Release kinetics of intact and degraded troponin I and T after irreversible cell damage.

PURPOSE: We characterized the release kinetics of cardiac troponin I and T in relation to lactate dehydrogenase (LDH) from cardiomyocytes before and after the transition from reversible to irreversible cell damage. METHODS: Cardiomyocytes were exposed to mild metabolic inhibition (1 mmol/L sodium azide) to induce a necrotic cell death process that is characterized by a reversible (0-12 h) and irreversible phase (12-30 h). At various time intervals cells and media were collected and analyzed for LDH activity, intact cTnI and cTnT, and their degradation products. RESULTS: During the first 12 h of metabolic inhibition, cell viability was unchanged with no release of intact cTnI and cTnT nor their degradation products. Between 12 and 30 h of azide treatment, cardiomyocytes showed progressive cell death accompanied by release of intact cTnI (29 kDa), intact cTnT (39 kDa), four cTnI degradation products of 26, 20, 17 and 12 kDa, and three cTnT degradation products of 37, 27 and 14 kDa. Possibly due to degradation, there is progressive loss of cTnI and cTnT protein that is obviously undetected by the antibodies used. CONCLUSIONS: Metabolic inhibition of cardiomyocytes induces a parallel release of intact cTnI and cTnT and their degradation products, starting only after onset of irreversible cardiomyocyte damage.

A continuous displacement immunoassay for human heart-type fatty acid-binding protein in plasma.

Human heart-type fatty acid-binding protein (FABP) is suggested as an early plasma marker of acute myocardial infarction (AMI), and several studies have proved that, for early diagnosis of AMI, FABP performs better than myoglobin, which is a more often used early marker protein. Because serial measurement of biochemical markers in plasma is now universally accepted as an important determinant in AMI diagnosis, a rapid and continuous measuring method for FABP would be desirable. The aim of the present study was to develop an immunoassay based on the principle of displacement and using a column for rapid and continuous measurement of FABP in plasma. Glass columns filled with Sepharose-bound FABP were loaded with a horseradish peroxidase (HRP)-labeled antibody (Ab) and equilibrated with human plasma. After reaching a stable baseline, human plasma spiked with FABP or plasma from AMI patients was added. The Ab-HRP complex dissociated due to the presence of FABP in the plasma and was subsequently quantified. For plasma from AMI patients (n=5), the Ab-HRP level thus measured correlated with the corresponding plasma FABP concentration (R=0.96). The results of this study show the feasibility of a sensor for continuous monitoring of FABP in plasma.

Development of a displacement immunoassay for human heart-type fatty acid-binding protein in plasma: the basic conditions.

To risk-stratify patients with chest pain who are admitted to emergency rooms and for whom initial evaluation is not conclusive, the use of cardiac markers has become a standard procedure. A recently introduced early plasma marker for acute myocardial infarction (AMI) is the 14.5-kDa cytoplasmic heart-type fatty acid-binding protein (FABP). To fully exploit its early release from injured myocardium, a rapid method for repeated measurements or continuous monitoring of FABP in plasma is desirable. Such an on-line method could be an immunosensor based on displacement. The aim of the present study was to further investigate the principles underlying the displacement assay of FABP, both in buffer and in plasma. Batches of sepharose-bound FABP were loaded with an antibody-horseradish peroxidase (HRP) conjugate (anti-FABP). Continuous measurement of FABP was mimicked by repeated addition of FABP containing solutions followed by several washing steps. In the presence of free FABP the antibody-HRP complex dissociated and was subsequently quantified. Significant displacement in the presence of free FABP was observed in both buffer and human plasma. Anti-FABP could be intermittently displaced in the same batch, for at least 9 h, and the displacement was concentration-dependent. These results show the feasibility of a sensor based on the displacement principle to be used for the diagnosis of AMI in emergency medicine.

Tissue cultures from adult human postmortem subcortical brain areas.

Animal models used to study human aging and neurodegeneration do not display all symptoms of these processes as they are found in humans. Recently, we have shown that many cells in neocortical slices from adult human postmortem brain may survive for extensive periods in vitro. Such cultures may enable us to study age and disease related processes directly in human brain tissue. Here, we present observations on subcortical brain tissue.

Continuous 48-h C1-inhibitor treatment, following reperfusion therapy, in patients with acute myocardial infarction.

