In vitro neurotoxic properties and excitatory aminoacids concentration in the cerebrospinal fluid of amyotrophic lateral sclerosis patients. Relationship with the degree of certainty of disease diagnoses.

OBJECTIVE: To determine glutamate and aspartate levels in the cerebrospinal fluid (CSF) in patients with sporadic amyotrophic lateral sclerosis (SALS) grouped according to El Escorial diagnostic criteria, and to perform an in vitro assessment of the neurotoxicity of the CSF in murine cortical neurons. METHODS: SALS patients were sorted according to El Escorial diagnostic criteria. Glutamate and aspartate were measured in the CSF using high performance liquid chromatography. Cultured cortical neuron viability was determined after exposure to CSF for 24 h. RESULTS: Glutamate levels were elevated in 28 out of the 29 patients with definite, probable or possible SALS. There were no differences in glutamate concentrations when the three clinical forms of the disease were compared; neither there were significant variation across disease duration and clinical presentation. In agreement with previous reports, we concluded that CSF-SALS-induced in vitro neurotoxicity is mediated by ionotropic glutamate receptors. We found no relationship between the degree of in vitro neurotoxicity and glutamate concentration in the CSF. CONCLUSIONS: Glutamate but not aspartate CSF levels may contribute to ALS pathogenesis. However, glutamate levels may not influence the degree of diagnosis certainty or lesion extension.

Discrepancies between the fate of myoblast xenograft in mouse leg muscle and NMR label persistency after loading with Gd-DTPA or SPIOs.

1H-NMR (nuclear magnetic resonance) imaging is regularly proposed to non-invasively monitor cell therapy protocols. Prior to transplantation, cells must be loaded with an NMR contrast agent (CA). Most studies performed so far make use of superparamagnetic iron oxide particles (SPIOs), mainly for favorable detection sensitivity. However, in the case of labeled cell death, SPIO recapture by inflammatory cells might introduce severe bias. We investigated whether NMR signal changes induced by preloading with SPIOs or the low molecular weight gadolinium (Gd)-DTPA accurately monitored the outcome of transplanted cells in a murine model of acute immunologic rejection. CA-loaded human myoblasts were grafted in the tibialis anterior of C57BL/6 mice. NMR imaging was repeated regularly until 3 months post-transplantation. Label outcome was evaluated by the size of the labeled area and its relative contrast to surrounding tissue. In parallel, immunohistochemistry assessed the presence of human cells. Data analysis revealed that CA-induced signal changes did not strictly reflect the graft status. Gd-DTPA label disappeared rapidly yet with a 2-week delay compared with immunohistochemical evaluation. More problematically, SPIO label was still visible after 3 months, grossly overestimating cell survival (<1 week). SPIOs should be used with extreme caution to evaluate the presence of grafted cells in vivo and could hardly be recommended for the long-term monitoring of cell transplantation protocols.

Periodic variation in R-R intervals and cardiovascular autonomic regulation in young adult Syrian hamsters.

Several hamster strains are commonly used as models for cardiomyopathic phenotypes evolving toward heart failure. However, little is known about heart rate variability (HRV) in this species. Prolonged surface ECG recording, a prerequisite to HRV studies, can be obtained either by telemetry or by restraints. Here, we performed long time ECG recording using telemetry on young adult Syrian hamsters and we analyzed time series of interbeat intervals. Standard statistics showed that the mean of normal R-R intervals slightly increased with age, with standard deviation of normal R-R intervals remaining stable over time. However, time domain analysis using Poincaré plots revealed dynamic changes in the HRV. Analysis of frequency domains revealed that the ratio of spectral components (low frequency/high frequency) exhibited a maturation pattern. Thus refined analysis of HRV revealed a more complex pattern than common statistical analysis would translate. Unlike other rodents, hamsters display a great spontaneous variability of their heart rate. As the complexity canvas of HRV might be the consequence of extracardiac regulation factors, we assessed the sympathovagal balance in both time and frequency domain of heart rate. Pharmacological tests revealed that both sympathetic and vagal tones contribute to HRV in Syrian hamsters. Thus Syrian hamsters have a broad intrinsic HRV with large influences of the neurovegetative system. However, the influence of the previous beat seems to prevail over the autonomic oscillators. These animals present a high sensitivity to artificially altered cardiac regulation and might be great models for the diagnosis of early alterations in the HRV related to pathology. Therefore, Syrian hamsters represent a unique model for HRV studies.

