxicity of trivalent and pentavalent inorganic and methylated arsenicals in rat and human cells.

Biomethylation is considered a major detoxification pathway for inorganic arsenicals (iAs). According to the postulated metabolic scheme, the methylation of iAs yields methylated metabolites in which arsenic is present in both pentavalent and trivalent forms. Pentavalent mono- and dimethylated arsenicals are less acutely toxic than iAs. However, little is known about the toxicity of trivalent methylated species. In the work reported here the toxicities of iAs and trivalent and pentavalent methylated arsenicals were examined in cultured human cells derived from tissues that are considered a major site for iAs methylation (liver) or targets for carcinogenic effects associated with exposure to iAs (skin, urinary bladder, and lung). To characterize the role of methylation in the protection against toxicity of arsenicals, the capacities of cells to produce methylated metabolites were also examined. In addition to human cells, primary rat hepatocytes were used as methylating controls. Among the arsenicals examined, trivalent monomethylated species were the most cytotoxic in all cell types. Trivalent dimethylated arsenicals were at least as cytotoxic as trivalent iAs (arsenite) for most cell types. Pentavalent arsenicals were significantly less cytotoxic than their trivalent analogs. Among the cell types examined, primary rat hepatocytes exhibited the greatest methylation capacity for iAs followed by primary human hepatocytes, epidermal keratinocytes, and bronchial epithelial cells. Cells derived from human bladder did not methylate iAs. There was no apparent correlation between susceptibility of cells to arsenic toxicity and their capacity to methylate iAs. These results suggest that (1) trivalent methylated arsenicals, intermediary products of arsenic methylation, may significantly contribute to the adverse effects associated with exposure to iAs, and (2) high methylation capacity does not protect cells from the acute toxicity of trivalent arsenicals.

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.

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.

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.

Metabolism of arsenic in primary cultures of human and rat hepatocytes.

The liver is considered a major site for methylation of inorganic arsenic (iAs). However, there is little data on the capacity of human liver to methylate iAs. This work examined the metabolism of arsenite (iAs(III)), arsenate (iAs(V)), methylarsine oxide (MAs(III)O), methylarsonic acid (MAs(V)), dimethylarsinous acid (DMAs(III)), and dimethylarsinic acid (DMAs(V)) in primary cultures of normal human hepatocytes. Primary rat hepatocytes were used as methylating controls. iAs(III) and MAs(III)O were metabolized more extensively than iAs(V) and MAs(V) by either cell type. Neither human nor rat hepatocytes metabolized DMAs(III) or DMAs(V). Methylation of iAs(III) by human hepatocytes yielded methylarsenic (MAs) and dimethylarsenic (DMAs) species; MAs(III)O was converted to DMAs. The total methylation yield (MAs and DMAs) increased over the range of 0.1 to 4 microM iAs(III). However, DMAs production was inhibited by iAs(III) in a concentration-dependent manner, and the DMAs/MAs ratio decreased. iAs(III) (10 and 20 microM) inhibited both methylation reactions. Inhibition of DMAs synthesis resulted in accumulation of iAs and MAs in human hepatocytes, suggesting that dimethylation is required for iAs clearance from cells. Methylation capacities of human hepatocytes obtained from four donors ranged from 3.1 to 35.7 pmol of iAs(III) per 10(6) cells per hour and were substantially lower than the methylation capacity of rat hepatocytes (387 pmol of iAs(III) per 10(6) cells per hour). The maximal methylation rates for either rat or human hepatocytes were attained between 0.4 and 4 microM iAs(III). In summary, (i) human hepatocytes methylate iAs, (ii) the capacities for iAs methylation vary among individuals and are saturable, and (iii) moderate concentrations of iAs inhibit DMAs synthesis, resulting in an accumulation of iAs and MAs in cells.

Advanced practice nursing in the Nordic countries.

Changes in the delivery of health care and changes in population characteristics and health care requirements mandate changing requirements in nursing education. This is necessary to meet patient and family needs and to deliver quality health care. This paper describes the background to nursing education in the Nordic countries and gives an account of an initiative in Norway to prepare advanced practice nurses for clinical practice in this dynamic environment.

The S-oxalin, N-acetyl-S-oxalylcysteamine, inhibits lymphocyte proliferation, IL-2 production and utilization.

S-Oxalins are a recently described class of cell metabolites that appear to function as negative regulators of proliferation. Previously we have shown that exogenous S-oxalylglutathione (GS-Ox) inhibits the proliferation of lymphocytes by inhibiting the production and utilization of IL-2. In the present study the synthetic S-oxalin, N-acetyl-S-oxalylcysteamine (ACS-Ox), was utilized in similar experiments to determine whether GS-Ox itself, or possibly some metabolite formed following initial conversion of GS-Ox by gamma-glutamyltransferase (GGT), is responsible for the effects (ACS-Ox is not metabolized by GGT). ACS-Ox inhibited DNA synthesis in lymphocytes stimulated by concanavalin A similarly to GS-Ox. IL-2 production and utilization and IL-2R expression were inhibited as well. ACS-Ox also inhibited the proliferation of IL-2 dependent cells at the same concentration as GS-Ox. Because the effects of GS-Ox and ACS-Ox are so similar, presumably the S-oxalin itself, rather than some metabolite, is responsible for the observed effects. Transfer of oxalyl groups from S-oxalins to various proteins thiols is the most likely mechanism involved.

Inhibition of proliferation and of IL-2 production and utilization in lymphocytes by S-oxalylglutathione.

Previously we have shown that S-oxalins (monothiolesters of oxalic acid) are ubiquitous mammalian metabolites whose concentrations decrease when lymphocytes are stimulated to proliferate. The present study was undertaken to further examine the role of S-oxalins in the proliferation process. When added to lymphocytes stimulated with concanavalin A, the S-oxalin, S-oxalylglutathione (GS-Ox), inhibited DNA synthesis by 50% when present at ca. 0.15 mM and virtually 100% at 0.5 mM. The inhibition was reversible. The presence of GS-Ox blocked IL-2 production, but addition of IL-2 did not permit DNA synthesis to proceed. GS-Ox also inhibited proliferation of an IL-2-dependent cell line, BT2. In primary lymphocytes GS-Ox reduced IL-2 receptor expression, but not in an IL-2-dependent blast cell line. Overall RNA synthesis and protein synthesis were not significantly altered by GS-Ox. Levels of the positive transcription factor, NF-kappaB, were decreased after incubation of lymphocytes with GS-Ox, but the amount of a negative transcription factor, NREA, was largely unchanged. The results not only provide further evidence that S-oxalins are small-molecule cell proliferation inhibitors, they also clarify to some extent the specific steps in the activation response modulated by S-oxalins.

Physical mapping of a centromere-proximal region of chromosome IV-L defines the placement of genes USO1, MBP1, PSA1 and SLC1.

A physical map of a 14.5 kb region close to the centromere on the left arm of chromosome IV of Saccharomyces cerevisiae is presented. This map has been constructed by restriction analysis of a clone from a YCp50 genomic library and by use of pre-existing and new sequence data from this region. The map reveals the following gene order (reading from the most centromere-distal to the most centromere-proximal locus): USO1/INT1-MBP1-PSA1-SLC1-YLA1 and defines the size of the open reading frames and intergenic regions.

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.

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.

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.

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.

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.