Kinetics of cardiac troponin and other biomarkers in patients with ST elevation myocardial infarction.

Objective: To examine changes in concentration, time-to-peak and the ensuing half-life of cardiac biomarkers in patients with myocardial infarction. Methods: Blood sampling was performed every third hour within 24 h after percutaneous coronary intervention (PCI) on a cohort of patients with ST elevation myocardial infarction. Cardiac troponin (cTn) was measured by the Dimension Vista, Vitros, Atellica, and Alinity high-sensitivity (hs) cTnI assays, and the Elecsys hs-cTnT assay. Further, creatine kinase (CK), myoglobin, creatine kinase MB (CKMB) and other biomarkers were analyzed. Results: A total of 36 patients completed blood sampling (median age 60 years, IQR 56.4-66.5 years; seven women, 19.4%). Hs-cTnI measured by the Vitros assay was the first hs-cTn to peak at 9.1 h (95%-CI 6.2-10.1) after PCI and 11.7 h (95%-CI 10.4-14.8) after symptoms onset. There were no notable differences between hs-cTn assays in regard to time-to-peak. Also, Vitros hs-cTnI reached the highest median ratio of concentration to upper reference level of nearly 2,000. The median half-life from peak concentration ranged from 7.6 h for myoglobin (CI 6.8-8.6) to 17.8 h for CK (CI 6.8-8.6). For hs-cTn assays the median T½ ranged from 12.4 h for the Vista hs-cTnI assay (95%-CI 11.0-14.1 h) to 17.3 h for the Elecsys hs-cTnT (95%-CI 14.9-20.8 h). Conclusions: This study updates knowledge on the kinetics of cardiac biomarkers in current clinical use. There was no notable difference in trajectories, time-to-peak or half-life between hs-cTn assays.

Transesophageal Doppler reliably tracks changes in cardiac output in comparison with intermittent pulmonary artery thermodilution in cardiac surgery patients.

In this study a comparison of cardiac output (CO) measurements obtained with CardioQ transesophageal Doppler (TED) and pulmonary artery catheter (PAC) thermodilution (TD) technique was done in a systematic set-up, with induced changes in preload, afterload and heart rate. Twenty-five patients completed the study. Each patient were placed in the following successive positions: (1) supine, (2) head-down tilt, (3) head-up tilt, (4) supine, (5) supine with phenylephrine administration, (6) pace heart rate 80 beats per minute (bpm), (7) pace heart rate 110 bpm. The agreement of compared data was investigated by Bland-Altman plots, and to assess trending ability a four quadrants plot and a polar plot were constructed. Both methods showed an acceptable precision 6.4 % (PAC TD) and 12.8 % (TED). In comparison with PAC TD, the TED was associated with a mean bias in supine position of -0.30 l min-1 (95 % CI -0.88; 0.27), wide limits of agreement, a percentage error of 69.5 %, and a trending ability with a concordance rate of 92 %, angular bias of 1.1° and a radial sector size of 40.0° corresponding to an acceptable trending ability. In comparison with PAC TD, the CardioQ TED showed a low mean bias, wide limits of agreement and a larger percentage error than should be expected from the precision of the two methods. However, an acceptable trending ability was found. Thus, the CardioQ TED should not replace CO measurements done by PAC TD, but could be a valuable tool in guiding therapy.

Targeted temperature management at 33°C versus 36°C and impact on systemic vascular resistance and myocardial function after out-of-hospital cardiac arrest: a sub-study of the Target Temperature Management Trial.

