Myocardial infarction does not affect circulating haematopoietic stem and progenitor cell self-renewal ability in a rat model.

NEW FINDINGS: What is the central question of this study? Although peripheral blood haematopoietic stem and progenitor cells are potentially important in regeneration after acute myocardial infarction, their self-renewal ability in the post-acute phase has not yet been addressed. What is the main finding and its importance? In rat peripheral blood, we show that myocardial infarction does not negatively affect circulating haematopoietic stem and progenitor cell self-renewal ability 2 weeks after acute infarction, which suggests a constant regenerative potential in the myocardial infarction post-acute phase. Given the importance of peripheral blood haematopoietic stem and progenitor cells (HPCs) in post-acute regeneration after acute myocardial infarction (MI), the aim of the present study was to investigate the number and secondary replating capacity/self-renewal ability of HPCs in peripheral blood before and 2 weeks after MI. In female Lewis inbred rats (n = 9), MI was induced by ligation of the left coronary artery, and another nine underwent sham surgery, without ligation, for control purposes. Myocardial infarction was confirmed by troponin I concentrations 24 h after surgery. Peripheral blood was withdrawn and fractional shortening and ejection fraction of the left ventricle were assessed before (day 0) and 14 days after MI or sham surgery (day 14). After mononuclear cell isolation, primary and secondary functional colony-forming unit granulocyte-macrophage (CFU-GM) assays were performed in order to detect the kinetics of functional HPC colony counts and cell self-renewal ability in vitro. The CFU-GM counts and cell self-renewal ability remained unchanged (P > 0.05) in both groups at day 14, without interaction between groups. In the intervention group, higher day 0 CFU-GM counts showed a relationship to lower fractional shortening on day 14 (ρ = -0.82; P < 0.01). Myocardial infarction did not negatively affect circulating HPC self-renewal ability, which suggests a constant regenerative potential in the post-acute phase. A relationship of cardiac contractile function 14 days after MI with circulating CFU-GM counts on day 0 might imply functional colony count as a predictive factor for outcome after infarction.

Combined transcriptome studies identify AFF3 as a mediator of the oncogenic effects of ß-catenin in adrenocortical carcinoma.

Adrenocortical cancer (ACC) is a very aggressive tumor, and genomics studies demonstrate that the most frequent alterations of driver genes in these cancers activate the Wnt/ß-catenin signaling pathway. However, the adrenal-specific targets of oncogenic ß-catenin-mediating tumorigenesis have not being established. A combined transcriptomic analysis from two series of human tumors and the human ACC cell line H295R harboring a spontaneous ß-catenin activating mutation was done to identify the Wnt/ß-catenin targets. Seven genes were consistently identified in the three studies. Among these genes, we found that AFF3 mediates the oncogenic effects of ß-catenin in ACC. The Wnt response element site located at nucleotide position -1408 of the AFF3 transcriptional start sites (TSS) mediates the regulation by the Wnt/ß-catenin signaling pathway. AFF3 silencing decreases cell proliferation and increases apoptosis in the ACC cell line H295R. AFF3 is located in nuclear speckles, which play an important role in RNA splicing. AFF3 overexpression in adrenocortical cells interferes with the organization and/or biogenesis of these nuclear speckles and alters the distribution of CDK9 and cyclin T1 such that they accumulate at the sites of AFF3/speckles. We demonstrate that AFF3 is a new target of Wnt/ß-catenin pathway involved in ACC, acting on transcription and RNA splicing.

Covalent immobilisation of VEGF on plasma-coated electrospun scaffolds for tissue engineering applications.