AIMS: Complement inhibition by C1-inhibitor has been shown to reduce myocardial ischaemia-reperfusion injury in animal models. We therefore studied the effects of intravenous C1-inhibitor, following reperfusion therapy, in patients with acute myocardial infarction. METHODS AND RESULTS: C1-inhibitor therapy was started not earlier than 6h after acute myocardial infarction, in order to prevent interference with thrombolytic therapy. A loading dose of C1-inhibitor was followed by a continuous infusion for 48 h, using three escalating dosage schemes. Efficacy of complement inhibition was estimated from C4 activation fragments. Plasma concentrations of myocardial proteins were compared to values measured in matched control patients. In 22 patients, C1-inhibitor was well tolerated and drug-related adverse events were not observed. Target plasma levels of C1-inhibitor were reached, with values of 48.2 ml.kg(-1) for distribution space and 35.5h for the half-life time of C1-inhibitor. A dose-dependent reduction of C4 fragments was found P=0.005). In 13 patients who received early thrombolytic therapy, release of troponin T and creatine kinase-MB(mass) was reduced by 36% and 57%P =0.001), compared to 18 controls. CONCLUSION: Continuous 48-h treatment with C1-inhibitor provides safe and effective inhibition of complement activation after reperfused acute myocardial infarction and may reduce myocardial injury.

Mutant ubiquitin expressed in Alzheimer's disease causes neuronal death.

Ubiquitin-B+1 (UBB+1) is a mutant ubiquitin that accumulates in the neurones of patients with Alzheimer's disease (AD). Here we report on the biochemical and functional differences between ubiquitin and UBB+1 and the effect of the mutant protein on neuronal cells. UBB+1 lacks the capacity to ubiquitinate, and although it is ubiquitinated itself, UBB+1 is not degraded by the ubiquitin-proteasomal system and is quite stable in neuronal cells. Overexpression of UBB+1 in neuroblastoma cells significantly induces nuclear fragmentation and cell death. Our results demonstrate that accumulation of UBB+1 in neurones is detrimental and may contribute to neuronal dysfunction in AD patients.

Is postoperative blood loss, loss of blood? A pilot study in cardiac surgical patients.

The effect of estimating the blood balance using changes in erythrocyte volumes (EVs) instead of the routinely used changes in haematocrit values was studied in 20 patients scheduled for cardiac surgery. We determined the mean haematocrit of the effluent from the postoperative thoracic drainage system at various time intervals. These data were used to more accurately calculate the blood balance. From 8h after surgery onwards, the haematocrit in the thoracic effluent was less than 10%. Total loss of thoracic effluent until 24h after removal of the aortic crossclamp (ACC) was 1,735 +/- 803 ml. Calculated blood loss until 24 h after ACC was only 58% of the total thoracic effluent. Plasma volumes in these patients increased from preoperative values of 2,505 +/- 499ml at admission to the hospital to maximum levels of 4,969 +/- 1,027 ml at 12 h after ACC (p < 0.05). Blood volume rose to 159% of the preoperative value at 12 h after ACC, whereas the EV remained relatively stable, decreasing to 95% of the preoperative value at 4 h after ACC and increasing to 107% of the baseline value at 24 h after ACC. In the meantime, patient haematocrit decreased to 78% of the reference value at the time of induction of anaesthesia at 4 h after ACC and then increased to 84% at 24 h after ACC. Thus, the use of patient haematocrit considerably overestimates blood loss. The EV appears to be a more appropiate variable than haematocrit in monitoring the blood balance in cardiac surgical patients. Future studies should reveal whether the EV is practicable in daily clinical practice.

Adenoviral vector-mediated expression of neurotrophin-3 increases neuronal survival in suprachiasmatic nucleus grafts.

To improve transplantation results of fetal suprachiasmatic nucleus (SCN) in SCN-lesioned (SCNX) rats, grafts were ex vivo transduced with an adenoviral vector encoding for neurotrophin-3 (AdNT-3) before implantation. Mock- and AdLacZ-transduced grafts were used as controls. First, transplants were evaluated microscopically and by image analysis for the presence of vasopressinergic (VPergic) and vasoactive intestinal polypeptidergic (VIPergic) SCN neurons at 10 weeks or later postgrafting. Ex vivo AdNT-3-transduced transplants displayed increased volume areas of VPergic and VIPergic SCN cells in comparison with those in mock- and AdLacZ-transduced transplants, but significantly improved graft-to-host VPergic and VIPergic SCN fiber growth was not reached (though AdNT-3-transduced transplants tended to grow more VPergic fibers into the brain of VP-deficient SCNX Brattleboro rat recipients, which were chosen as recipients to circumvent the presence of non-SCN VP fiber staining). Second, a small group of arrhythmic Wistar rats received AdNT-3- or control-treated SCN grafts while continuously on-line for the monitoring of overt circadian activities in the pre- and postgrafting periods. The results indicated that ex vivo transduced SCN grafts can still restore arrhythmia, but that the NT-3-mediated anatomical improvements of the grafting results were not sufficient to enhance efficacy of reinstatement of circadian rhythm in SCN-lesioned rats. However, in this group VIP staining volume area, not VP staining volume area, correlated significantly with reinstatement of circadian rhythm.