BACKGROUND: Cardiovascular dysfunction is common after out-of-hospital cardiac arrest as part of the postcardiac arrest syndrome, and hypothermia may pose additional impact on hemodynamics. The aim was to investigate systemic vascular resistance index (SVRI), cardiac index, and myocardial performance at a targeted temperature management of 33°C (TTM33) versus 36°C (TTM36). METHODS AND RESULTS: Single-center substudy of 171 patients included in the Target Temperature Management Trial (TTM Trial) randomly assigned to TTM33 or TTM36 for 24 hours after out-of-hospital cardiac arrest. Mean arterial pressure ≥65 mm Hg and central venous pressure of 10 to 15 mm Hg were hemodynamic treatment goals. Hemodynamic evaluation was performed by serial right heart catheterization and transthoracic echocardiography. Primary end point was SVRI after 24 hours of cooling and secondary end points included mean SVRI, cardiac index, systolic function, and lactate levels. The TTM33 group had a significant increase in SVRI compared with TTM36 (2595; 95% confidence interval, 2422-2767) versus 1960 (95% confidence interval, 1787-2134) dynes m(2)/s per cm(5); P<0.0001, respectively) after 24 hours of cooling with an overall difference of 556 dynes m(2)/s per cm(5) (P(group) <0.0001). TTM33 was associated with decreased cardiac index (-0.4 L/min per m(2); P(group) <0.0001), decreased heart rate (P(group)=0.01), and stroke volume index (P(group)=0.004) compared with TTM36. Left ventricular ejection fraction (P=0.39) and peak systolic myocardial velocity (P=0.62) did not differ between TTM groups. Lactate levels were significantly higher in the TTM33 group (P=0.0008). CONCLUSIONS: Targeted temperature management at 33°C with target mean arterial pressure ≥65 mm Hg is associated with increased SVRI and lower cardiac index because of lower heart rate with unaffected left ventricular systolic function compared with 36°C. CLINICAL TRIAL REGISTRATION URL: http://www.clinicaltrials.gov. Unique identifier: NCT01020916.

Increased intrathoracic pressure affects cerebral oxygenation following cardiac surgery.

BACKGROUND: Cerebral oximetry reflects circulatory stability during surgery. We evaluated whether frontal lobe oxygenation is influenced by a transient increase in intrathoracic pressure as induced by a lung recruitment manoeuvre. METHODS: Intrathoracic pressure was increased to 40 cm H(2)O for 20 s immediately after cardiac surgery in ten patients (age 64 ± 10 year, mean ± SD) with frontal lobe oxygenation assessed by near-infrared spectroscopy and cardiac output by thermodilution. RESULTS: The lung recruitment manoeuvre increased arterial O(2) pressure (from 29 ± 15 to 40 ± 12 kPa) with a decrease in mean arterial pressure (MAP) (from 69 ± 7 to 55 ± 11 mmHg), cardiac output (from 5·4 ± 0·6 to 5·0 ± 0·7 l min(-1)) and frontal lobe oxygenation (from 68 ± 9 to 60 ± 13%; all P<0·05). A reduction in MAP by more than 15 mmHg caused cerebral desaturation by 10%, the lowest cerebral oxygenation (44%) was with a reduction in MAP by 24 mmHg, and according to multiple linear regression, only MAP predicted cerebral deoxygenation (P = 0·03). Following the lung recruitment manoeuvre, hemodynamic variables and frontal lobe oxygenation were restored. CONCLUSIONS: A lung recruitment manoeuvre decreases frontal lobe oxygenation when MAP is low suggesting that with increased intrathoracic pressure, arterial pressure should be kept within the limits of cerebral autoregulation.

Identification and characterization of endonuclein binding proteins: evidence of modulatory effects on signal transduction and chaperone activity.

BACKGROUND: We have previously identified endonuclein as a cell cycle regulated WD-repeat protein that is up-regulated in adenocarcinoma of the pancreas. Now, we aim to investigate its biomedical functions. RESULTS: Using the cDNA encoding human endonuclein, we have expressed and purified the recombinant protein from Escherichia coli using metal affinity chromatography. The recombinant protein was immobilized to a column and by affinity chromatography several interacting proteins were purified from several litres of placenta tissue extract. After chromatography the eluted proteins were further separated by two-dimensional gel electrophoresis and identified by tandem mass spectrometry. The interacting proteins were identified as; Tax interaction protein 1 (TIP-1), Aalpha fibrinogen transcription factor (P16/SSBP1), immunoglobulin heavy chain binding protein (BiP), human ER-associated DNAJ (HEDJ/DNAJB11), endonuclein interaction protein 8 (EIP-8), and pregnancy specific beta-1 glycoproteins (PSGs). Surface plasmon resonance analysis and confocal fluorescence microscopy were used to further characterize the interactions. CONCLUSIONS: Our results demonstrate that endonuclein interacts with several proteins indicating a broad function including signal transduction and chaperone activity.