Myoblast xenotransplantation as a tool to evaluate the appropriateness of nanoparticular versus cellular trackers.

Myoblast transplantation is being considered as a potential strategy to improve muscle function in myopathies; hence, it is important to identify the transplanted cells and to have available efficient reagents to track these cells. We first validated a human to mouse xenotransplantation model warranting the complete and rapid rejection of the cells. We then used this model to assess the appropriateness of a nanoparticle reagent to track the transplanted cells. Human myoblasts were loaded with ferrite nanoparticles and injected into the tibialis muscle of immunocompetent mice. Upon collection and histological analysis of muscle sections at different time points, we observed the total disappearance of the human cells within 6 days while ferrite particles remained detectable and colocalized with mouse infiltrating and neighboring cells at the injection site. These results suggest that the use of exogenous markers such as ferrite nanoparticles may lead to false-positive results and misinterpretation of cell fate.

Non-invasive restrained ECG recording in conscious small rodents: a new tool for cardiac electrical activity investigation.

In vivo electrophysiology remains a suitable method to monitor cardiac activity; however, surface electrocardiogram (ECG) monitoring remains complicated in the case of small animals. Sedation has helped to maintain the animal still; however, it is known that anesthetic drugs impair the regulation of the cardiac electrical activity. To circumvent this problem, ECG monitoring using telemetry or restraints has been developed. This study reports a new methodology, based on a restraining system without further sedation, for recording ECGs on small animal models. We investigated its efficacy in Syrian hamsters and in several strains of mice, and we compared these data to those obtained with telemetry devices. We show that this new system can easily be used in animals of different sizes ranging from adult hamsters to newborn mice. When compared to telemetry, this restrained ECG monitoring method shows a very good yield, as 65% of total beats can be used for further analysis. When recorded in the same animals, RR intervals distributions are identical for both techniques. In conclusion, this restrained ECG monitoring technique is a well-suited tool for exploring various aspects of cardiac electrophysiology in a wide variety of small animals including very young mice.

Intrapericardial administration of novel DNA formulations based on thermosensitive Poloxamer 407 gel.

Inherited cardiopathies are leading to life-threatening conditions such as heart failure. Moreover, treatments currently available fail in altering the cardiac phenotype. Thus, gene therapy appears as an attracting alternative to conventional treatments. However, gene delivery remains a major hurdle in achieving this goal. To obtain regional delivery of plasmid DNA, intrapericardial administration seems to be an interesting approach. In order to improve retention time at the site of injection, formulations based on a thermosensitive gel of Poloxamer 407 were assessed. Protection and condensation of plasmid DNA was initially performed through complexation with polyethyleneimine (PEI), a widely used polymer. Characterization of the size and zeta potential of the complexes suggested interactions between the polyplexes and the Poloxamer gel through significant increase of the size of the polyplexes and shielding of the surface charges. In vivo evaluation has highlighted the toxicity of PEI/DNA polyplexes toward the myocardium. However, feasibility of intrapericardial injection of Poloxamer based formulations as well as their very low toxicity has been established.

Role of allopregnanolone on cerebellar granule cells neurogenesis.

The role of allopregnanolone on immature cerebellar granule cells (CGC) proliferation was studied. Allopregnanolone (0.1-1 microM) increased [(3)H]thymidine incorporation and cell number determined by neuronal counting and by an MTT colorimetric assay. The effect of the neurosteroid was completely prevented by preincubation with 10 mM MgCl(2), 10 microM nifedipine, 10 microM picrotoxin or by 50 microM bicuculine. We conclude that ALLO affects cerebellar neurogenesis by increasing calcium influx through voltage-gated calcium channels and GABA(A) receptors activation.

