Effectiveness of a Transesophageal Echocardiography Course.

BACKGROUND: Transesophageal echocardiography has important applications for the management of the critically ill patient. There is a need to develop effective training programs for the critical care community in acquiring skill at critical care transesophageal echocardiography. OBJECTIVE: We studied the effectiveness of a 1-day simulation-based course that focused on the acquisition of skill in the performance of critical care transesophageal echocardiography. METHODS: Learners received training in image acquisition with a transesophageal simulator and training in image interpretation in small group sessions. Skill at image acquisition and image interpretation was assessed at the beginning and at the completion of the course. RESULTS: There were 27 learners who attended the course. Pre and post knowledge scores were 55 (19; mean [SD]) and 88 (9; P < .0005), respectively. Pre and post image acquisition scores were 3.6 (3.7) and 9.9 (0.3; P < .0001), respectively. CONCLUSIONS: A 1-day course in critical care transesophageal echocardiography that combined case-based image interpretation with image acquisition training using a simulator improved technical skills and knowledge base.

Activation of caspase-3 in the skeletal muscle during haemodialysis.

BACKGROUND AND OBJECTIVE: Muscle atrophy in end-stage renal disease (ESRD) may be due to the activation of apoptotic and proteolytic pathways. We hypothesized that activation of caspase-3 in the skeletal muscle mediates apoptosis and proteolysis during haemodialysis (HD). MATERIALS AND METHODS: Eight ESRD patients were studied before (pre-HD) and during HD and the findings were compared with those from six healthy volunteers. Protein kinetics was determined by primed constant infusion of L-(ring (13)C(6) ) Phenylalanine. RESULTS: Caspase-3 activity in the skeletal muscle was higher in ESRD patients pre-HD than in controls (24966·0 ± 4023·9 vs. 15293·3 ± 2120·0 units, P<0·01) and increased further during HD (end-HD) (37666·6 ± 4208·3 units) (P<0·001). Actin fragments (14 kDa) generated by caspase-3 mediated cleavage of actomyosin was higher in the skeletal muscle pre-HD (68%) and during HD (164%) compared with controls. The abundance of ubiquitinized carboxy-terminal actin fragment was also significantly increased during HD. Skeletal muscle biopsies obtained at the end of HD exhibited augmented apoptosis, which was higher than that observed in pre-HD and control samples (P<0·001). IL-6 content in the soluble fraction of the muscle skeletal muscle was increased significantly during HD. Protein kinetic studies showed that catabolism was higher in ESRD patients during HD compared with pre-HD and control subjects. Muscle protein catabolism was positively associated with caspase-3 activity and skeletal muscle IL-6 content. CONCLUSION: Muscle atrophy in ESRD may be due to IL-6 induced activation of caspase-3 resulting in apoptosis as well as muscle proteolysis during HD.

Etiology of decreased gastric nitric oxide in the critically ill.

BACKGROUND: Nitric oxide (NO) is present in the gas phase of the normal human stomach at a high concentration (1-10 ppm). The majority of this NO is produced from the reduction of dietary nitrate to nitrite and finally NO. Generation of this nonenzymatically produced gastric NO occurs only in an acidic environment. We examined NO concentrations in critically ill subjects and the mechanism for the observed perturbations. METHODS: Seven critically ill, intubated intensive care unit (ICU) patients (mean APACHE II score 16) and seven control patients were studied. Gastric NO concentrations were measured with a Sievers NO analyzer (GE, Boulder, CO). Nitrate and nitrite concentrations were determined by a modified Griess assay. Bacterial counts were determined by optical density at 600 nm. RESULTS: Gastric NO concentration was significantly lower in the critically ill group (102.7 ppb) compared with the control group (953.2 ppb), although this difference was abolished by treating the control group with omeprazole (54 ppb). Gastric nitrate and nitrite concentrations were similar in the control and ICU groups, suggesting that substrate deficiency was not a cause of the low intragastric NO. Gastric pH was significantly lower in the control subjects (3.0) compared with the ICU patients (6.3) and the control subjects after receiving omeprazole (6.5). ICU patients had a trend toward higher gastric bacterial load. CONCLUSION: In critically ill patients, markedly decreased NO concentrations are found in the gas of the stomach owing to a failure of gastric acidification.