Proteomic analysis identifies mitochondrial metabolic enzymes as major discriminators between different stages of the failing human myocardium.

OBJECTIVES: Our aim was to identify patterns in differentially regulated proteins associated with the progression of chronic heart failure. We specifically studied proteomics in chronic reversibly (RDM) and irreversibly dysfunctional myocardium (IRDM), as well as end-stage failing myocardium (ESFM). METHODS: We studied biopsies from 9 patients with stable chronic heart failure undergoing coronary artery bypass surgery (CABG) (EF 34% +/- 3%) and from 4 patients with ESFM undergoing heart transplantation (EF 17% +/- 5%). In CABG patients paired echocardiographic studies before and 6 months after revascularization classified dysfunctional myocardium as RDM or IRDM. Regions with preserved contractile function served as control. We used two-dimensional gel electrophoresis (2D-PAGE) and computerized image analysis to investigate myocardial protein expression. Proteins were identified by in-gel digestion and subsequent liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS: Among 3 significantly altered protein spots in RDM we identified 2 up-regulated glycolytic enzymes. In IRDM 15 proteins were signficantly altered of which we identified 10, among these 6 were down-regulated mitochondrial enzymes. In ESFM 9 of 12 significantly altered protein spots were identified. Six were down-regulated mitochondrial enzymes. CONCLUSION: Myocardial metabolism may be involved in the progression of heart failure to irreversible dysfunction and end-stage heart failure.

Pulse contour cardiac output: an evaluation of the FloTrac method.

BACKGROUND AND OBJECTIVE: The aim of this study was to determine the agreement between pulmonary artery thermodilution (PA-TD) and a new pulse contour method (PCM), FloTrac/Vigileo version 1.0, and to asses the ability of FloTrac to track sudden changes in cardiac output. METHODS: Cardiac output was determined twice after induction of anaesthesia, but before cardiac surgery, with both PA-TD and a PCM in order to determine the precision of both methods. The bias and agreement between the two methods were calculated using Bland-Altman analysis. Postoperatively, in patients with heart rates under 60 beats min(-1), atrial pacing was initiated and cardiac output was determined before and after with both methods. RESULTS: Twenty-five patients were investigated. The precisions of PA-TD and the PCM were 0.35 (95% confidence interval +/-0.12) and 0.6 l min(-1) (95% confidence interval +/-0.21%). The bias between PA-TD and the PCM was -0.51 l min(-1) and the limits of agreement were +/-1.87 l min(-1) (95% confidence interval +/-0.39 and +/-0.66). The percentage error was 48%. The changes in cardiac output with atrial pacing were in the same direction in all nine patients. CONCLUSION: In this study, agreement between PA-TD and the PCM was poor, but the PCM was able to track the direction of pace-induced changes in cardiac output.

Differences in the protein expression in limbal versus central human corneal epithelium--a search for stem cell markers.

In the search for potential limbal stem cell protein markers, the purpose of this study was to characterize differences in protein expression between human central and limbal corneal epithelium by a proteomic approach using two-dimensional polyacrylamide gel electrophoresis (2D PAGE) combined with mass spectrometry (LC-MS/MS). The results were subsequently confirmed by Western blotting and immunohistochemistry. We detected more than 1000 protein spots in each gel. Thirty-two spots were significantly over-expressed in the central part and 70 spots were significantly over-expressed in the limbal part. We identified 25 different proteins. Among these 11 proteins representing different cellular locations and functions were selected for further investigations. Most interestingly, superoxide dismutase 2 (SOD2), was expressed in clusters of cells in the basal limbal epithelium. Heat shock protein 70 protein 1 (HSP70.1) and annexin I were highly abundant in limbal epithelium, although they were also present in the central epithelium to a minor extent. Among the proteins primarily expressed in the limbal fraction we further identified cytokeratin (CK) 15, CK19 and alpha enolase, which have been reported previously to be related to the limbal basal epithelium. The basal limbal epithelium consists of clusters of slow cycling limbal stem cells and rapid cycling transient amplifying cells. Ideally, proteins exclusively expressed in the limbal part of the epithelium may serve as markers for the basal limbal cells. SOD2 and CK15 identify clusters of limbal basal cells and therefore they may serve as markers for limbal stem cells in conjunction with the earliest transient amplifying cells.