Cell transplantation for post-ischemic heart failure.

Post-ischemic heart failure is becoming a major issue for public health in occidental countries and therapeutical options are limited. Therefore cell transplantation was developed as an alternative strategy to improve cardiac structure and function. This review describes the multiple cell types and clinical trials considered for use in this indication. The transplantation of fetal or neonatal cardiomyocytes has proven to be functionally successful, but ethical as well as technical reasons make their clinical use limited. Recent reports, however, suggested that adult autologous cardiomyocytes could be prepared from stem cells present in various mesenchymal tissues. Alternatively, endothelial progenitors originating from bone marrow or peripheral blood could promote the neoangiogenesis within the scar tissue. Finally, the transplantation of skeletal muscle cells (SMC) in the infarcted area improved myocardial function, in correlation with the development of skeletal muscle tissue in various animal models. The latter results paved the way for the development of a first phase I clinical trial of SMC transplantation in patients with severe ischemic heart failure. It required the scale-up of human cell production according to Good Manufacturing Procedures, it started in June 2000 in Paris and was terminated in November 2001, and it was followed by several others. The results were encouraging and prompted the onset of a blinded, multicentric phase II clinical trial for SMC transplantation. Meanwhile, clinical trials also evaluate the safety and efficacy of various cells types originating from the bone marrow.

Automatic identification of patients eligible for a pneumonia guideline: comparing the diagnostic accuracy of two decision support models.

BACKGROUND: In busy clinical settings, physicians often do not have enough time to identify patients for specific therapeutic guidelines. As a solution, decision support systems could automatically identify eligible patients and trigger computerized guidelines for specific diseases. Applying this idea to community-acquired pneumonia (CAP), we developed a Bayesian network (BN) and an artificial neural network (ANN) for identifying patients who have CAP and are eligible for a pneumonia guideline. OBJECTIVE: The aim of this study was to determine whether the diagnostic accuracy of these two decision support models differs in terms of identifying CAP patients. METHODS: We trained and tested the networks with a data set of 32,662 adult patients. For each network, we (1) calculated the specificity, the positive predictive value (PPV), and the negative predictive value (NPV) at a sensitivity of 95%, and (2) determined the area under the receiver operating characteristic curve (AUC) as a measure of overall accuracy. We tested for statistical difference between the AUCs using the correlated area z statistic. RESULTS: At a sensitivity of 95%, the respective values for specificity, PPV, and NPV were: 92.3%, 15.1%, and 99.9% for the BN, and 94.0%, 18.6%, and 99.9% for the ANN. The BN had an AUC of 0.9795 (95% CI: 0.9736, 0.9843), and the ANN had an AUC of 0.9855 (95% CI: 0.9805, 0.9894). The difference between the AUCs was statistically significant (p=0.0044). CONCLUSIONS: The networks achieved high overall accuracies on the testing data set. Because the difference in accuracies is statistically significant but not clinically significant, both networks are equally suited to drive a guideline.

Hydrogen peroxide-induced neurotoxicity in cultured cortical cells grown in serum-free and serum-containing media.

To compare different culture conditions for neuroprotection assays in cultured cortic neurons, we evaluated cell viability after H2O2 exposure in cells cultured with standard N2 and with the enriched B-27 developed by GIBCO, both serum-free supplements. The following additives/associations were compared: N2 (+N2), B-27 (+B-27), 10% FBS (+FBS), 1% FBS in combination with N2 (FBS/N2) or N2 supplement preceded by an 1 hour precoating with 10% FBS (N2 + precoated). Our data demonstrated that B-27 is as efficient as 10% FBS to support neuronal growth for more than a week. As shown by phase-contrast optics cells grown in N2 started degenerating within 24-48 hours although measurable absorbance was seen with MTT. The pre-coating procedure failed to modify substantially cell viability as compared with N2 alone. Dose-response curves for H2O2 to induce neuronal apoptosis were almost identical for B-27 and serum supplemented samples. Catalase (100 U/ml) or vitamin E (200 microM) prevented cell death in both culture conditions. Our results indicate that DMEM/B-27 provides a serum-free cell culture environment that allows neurons to grow with optimal cell viability, comparable to that obtained with serum. We conclude that this culture condition reveals as a useful tool to test the efficacy of neuroprotectants when a serum free medium is required.