Assessment of coronary reperfusion in patients with myocardial infarction using fatty acid binding protein concentrations in plasma.

OBJECTIVE: To examine whether successful coronary reperfusion after thrombolytic treatment in patients with confirmed acute myocardial infarction can be diagnosed from the plasma marker fatty acid binding protein (FABP), for either acute clinical decision making or retrospective purposes. DESIGN: Retrospective substudy of the GUSTO trial. SETTING: 10 hospitals in four European countries. PATIENTS: 115 patients were treated with thrombolytic agents within six hours after the onset of acute myocardial infarction. Patency of the infarct related artery was determined by angiography within 120 minutes of the start of thrombolysis. MAIN OUTCOME MEASURES: First hour rate of increase in plasma FABP concentration after thrombolytic treatment, compared with increase in plasma myoglobin concentration and creatine kinase isoenzyme MB (CK-MB) activity. Infarct size was estimated from the cumulative release of the enzyme alpha hydroxybutyrate dehydrogenase in plasma during 72 hours, or from the sum of ST segment elevations on admission. Logistic regression analyses were performed to construct predictive models for patency. RESULTS: Complete reperfusion (TIMI 3) occurred in 50 patients, partial reperfusion (TIMI 2) in 36, and no reperfusion (TIMI 0+1) in 29. Receiver operating characteristic (ROC) curve analyses showed that the best performance of FABP was obtained when TIMI scores 2 and 3 were grouped and compared with TIMI score 0+1. The performance of FABP as a reperfusion marker was improved by combining it with alpha hydroxybutyrate dehydrogenase infarct size, but not with an early surrogate of infarct size (ST segment elevation on admission). In combination with infarct size FABP performed as well as myoglobin (areas under the ROC curve 0.868 and 0.857, respectively) and better than CK-MB (area = 0.796). At optimum cut off levels, positive predictive values were 97% for FABP, 95% for myoglobin, and 89% for CK-MB (without infarct size, 87%, 88%, and 87%, respectively), and negative predictive values were 55%, 52%, and 50%, respectively (without infarct size, 44%, 42%, and 34%). CONCLUSIONS: FABP and myoglobin perform equally well as reperfusion markers, and successful reperfusion can be assessed, with positive predictive values of 87% and 88%, or even 97% and 95% when infarct size is also taken into account. However, identification of non-reperfused patients remains a problem, as negative predictive values will generally remain below 70%.

Tropism of AAV-2 vectors for neurons of the globus pallidus.

A recombinant AAV-2 vector encoding the green fluorescent protein (gfp) under the control of the cytomegalovirus (CMV) promoter was injected into the striatum at varying antero-posterior coordinates. When the virus was delivered to the anterior part of the striatum, transduction efficiency was low and limited to the vicinity of the needle tract. In contrast, after injection into the posterior part of the striatum, in addition to a localized transduced area in the striatum, efficient and widespread transduction was observed at distance from the injection site, in the globus pallidus. In the latter case, labelled cells were also detected in the internal capsule and in the stria terminalis. The number of transduced cells in the striatum increased up to I month and then decreased whereas in the globus pallidus, transduction was maximal as early as 2 weeks post-injection. In the striatum and in the globus pallidus, the labelled cells had a neuron-like morphology. In contrast, in the internal capsule, labelled cells had a glial-like morphology.

The use of adenoviral vectors and ex vivo transduced neurotransplants: towards promotion of neuroregeneration.

Regeneration of injured axons following injury depends on a delicate balance between growth-promoting and growth-inhibiting factors. Overexpression of neurotrophin genes seems a promising strategy to promote regeneration. Trophic genes can be overexpressed at the site of injury at the axonal stumps, or at the perikaryal level of the injured neuron. Transduction of the neural cells can be achieved by applying adenoviral vectors, either directly in vivo or-in the case of neurotransplantation as an ex vivo approach. In both cases it would create a more permissive environment for axonal growth and therefore in functional regeneration. In this article, the feasibility of the use of adenoviral vectors in several neuroregeneration models--in particularly in spinal cord lesion models and the biological clock transplantation model--is illustrated. The results show that the adenoviral vectors can be a powerful tool to study the effects of overexpression of genes in an in vivo paradigm of nerve regeneration or nerve outgrowth. The potential use of adenoviral vectors and ex vivo transduced neurotransplants is discussed.