Identification of differentially regulated proteins in a patient with Leber's Congenital Amaurosis--a proteomic study.

BACKGROUND: To identify the pattern of protein expression in the retina from a patient with Leber's Congenital Amaurosis (LCA) secondary to a mutation in the AIPL1 gene. The retina from one eye of a patient with LCA and 7 control eyes were studied. The tissue was subjected to high resolution two-dimensional gel electrophoresis, image analysis and mass spectrometry, in an effort to identify differentially regulated proteins. RESULTS: In the LCA retina seven protein spots were differentially expressed. Six proteins were significantly up-regulated of which three could be identified as: alphaA-crystallin, triosephophate isomerase, and an N-terminal fragment of the beta-chain of ATP synthase. One protein spot that was down-regulated in the LCA retina was identified as a C-terminal fragment of beta-tubulin. CONCLUSION: Retinal tissue in LCA is characterised by an up-regulation of alphaA-crystallin, triosephosphate isomerase, and ATP synthase (beta-chain fragment) and down-regulation of a fragment of beta-tubulin. These proteins/protein fragments may play a crucial role for the retinal degeneration processes in LCA and other retinal dystrophies.

Proteomic profiling of fibroblasts reveals a modulating effect of extracellular calumenin on the organization of the actin cytoskeleton.

CREC proteins constitute a family of EF-hand calcium binding proteins localized to the secretory pathway. Calumenin is the only member known to be secreted. Recently, it was shown that thrombin-activated thrombocytes liberate calumenin, which also is found in atherosclerotic lesions but not in normal vasculature. To study the possible effects of calumenin extracellularly, we used proteomic profiling of fibroblasts cultured in absence and in presence of calumenin. Using 2-DE and MS/MS, we show that normal fibroblasts contain several 28-29-kDa N-terminal and a 16-kDa C-terminal fragment of beta- or gamma-actin. Extracellularly added calumenin decreases the levels of both the N-terminal and C-terminal actin fragments, and, in addition, decreases the expression level of septin 2, which interacts with the actin cytoskeleton and is involved in cytokinesis. Labeling of S-phase fibroblasts with bromo-2'deoxy-uridine indicates that calumenin added to the medium also modulates the cell cycle. Our study thus indicates that calumenin may have an autocrine or a paracrine effect on the cells in its vicinity, and, therefore, may be involved in the pathophysiology of thrombosis or in wound healing.

Lung recruitment maneuver depresses central hemodynamics in patients following cardiac surgery.

OBJECTIVE: To assess the impact of the lung recruitment maneuver on circulation following cardiac surgery. DESIGN AND SETTING: Prospective randomized cross-over study at the Departments of Anesthesia and Thoracic Surgery, Copenhagen University Hospital. PATIENTS: Ten adult undergoing coronary artery bypass surgery. INTERVENTIONS: Patients were randomized to two durations of lung recruitment maneuvers (40 cmH2O airway pressure for 10 s and 20 s or vice versa after 5 min) administered immediately after surgery. MEASUREMENTS AND RESULTS: Transesophageal echocardiography (left ventricular short axis view), pulse contour cardiac output, and arterial blood pressure were monitored continuously. Systemic and pulmonary arterial blood gases were sampled before and after each lung recruitment maneuver to calculate the intrapulmonary shunt. Left ventricular end-diastolic areas decreased significantly during both the 10-s and the 20-s lung recruitment maneuvers. Cardiac output was 5.6+/-0.8 l/min at baseline, decreasing by 3.0+/-1.1 l/min and 3.6+/-1.2 l/min during lung recruitment maneuvers of 10 and 20 s, respectively. Shunt decreased from 20+/-5% to 15+/-6% after the first lung recruitment maneuver and from 15+/-6% to 12+/-5% after the second. CONCLUSIONS: Lung recruitment maneuvers markedly reduced cardiac output and left ventricular end-diastolic areas in hemodynamically stable patients following cardiac surgery.

Functional genomics studied by proteomics.