Electrotransfer of naked DNA in the skeletal muscles of animal models of muscular dystrophies.

The electrotransfer of naked DNA has recently been adapted to the transduction of skeletal muscle fibers. We investigated the short- and long-term efficacy of this methodology in wild-type animals and in mouse models of congenital muscular dystrophy (dy/dy, dy(2J)/dy(2J)), or Duchenne muscular dystrophy (mdx/mdx). Using a reporter construct, the short-term efficacy of fiber transduction reached 40% and was similar in wild-type, dy/dy and dy(2J)/dy(2J) animals, indicating that ongoing muscle fibrosis was not a major obstacle to the electrotransfer-mediated gene transfer. Although the complete rejection of transduced fibers was observed within 3 weeks in the absence of immunosuppression, the persistency was prolonged over 10 weeks when transient or continuous immunosuppressive regimens were used. Using therapeutic plasmids, we demonstrated that electrotransfer also allowed the transduction of large constructs encoding the laminin alpha2 chain in dy/dy mouse, or a chimeric dystrophin-EGFP protein in mdx/mdx mouse. The correct sarcolemmal localization of these structural proteins demonstrated the functional relevance of their expression in vivo, with a diffusion domain estimated to be 300 to 500 microm. However, degeneration-regeneration events hampered the long-term stability of transduced fibers. Given its efficacy for naked DNA transfer in these models of muscular dystrophies, and despite some limitations, gene electrotransfer methodology should be further explored as a potential avenue for treatment of muscular dystrophies.

A single-cell model to study changes in neuronal fractal dimension.

Many methods have been developed to quantify neuronal morphology: measurement of neurite length, neurite number, etc. However, none of these approaches provides a comprehensive view of the complexity of neuronal morphology. In this work we have analyzed the evaluation of fractal dimension (D) as a tool to represent and quantify changes in complexity of the dendritic arbor, in in vitro cultures grown under low-density conditions. Neurons grown in isolation developed a bipolar morphology corresponding to a fractal dimension close to the unit. The analysis showed that neuronal complexity increased when cells were incubated with a depolarizing potassium concentration and there was a correlation with an increase in fractal dimension (D5 mM KCl = 1.08 +/- 0.01, D25 mM KCl =1.25 +/- 0.01). We conclude that fractal dimension is a suitable parameter to quantify changes in neuronal morphological complexity.

COOH-terminal truncated human cardiac MyBP-C alters myosin filament organization.

Myosin-binding protein C (MyBP-C) is thought to play structural and/or regulatory role in striated muscles. The cardiac isoform of MyBP-C is one of the disease genes associated with familial hypertrophic cardiomyopathy and most of the mutations produce COOH truncated proteins. In order to determine the consequences of these mutations on myosin filament organization, we have characterized the effect of a 52-kDa NH2-terminal peptide of human cardiac MyBP-C on the alpha-myosin heavy chain (alpha-MyHC) filament organization. This peptide lacks the COOH-terminal MyHC-binding site and retains the two MyHC-binding domains located in the N-terminal part of MyBP-C. For this characterization, cDNA constructs (rat alpha-MyHC, full-length and truncated human cardiac MyBP-C) were transiently expressed singly or in pairwise combination in COS cells. In conformity with previous works performed on the skeletal isoform of MyBP-C, we observed that full-length cardiac MyBP-C organizes the MyHC into dense structures of uniform width. While the truncated protein is stable and can interact with MyHC in COS cells, it does not result in the same organization of sarcomeric MyHC that is seen with the full-length MyBP-C. These results suggest that the presence of truncated cardiac MyBP-C could, at least partly, disorganize the sarcomeric structure in patients with familial hypertrophic cardiomyopathy.