Heart fatty acid binding protein and cardiac troponin T plasma concentrations as markers for myocardial infarction after coronary artery ligation in mice.

Ligation of the main left coronary artery in mice serves as a model for myocardial infarction (MI). We tested whether plasma concentrations of heart-type fatty acid-binding protein (H-FABP) and/or cardiac troponin T (cTnT) discriminate between infarcted and sham-operated mice and allow estimation of infarct size. Mice were subjected to coronary artery ligation or sham surgery and release curves of H-FABP and cTnT were determined. At 4 h after surgery the mean (+/-SD) H-FABP plasma concentration was 461+/-134 microg/l (n=10) in MI and 185+/-51 microg/l (n=6; P<0.001) in sham-operated mice. By 24 h after surgery H-FABP levels had returned to normal in both groups. cTnT plasma concentrations increased up to 48 h after MI to 13.5+/-6.2 microg/l (n=6; P<0.001) compared with 0.031+/-0.063 microg/l (n=7) in sham-operated mice. Linear regression analysis revealed a significant correlation between plasma H-FABP at 4 h and infarct size assessed 7 days after surgery. Plasma cTnT did not correlate significantly with infarct size. In conclusion, plasma cTnT concentration at 48 h after infarction can be used to distinguish MI from sham mice, whereas H-FABP concentration at 4 h can be used for stratification of animals according to infarct size.

Peri-operative myocardial tissue injury and the release of inflammatory mediators in coronary artery bypass graft patients.

OBJECTIVE: This study was conducted to evaluate to what extent the ischemia-reperfusion injury resulting from the cardiopulmonary bypass (CPB) and aortic cross-clamping procedures during coronary artery bypass grafting (CABG) contributes to the systemic inflammatory response generally found in these patients. METHODS: Serum levels of enzymes (CK and CK-MB) and non-enzymatic proteins (FABP and myoglobin) as markers of myocardial tissue injury, bactericidal permeability increasing protein (BPI) as an indicator of neutrophil activation, interleukin-6 (IL-6) as inducer of the acute phase response and lipopolysaccharide binding protein (LBP) as parameter of the acute phase response were measured in 15 low-risk CABG patients with cardiopulmonary bypass (CPB), and 17 low-risk CABG patients without CPB. RESULTS: Already 0.5 h after reperfusion significantly increased plasma levels of all markers of myocardial tissue injury were noted in patients having surgery with CPB, but not in non-CPB patients. No significant differences were found between both groups for BPI and IL-6 levels in the early reperfusion period. BPI and IL-6 levels were higher in the non-CPB group on the first post-operative day (P < 0.05). However, no correlations were found for any marker of peri-operative tissue damage with either early neutrophil activation, or acute phase reactants. CONCLUSIONS: Perioperative myocardial injury resulting from CPB and aortic cross-clamping in low-risk CABG patients does not contribute to the release of inflammatory mediators in these patients.

Effect of acute phase response on cumulative troponin T release.

UNLABELLED: We studied a possible effect of the extent of the acute phase response after acute myocardial infarction on the cumulative release of troponin T. The height of the acute phase response might influence the cumulative release of troponin T, bound to the myofibrillar structures of the heart, in a different way compared to the free cytoplasmic cardiac marker hydroxybutyrate dehydrogenase (EC 1.1.1.27). To investigate this, the cumulative amount of C-reactive protein in plasma, i.e. the quantified acute phase response, was related to the cumulative plasma release of hydroxybutyrate dehydrogenase (an established method for infarct sizing) on the one hand and to that of troponin T on the other hand. The study was performed in patients receiving (n=16) and in patients not receiving (n=6) thrombolytic therapy. Cumulative protein release was calculated using a two-compartment model for circulating proteins. CONCLUSIONS: The cumulative amount of plasma C-reactive protein is significantly higher in the patients not receiving thrombolytic therapy, as is in accordance with earlier studies. The cumulative amount of troponin T released is significantly related to the cumulated concentration of C-reactive protein, especially in patients not receiving thrombolytic therapy. The intensity of the acute phase response, estimated from cumulative plasma C-reactive protein response, has no effect on the relative proportions of troponin T and hydroxybutyrate dehydrogenase released into plasma.

Ectopic adenoviral vector-directed expression of Sema3A in organotypic spinal cord explants inhibits growth of primary sensory afferents.