Erythromycin accelerates gastric emptying in a dose-response manner in healthy subjects.

STUDY OBJECTIVES: To evaluate whether a dose-response curve exists for erythromycin, determine the lowest effective dose of erythromycin needed to improve gastric motility, and compare erythromycin's effectiveness with that of metoclopramide in improving gastric emptying. DESIGN: Randomized, crossover, multiintervention trial. SETTING: Inpatient clinical research center. SUBJECTS: Ten healthy volunteers (four men, six women) from the general population. INTERVENTION: On each study day, the subjects were infused with erythromycin 0.75 mg/kg, 1.5 mg/kg, or 3.0 mg/kg; metoclopramide 10 mg; or placebo, in random order. Subjects then drank Ensure 200 ml mixed with acetaminophen 1.5 g. Gastric emptying was estimated by comparing the area under the curve after 60 minutes for acetaminophen absorption using four timed blood draws. MEASUREMENTS AND MAIN RESULTS: Erythromycin increased gastric emptying in a dose-response manner. Erythromycin 3.0 mg/kg and metoclopramide 10 mg were associated with statistically significant increases in liquid gastric emptying compared with placebo. During infusion, nausea and stomach cramping were associated with the 3.0-mg/kg dose of erythromycin; drowsiness was associated with metoclopramide. CONCLUSION: In patients requiring intravenous erythromycin for gastric motility, the 3.0-mg/kg dose seems the most effective, with a reasonable side effect profile.

Mechanism of interferon-gamma-induced increase in T84 intestinal epithelial tight junction.

Interferon-gamma (IFN-gamma) is an important proinflammatory cytokine that plays a central role in the intestinal inflammatory process of inflammatory bowel disease. IFN-gamma induced disturbance of the intestinal epithelial tight junction (TJ) barrier has been postulated to be an important mechanism contributing to intestinal inflammation. The intracellular mechanisms that mediate the IFN-gamma induced increase in intestinal TJ permeability remain unclear. The aim of this study was to examine the role of the phosphatidylinositol 3-kinase (PI3-K) pathway in the regulation of the IFN-gamma induced increase in intestinal TJ permeability using the T84 intestinal epithelial cell line. IFN-gamma caused an increase in T84 intestinal epithelial TJ permeability and depletion of TJ protein, occludin. The IFN-gamma induced increase in TJ permeability and alteration in occludin protein was associated with rapid activation of PI3-K; and inhibition of PI3-K activation prevented the IFN-gamma induced effects. IFN-gamma also caused a delayed but more prolonged activation of nuclear factor-kappaB (NF-kappaB); inhibition of NF-kappaB also prevented the increase in T84 TJ permeability and alteration in occludin expression. The IFN-gamma induced activation of NF-kappaB was mediated by a cross-talk with PI3-K pathway. In conclusion, the IFN-gamma induced increase in T84 TJ permeability and alteration in occludin protein expression were mediated by the PI3-K pathway. These results show for the first time that the IFN-gamma modulation of TJ protein and TJ barrier function is regulated by a cross-talk between PI3-K and NF-kappaB pathways.

Mechanism of glucocorticoid regulation of the intestinal tight junction barrier.