The human genome contains about 30,000 genes, each creating several transcripts per gene. Transcript structures and expression are studied by high-throughput transcriptomic techniques using microarrays. Generally, transcripts are not directly operating molecules, but are translated into functional proteins, post-translationally modified by proteolysis, glycosylation, phosphorylation, etc., sometimes with great functional impact. Proteins need to be analyzed by proteomic techniques, less suited for high-throughput. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), separating thousands of proteins has developed slowly over the past quarter of a century. This technique is now quite reproducible and suitable for differential proteomics, comparing normal and diseased cells/tissues revealing differentially regulated proteins. 2D-PAGE is combined with protein-identification methods, currently mass spectrometry (MS), which has been significantly improved over the last decade. Other proteomic techniques studying protein-protein interactions are now either established or still being developed, such as peptide or protein arrays, phage display, and the yeast two-hybrid system. The strengths and weaknesses of these techniques are discussed.

Proteomic analysis of hyperoxia-induced responses in the human choriocarcinoma cell line JEG-3.

Living cells exposed to changes in the surrounding oxygen tension, have the ability to adapt to the new environment through the regulatory effect of intracellular mediators. In an effort to identify important proteins that may be involved in the hyperoxic response, we performed proteomic analysis on the human choriocarcinoma cell line JEG-3, incubated under high oxygen tension (carbogen, 95% O2/5% CO(2)) or air (21% oxygen/5% CO(2)). We identified 13 protein spots that were significantly down-regulated (p < 0.05) in JEG-3 cells incubated under hyperoxic conditions compared to standard conditions. Ten of these spots were positively identified by matrix-assisted laser desorption/ionization time of flight-mass spectrometry as nine different proteins: Villin 2, tublin beta, profilin I, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate mutase, peroxiredoxin 1, neuroplypeptide h3, poly(rC)-binding protein 1 and cyclophilin A. These proteins have been implicated in regulating cytoskeletal structure, glycolysis, redox status, signal transduction, transcription and protein folding. The data obtained are consistent with the roles of these proteins in mediating cellular response to oxidative stress and in regulating cell proliferation and motility.

AQP1 and AQP3, psoriasin, and nitric oxide synthases 1-3 are inflammatory mediators in erythema toxicum neonatorum.

Erythema toxicum neonatorum is a common, inflammatory skin reaction in healthy newborn infants characterized by an accumulation of activated immune cells in the lesions. Its etiology and physiologic significance are still unclear. The purpose of this study was to extend the search for possible inflammatory mediators of the rash. We performed immunohistochemistry on punch biopsy cryosections from lesions of four, 1-day-old infants and from four matched controls without rash, using antibodies against the water channel proteins aquaporin-1 (AQP1) and aquaporin-3 (AQP3), psoriasin, and the nitric oxide synthase (NOS) enzymes, neuronal NOS (nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS). All sections from the lesions showed a dense, nodular cellular infiltrate located near the hair follicle. The vessels in the dermis showed a high incidence of AQP1 and eNOS. Strong staining for AQP1, AQP3, and psoriasin, as well as nNOS, iNOS, and eNOS were seen in the entire epidermal layer. The infiltrate in the dermis contained numerous cells expressing AQP1, AQP3, nNOS, iNOS, and eNOS. Double immunofluorescence staining showed that AQP3 was located in CD1a-expressing Langerhans cells and other dendritic cells in the dermis, as well as in CD14-expressing macrophages, CD15-expressing neutrophils, and EG2-expressing eosinophils surrounding the hair follicle. Our findings show that AQP1 and AQP3, psoriasin, and NOSs are involved in the activation of the skin immune system at birth.

Protein profiling of the human epidermis from the elderly reveals up-regulation of a signature of interferon-gamma-induced polypeptides that includes manganese-superoxide dismutase and the p85beta subunit of phosphatidylinositol 3-kinase.