Factors affecting functional outcome after autologous skeletal myoblast transplantation.

BACKGROUND: This study assessed the extent to which the initial degree of functional impairment and the number of injected cells may influence the functional improvement provided by autologous skeletal myoblast transplantation into infarcted myocardium. METHODS: One week after left coronary artery ligation, 44 rats received into the infarcted scar, autologous skeletal myoblasts expanded in vitro for 7 days (mean, 3.5 x 10(6), n = 21), or culture medium alone (controls, n = 23). Left ventricular function was assessed by two-dimensional echocardiography. RESULTS: When transplanted hearts were stratified according to their baseline ejection fraction, a significant improvement occurred at 2 months in the less than 25% (from 21.4% to 37%), 25% to 35% (from 29% to 43.8%), and in the 35% to 40% (from 37.2% to 41.7%) groups, compared to controls (p = 0.048, 0.0057, and 0.034, respectively), but not in the more than 40% stratum. A significant linear relationship was found between the improvement in ejection fraction and the number of injected myoblasts, both at 1 and 2 months after transplantation (p < 0.0001). CONCLUSIONS: Autologous myoblast transplantation is functionally effective over a wide range of postinfarct ejection fractions, including in the sickest hearts provided that they are injected with a sufficiently high number of cells.

Quantifying the characteristics of unambiguous chest radiography reports in the context of pneumonia.

RATIONALE AND OBJECTIVES: The purpose of this study was to statistically identify some characteristics of unambiguous (ie, clear) chest radiography reports in the context of acute bacterial pneumonia. MATERIALS AND METHODS: Seven physicians individually read 292 chest radiography reports to determine if they contained radiologic evidence of pneumonia. Unambiguous reports were defined as those that physicians unanimously classified as supporting or not supporting the diagnosis of pneumonia. Ambiguous reports were assigned degrees of ambiguity on the basis of how much disagreement they caused among the physicians. Characteristics of unambiguous reports as described in the literature were manually quantified and assigned to every report. To identify characteristics that statistically distinguished unambiguous from ambiguous reports, the authors performed an ordinal logistic regression analysis for which the dependent variable was the number of dissenting votes the report received and the independent variables were the quantified characteristics of the report. RESULTS: Six independent variables were statistically significantly associated with unambiguous reports (P < .05). Three were positively associated: an interpretation of whether findings supported the diagnosis of pneumonia in reports with pneumonia-related observations, short sentences, and redundancy of pneumonia-related observations. Three were negatively associated: high use of uncertainty modifiers for pneumonia-related observations, use of only descriptive terms to describe pneumonia-related observations, and insufficient amount of pneumonia-related information. CONCLUSION: The most influential characteristic of an unambiguous chest radiography report was an interpretation of whether the radiograph supported the diagnosis of pneumonia when findings could be indicative.

Combining decision support methodologies to diagnose pneumonia.

OBJECTIVE: To evaluate the performance of a computerized decision support system that combines two different decision support methodologies (a Bayesian network and a natural language understanding system) for the diagnosis of patients with pneumonia. DESIGN: Evaluation study using data from a prospective, clinical study. PATIENTS: All patients 18 years and older who presented to the emergency department of a tertiary care setting and whose chest x-ray report was available during the encounter. METHODS: The computerized decision support system calculated a probability of pneumonia using information provided by the two systems. Outcome measures were the area under the receiver operating characteristic curve, sensitivity, specificity, predictive values, likelihood ratios, and test effectiveness. RESULTS: During the 3-month study period there were 742 patients (45 with pneumonia). The area under the receiver operating characteristic curve was 0.881 (95% CI: 0.822, 0.925) for the Bayesian network alone and 0.916 (95% CI: 0.869, 0.949) for the Bayesian network combined with the natural language understanding system (p=0.01). CONCLUSION: Combining decision support methodologies that process information stored in different data formats can increase the performance of a computerized decision support system.