A defective intestinal epithelial tight junction (TJ) barrier has been proposed as an important pathogenic factor contributing to the intestinal inflammation of Crohn's disease. Glucocorticoids are first-line therapeutic agents for the treatment of moderate to severe Crohn's disease. Glucocorticoid treatment has been shown to induce retightening of the intestinal TJ barrier defect in Crohn's disease patients. However, the mechanisms that mediate the glucocorticoid therapeutic action on intestinal TJ barrier function remain unknown. The aim of this study was to elucidate the mechanism of glucocorticoid modulation of the intestinal epithelial TJ barrier using an in vitro model system. Filter-grown Caco-2 intestinal epithelial cells were used as an in vitro model to examine the effects of glucocorticoids on basal intestinal epithelial TJ barrier function and on TNF-alpha-induced disruption of the TJ barrier. Glucocorticoids (prednisolone and dexamethasone) did not have a significant effect on baseline Caco-2 TJ barrier function but prevented the TNF-alpha-induced increase in Caco-2 TJ permeability. The glucocorticoid protective effect against the TNF-alpha-induced increase in Caco-2 TJ permeability required activation of the glucocorticoid receptor (GR) complex. The activation of the GR complex resulted in GR complex binding to the glucocorticoid response element (GRE) site on DNA and activation of a GR-responsive promoter. Glucocorticoids inhibited the TNF-alpha-induced increase in myosin light chain kinase (MLCK) protein expression, a key process mediating the TNF-alpha increase in intestinal TJ permeability. The glucocorticoid inhibition of the TNF-alpha-induced increase in MLCK protein expression was due to the binding of the GR complex to a GRE binding site on the MLCK promoter region suppressing the TNF-alpha-induced activation. Glucocorticoids inhibit the TNF-alpha-induced increase in Caco-2 TJ permeability. The prednisolone protective action was mediated by binding of activated GR complex to the GRE site on the MLCK promoter, suppressing the TNF-alpha-induced increase in MLCK gene activity, protein expression, and subsequent opening of the intestinal TJ barrier.

Amino acid repletion does not decrease muscle protein catabolism during hemodialysis.

Intradialytic protein catabolism is attributed to loss of amino acids in the dialysate. We investigated the effect of amino acid infusion during hemodialysis (HD) on muscle protein turnover and amino acid transport kinetics by using stable isotopes of phenylalanine, leucine, and lysine in eight patients with end-stage renal disease (ESRD). Subjects were studied at baseline (pre-HD), 2 h of HD without amino acid infusion (HD-O), and 2 h of HD with amino acid infusion (HD+AA). Amino acid depletion during HD-O augmented the outward transport of amino acids from muscle into the vein. Increased delivery of amino acids to the leg during HD+AA facilitated the transport of amino acids from the artery into the intracellular compartment. Increase in muscle protein breakdown was more than the increase in synthesis during HD-O (46.7 vs. 22.3%, P < 0.001). Net balance (nmol.min(-1).100 ml (-1)) was more negative during HD-O compared with pre-HD (-33.7 +/- 1.5 vs. -6.0 +/- 2.3, P < 0.001). Despite an abundant supply of amino acids, the net balance (-16.9 +/- 1.8) did not switch from net release to net uptake. HD+AA induced a proportional increase in muscle protein synthesis and catabolism. Branched chain amino acid catabolism increased significantly from baseline during HD-O and did not decrease during HD+AA. Protein synthesis efficiency, the fraction of amino acid in the intracellular pool that is utilized for muscle protein synthesis decreased from 42.1% pre-HD to 33.7 and 32.6% during HD-O and HD+AA, respectively (P < 0.01). Thus amino acid repletion during HD increased muscle protein synthesis but did not decrease muscle protein breakdown.

Mechanism of TNF-{alpha} modulation of Caco-2 intestinal epithelial tight junction barrier: role of myosin light-chain kinase protein expression.