Aging of the human skin is a complex process that consists of chronological and extrinsic aging, the latter caused mainly by exposure to ultraviolet radiation (photoaging). Here we present studies in which we have used proteomic profiling technologies and two-dimensional (2D) PAGE database resources to identify proteins whose expression is deregulated in the epidermis of the elderly. Fresh punch biopsies from the forearm of 20 pairs of young and old donors (21-30 and 75-92 years old, respectively) were dissected to yield an epidermal fraction that consisted mainly of differentiated cells. One- to two-mm3 epidermal pieces were labeled with [35S]methionine for 18 h, lysed, and subjected to 2D PAGE (isoelectric focusing and non-equilibrium pH gradient electrophoresis) and phosphorimage autoradiography. Proteins were identified by matching the gels with the master 2D gel image of human keratinocytes (proteomics.cancer.dk). In selected cases 2D PAGE immunoblotting and/or mass spectrometry confirmed the identity. Quantitative analysis of 172 well focused and abundant polypeptides showed that the level of most proteins (148) remains unaffected by the aging process. Twenty-two proteins were consistently deregulated by a factor of 1.5 or more across the 20 sample pairs. Among these we identified a group of six polypeptides (Mx-A, manganese-superoxide dismutase, tryptophanyl-tRNA synthetase, the p85beta subunit of phosphatidylinositol 3-kinase, and proteasomal proteins PA28-alpha and SSP 0107) that is induced by interferon-gamma in primary human keratinocytes and that represents a specific protein signature for the effect of this cytokine. Changes in the expression of the eukaryotic initiation factor 5A, NM23 H2, cyclophilin A, HSP60, annexin I, and plasminogen activator inhibitor 2 were also observed. Two proteins exhibited irregular behavior from individual to individual. Besides arguing for a role of interferon-gamma in the aging process, the biological activities associated with the deregulated proteins support the contention that aging is linked with increased oxidative stress that could lead to apoptosis in vivo.

Human proteomic databases: a powerful resource for functional genomics in health and disease.

Decoding of the genome information in terms of regulation and function will be the next great challenge in the life sciences in this millennium and indeed, today we are experiencing a rapid explosion of technology for the high throughput expression analysis of genes and their products (functional genomics). In particular, the field of proteomics is booming as proteins are often the functional molecules and represent important targets for the pharmaceutical industry. The proteomic technology is complex, and comprises a plethora of state-of-the-art techniques to resolve, identify and detect their interacting partners, as well as to store and communicate protein information in comprehensive two-dimensional polyacrylamide gel electrophoresis (2D PAGE) databases. Besides annotating the genome, these databases will offer a global approach to the study of gene expression both in health and disease. Here, we review the current status of human 2D PAGE databases that we are systematically constructing for the study of bladder cancer and skin ageing.

Proteomic strategies to reveal tumor heterogeneity among urothelial papillomas.

Proteomics and immunohistochemistry were used to reveal tumor heterogeneity among urothelial papillomas (UPs) with the long term goal of predicting their biological potential in terms of outcome. First, we identified proteins that were deregulated in invasive fresh lesions as compared with normal urothelium, and thereafter we immunostained UPs with a panel of antibodies against some of the markers. Twenty-two major proteins showing variations of 2-fold or more in at least one-third of the invasive lesions were selected. Specific antibodies against several of the proteins were obtained, but only a few reacted positively in immunostaining. A panel consisting of antibodies against keratinocytes (CKs) 5, 13, 18, and 20 and markers of squamous metaplasia (CKs 7, 8, and 14) was used to probe normal urothelium and 30 UPs collected during a period of five years. Four UPs showed a normal phenotype, whereas the rest could be grouped in five major types that shared aberrant staining with the CK20 antibody. Type 1 heterogeneity (n = 4) showed preferred staining of the umbrella cells with the CK8 antibody. Type 2 (n = 11) was typified by the staining of the basal and intermediate layers with the CK20 antibody. Type 3 (n = 7) was characterized by the predominant staining of the basal cell layer with the CK5 antibody. Type 4 (n = 1) showed areas of CK7 negative cells, whereas type 5 (n = 3) showed loss of staining of the basal cells with the CK20. 29% of the patients experienced recurrences, but none progressed to invasive disease. Patients harboring phenotypic alterations in the basal cell compartment (types 3 and 5) showed the highest number of recurrences (4/7 and 2/3, respectively), and all type 3 lesions progressed to a higher degree of dedifferentiation. Even though a long term prospective study involving a larger sample size is required to assess the biological potential of these lesions, we believe that this approach will prove instrumental for revealing early phenotypic changes in different types of cancer.