Automatic detection of acute bacterial pneumonia from chest X-ray reports.

OBJECTIVE: To evaluate the performance of a natural language processing system in extracting pneumonia-related concepts from chest x-ray reports. DESIGN: Four physicians, three lay persons, a natural language processing system, and two keyword searches (designated AAKS and KS) detected the presence or absence of three pneumonia-related concepts and inferred the presence or absence of acute bacterial pneumonia from 292 chest x-ray reports. Gold standard: Majority vote of three independent physicians. Reliability of the gold standard was measured. OUTCOME MEASURES: Recall, precision, specificity, and agreement (using Finn's R: statistic) with respect to the gold standard. Differences between the physicians and the other subjects were tested using the McNemar test for each pneumonia concept and for the disease inference of acute bacterial pneumonia. RESULTS: Reliability of the reference standard ranged from 0.86 to 0.96. Recall, precision, specificity, and agreement (Finn R:) for the inference on acute bacterial pneumonia were, respectively, 0.94, 0.87, 0.91, and 0.84 for physicians; 0.95, 0.78, 0.85, and 0.75 for natural language processing system; 0.46, 0.89, 0.95, and 0.54 for lay persons; 0.79, 0.63, 0.71, and 0.49 for AAKS; and 0.87, 0.70, 0.77, and 0.62 for KS. The McNemar pairwise comparisons showed differences between one physician and the natural language processing system for the infiltrate concept and between another physician and the natural language processing system for the inference on acute bacterial pneumonia. The comparisons also showed that most physicians were significantly different from the other subjects in all pneumonia concepts and the disease inference. CONCLUSION: In extracting pneumonia related concepts from chest x-ray reports, the performance of the natural language processing system was similar to that of physicians and better than that of lay persons and keyword searches. The encoded pneumonia information has the potential to support several pneumonia-related applications used in our institution. The applications include a decision support system called the antibiotic assistant, a computerized clinical protocol for pneumonia, and a quality assurance application in the radiology department.

Intramyocardial transplantation of autologous myoblasts: can tissue processing be optimized?

BACKGROUND: Autologous skeletal myoblast (SM) transplantation improves function of infarcted myocardium, but pretransplantation cultures remain a complex process. This study assessed whether it could be optimized by muscle preconditioning with the local anesthetic bupivacaine or even bypassed with the use of the so-called mince technique. METHODS AND RESULTS: Muscle preconditioning consisted of intramuscular injections of the tibialis anterior of rats, 2 days before harvest. After 7 days of culture, the number of available myoblasts was significantly increased compared with nonconditioned controls (1 683 147 versus 85 300, P:=0.0013). The mince technique was then assessed. A myocardial infarction was created in 66 rats by coronary artery ligation. One week later, rats were reoperated on and intramyocardially injected with culture medium alone (controls, n=23), autologous cultured SM (3.5 x 10(6), n=21), or autologous muscle minced into a fine slurry, which was immediately transplanted (n=22). All muscles had been preconditioned. Left ventricular function was assessed by 2D echocardiography. Whereas end-diastolic volumes expanded over time in all groups, left ventricular ejection fraction (%, mean+/-SEM) was increased only in the cultured SM-transplanted group at 1 (P:=0. 0006) and 2 months (P:=0.0008) versus baseline (37.52+/-1.92 and 40. 92+/-2.17 versus 30.34+/-1.74), with a significant additional benefit between 1 and 2 months (P:=0.0069). CONCLUSIONS: Cell culture remains mandatory for SM transplantation to be successful but, in a clinical perspective, this process can be made more expeditious by preharvest muscle conditioning with bupivacaine, which greatly enhances the baseline cell yield.