Sema3A (Sema III, SemD, collapsin-1) can induce neuronal growth cone collapse and axon repulsion of distinct neuronal populations. To study Sema3A function in patterning afferent projections into the developing spinal cord, we employed the recombinant adenoviral vector technique in embryonic rat spinal cord slices. Virus solution was injected in the dorsal aspect of organotypic spinal cord cultures with segmentally attached dorsal root ganglia (sc-DRG). In cultures grown in the presence of nerve growth factor (NGF), injected either with the control virus AdCMVLacZ or with vehicle only, afferent innervation patterns were similar to those of control. However, unilateral injection of AdCMVSema3A/AdCMVLacZ in sc-DRG slices revealed a strong inhibitory effect on NGF-dependent sensory afferent growth. Ectopic Sema3A in the dorsal spinal cord, the target area of NGF-responsive DRG fibers in vivo, created an exclusion zone for these fibers and as a result they failed to reach and innervate their appropriate target zones. Taken together, gain of Sema3A function in the dorsal aspect of sc-DRG cultures revealed a dominant inhibitory effect on NGF-dependent, nociceptive sensory DRG afferents, an observation in line with the model proposed by E. K. Messersmith et al. (1995, Neuron 14, 949-959), suggesting that Sema3A secreted by spinal cord cells can act to repel central sensory fibers during the formation of lamina-specific connections in the spinal cord.

Purification of recombinant adeno-associated virus by iodixanol gradient ultracentrifugation allows rapid and reproducible preparation of vector stocks for gene transfer in the nervous system.

Recombinant adeno-associated virus (rAAV) vectors have become attractive tools for in vivo gene transfer. The production and purification of high-titer rAAV vector stocks for experimental and therapeutic gene transfer continue to undergo improvement. Standard rAAV vector purification protocols include the purification of the vector by cesium chloride (CsCl)-density gradient centrifugation followed by extensive desalination via dialysis against a physiological buffer for in vivo use. These procedures are extremely time consuming and frequently result in a substantial loss of the infectious vector titer. As an alternative to CsCl we have investigated the use of Iodixanol, an X-ray contrast solution, as the density-gradient medium. Purification of rAAV vectors by Iodixanol shortened the centrifugation period to 3 hr and resulted in reproducible concentration and purification of rAAV-vector stocks. We show that injection of rAAV derived from an Iodixanol gradient can be used for in vivo gene transfer applications in the brain and spinal cord without detectable cytopathic effects and directing stable transgene expression for at least 2 months.

C-reactive protein as a cardiovascular risk factor: more than an epiphenomenon?

BACKGROUND: Circulating levels of C-reactive protein (CRP) may constitute an independent risk factor for cardiovascular disease. How CRP as a risk factor is involved in cardiovascular disease is still unclear. METHODS AND RESULTS: By reviewing available studies, we discuss explanations for the associations between CRP and cardiovascular disease. CRP levels within the upper quartile/quintile of the normal range constitute an increased risk for cardiovascular events, both in apparently healthy persons and in persons with preexisting angina pectoris. High CRP responses after acute myocardial infarction indicate an unfavorable outcome, even after correction for other risk factors. This link between CRP and cardiovascular disease has been considered to reflect the response of the body to the inflammatory reactions in the atherosclerotic (coronary) vessels and adjacent myocardium. However, because CRP localizes in infarcted myocardium (with colocalization of activated complement), we hypothesize that CRP may directly interact with atherosclerotic vessels or ischemic myocardium by activation of the complement system, thereby promoting inflammation and thrombosis. CONCLUSIONS: CRP constitutes an independent cardiovascular risk factor. Unraveling the molecular background of this association may provide new directions for prevention of cardiovascular events.

A new principle for rapid immunoassay of proteins based on in situ precipitate-enhanced ellipsometry.

A new technique is presented that allows measurement of protein concentrations in the picomolar range with an assay time of only 10-20 min. The method is an enzyme-linked immunosorbent assay (ELISA), but uses in-situ ellipsometric measurement of a precipitating enzyme product instead of the usual colorimetric detection of accumulating enzyme product in solution. Quantitative validation was obtained by use of annexin V, a protein with high binding affinity for phosphatidylserine-containing phospholipid membranes, resulting in a transport-limited adsorption rate. This property was exploited to obtain a range of low surface concentrations of annexin V by timed exposures of phospholipid bilayers to known concentrations of annexin V. Using polyvinylchloride (PVC)-coated and silanized silicon slides, various versions of this technique were used for the rapid assay of fatty acid-binding protein (FABP), a recently introduced early marker for acute myocardial infarction with a normal plasma concentration below 1 nmol/l, interleukin 6 (IL-6), a cytokine with normal plasma concentrations below 1 pmol/l, and again, annexin V. A possible future application of the method in the development of a one-step ELISA is discussed.