TNF-alpha plays a central role in the intestinal inflammation of various inflammatory disorders including Crohn's disease (CD). TNF-alpha-induced increase in intestinal epithelial tight junction (TJ) permeability has been proposed as one of the proinflammatory mechanisms contributing to the intestinal inflammation. The intracellular mechanisms involved in the TNF-alpha-induced increase in intestinal TJ permeability remain unclear. The purpose of this study was to investigate the possibility that the TNF-alpha-induced increase in intestinal epithelial TJ permeability was regulated by myosin light-chain kinase (MLCK) protein expression, using an in vitro intestinal epithelial model system consisting of the filter-grown Caco-2 intestinal epithelial monolayers. TNF-alpha (10 ng/ml) produced a time-dependent increase in Caco-2 MLCK expression. The TNF-alpha increase in MLCK protein expression paralleled the increase in Caco-2 TJ permeability, and the inhibition of the TNF-alpha-induced MLCK expression (by cycloheximide) prevented the increase in Caco-2 TJ permeability, suggesting that MLCK expression may be required for the increase in Caco-2 TJ permeability. The TNF-alpha increase in MLCK protein expression was preceded by an increase in MLCK mRNA expression but not an alteration in MLCK protein degradation. Actinomycin-D prevented the TNF-alpha increase in MLCK mRNA expression and the subsequent increase in MLCK protein expression and Caco-2 TJ permeability, suggesting that the increase in MLCK mRNA transcription led to the increase in MLCK expression. The TNF-alpha increase in MLCK protein expression was also associated with an increase in Caco-2 MLCK activity. The cycloheximide inhibition of MLCK protein expression prevented the TNF-alpha increase in MLCK activity and Caco-2 TJ permeability. Moreover, inhibitors of MLCK, Mg(2+)-myosin ATPase, and metabolic energy prevented the TNF-alpha increase in Caco-2 TJ permeability, suggesting that the increase in MLCK activity was required for the TNF-alpha-induced opening of the Caco-2 TJ barrier. In conclusion, our results indicate for the first time that 1) the TNF-alpha increase in Caco-2 TJ permeability was mediated by an increase in MLCK protein expression, 2) the increase in MLCK protein expression was regulated by an increase in MLCK mRNA transcription, and 3) the increase in Caco-2 TJ permeability required MLCK protein expression-dependent increase in MLCK activity.

TNF-alpha-induced increase in intestinal epithelial tight junction permeability requires NF-kappa B activation.

Crohn's disease (CD) patients have an abnormal increase in intestinal epithelial permeability. The defect in intestinal tight junction (TJ) barrier has been proposed as an important etiologic factor of CD. TNF-alpha increases intestinal TJ permeability. Because TNF-alpha levels are markedly increased in CD, TNF-alpha increase in intestinal TJ permeability could be a contributing factor of intestinal permeability defect in CD. Our purpose was to determine some of the intracellular mechanisms involved in TNF-alpha modulation of intestinal epithelial TJ permeability by using an in vitro intestinal epithelial system consisting of filter-grown Caco-2 monolayers. TNF-alpha produced a concentration- and time-dependent increase in Caco-2 TJ permeability. TNF-alpha-induced increase in Caco-2 TJ permeability correlated with Caco-2 NF-kappa B activation. Inhibition of TNF-alpha-induced NF-kappa B activation by selected NF-kappa B inhibitors, curcumin and triptolide, prevented the increase in Caco-2 TJ permeability, indicating that NF-kappa B activation was required for the TNF-alpha-induced increase in Caco-2 TJ permeability. This increase in Caco-2 TJ permeability was accompanied by down-regulation of zonula occludens (ZO)-1 proteins and alteration in junctional localization of ZO-1 proteins. TNF-alpha modulation of ZO-1 protein expression and junctional localization were also prevented by NF-kappa B inhibitors. TNF-alpha did not induce apoptosis in Caco-2 cells, suggesting that apoptosis was not the mechanism involved in TNF-alpha-induced increase in Caco-2 TJ permeability. These results demonstrate for the first time that TNF-alpha-induced increase in Caco-2 TJ permeability was mediated by NF-kappa B activation. The increase in permeability was associated with NF-kappa B-dependent downregulation of ZO-1 protein expression and alteration in junctional localization.