Recent findings in the field of biomaterials and tissue engineering provide evidence that surface immobilised growth factors display enhanced stability and induce prolonged function. Cell response can be regulated by material properties and at the site of interest. To this end, we developed scaffolds with covalently bound vascular endothelial growth factor (VEGF) and evaluated their mitogenic effect on endothelial cells in vitro. Nano- (254±133 nm) or micro-fibrous (4.0±0.4 µm) poly(ɛ-caprolactone) (PCL) non-wovens were produced by electrospinning and coated in a radio frequency (RF) plasma process to induce an oxygen functional hydrocarbon layer. Implemented carboxylic acid groups were converted into amine-reactive esters and covalently coupled to VEGF by forming stable amide bonds (standard EDC/NHS chemistry). Substrates were analysed by X-ray photoelectron spectroscopy (XPS), enzyme-linked immuno-assays (ELISA) and immunohistochemistry (anti-VEGF antibody and VEGF-R2 binding). Depending on the reaction conditions, immobilised VEGF was present at 127±47 ng to 941±199 ng per substrate (6mm diameter; concentrations of 4.5 ng mm(-2) or 33.3 ng mm(-2), respectively). Immunohistochemistry provided evidence for biological integrity of immobilised VEGF. Endothelial cell number of primary endothelial cells or immortalised endothelial cells were significantly enhanced on VEGF-functionalised scaffolds compared to native PCL scaffolds. This indicates a sustained activity of immobilised VEGF over a culture period of nine days. We present a versatile method for the fabrication of growth factor-loaded scaffolds at specific concentrations.

Evaluation of the PAH and water-extractable phenols content in used cross ties from the French rail network.

Recycling used railway sleepers is a major economic and environmental issue since nearly 50000 tons of those are incinerated every year in France. Therefore, it appeared essential to determine the real toxicity of sleepers and particularly for very old one. They are treated with creosote, which contains toxic and carcinogen compounds such as polycyclic aromatic hydrocarbons (PAHs). This study aims at measuring the amount of 16 priority PAHs and water extractable phenols in 12 sleepers implemented between 1936 and 1978. Results showed that the creosote content was systematically far above 1000mgkg(-1), even after 76years ageing. Crossties should then be considered as a hazardous waste according to European regulations. Less creosote and PAHs were detected in the sleepers centers. Moreover, the fraction of volatile PAHs was lower in the surface part, due to their evaporation. It appeared that a long ageing process was not sufficient to remove the major part of volatile PAHs and that they could be yet released in the atmospheric environment. Moreover, most of the treated crossties contained huge amount of the highly toxic benzo[a]pyrene, between 179mgkg(-1) and up to 853mgkg(-1) in wood. In contrast, the study revealed that concentrations of water extractable phenols were well below European regulations (3% by mass of creosote).

Fine-tuning of substrate architecture and surface chemistry promotes muscle tissue development.

Tissue engineering has been increasingly brought to the scientific spotlight in response to the tremendous demand for regeneration, restoration or substitution of skeletal or cardiac muscle after traumatic injury, tumour ablation or myocardial infarction. In vitro generation of a highly organized and contractile muscle tissue, however, crucially depends on an appropriate design of the cell culture substrate. The present work evaluated the impact of substrate properties, in particular morphology, chemical surface composition and mechanical properties, on muscle cell fate. To this end, aligned and randomly oriented micron (3.3±0.8 µm) or nano (237±98 nm) scaled fibrous poly(ε-caprolactone) non-wovens were processed by electrospinning. A nanometer-thick oxygen functional hydrocarbon coating was deposited by a radio frequency plasma process. C2C12 muscle cells were grown on pure and as-functionalized substrates and analysed for viability, proliferation, spatial orientation, differentiation and contractility. Cell orientation has been shown to depend strongly on substrate architecture, being most pronounced on micron-scaled parallel-oriented fibres. Oxygen functional hydrocarbons, representing stable, non-immunogenic surface groups, were identified as strong triggers for myotube differentiation. Accordingly, the highest myotube density (28±15% of total substrate area), sarcomeric striation and contractility were found on plasma-coated substrates. The current study highlights the manifold material characteristics to be addressed during the substrate design process and provides insight into processes to improve bio-interfaces.

[MR imaging of the pancreas]. / IRM et maladies pancréatiques.

Magnetic resonance (MR) imaging of the pancreas has undergone a major change because it can provide noninvasive images of the pancreatic ducts and the parenchyma. MR cholangiopancreatography (MRCP) enables detection of anatomic variants such as pancreas divisum. Although contrast material-enhanced CT is still considered the gold standard in acute pancreatitis and for the detection of calcifications in chronic pancreatitis, MR imaging and secretin-enhanced MRCP are useful in evaluating pseudocysts and pancreatic disruption. The role of MR is still debated in pancreatic neoplasms except the cystic lesions where MR imaging provides critical information regarding the lesion's content and a possible communication with the pancreatic ducts. MRCP and MR of the pancreas are also useful in identifying other pancreatic diseases such as lymphoplasmocytic pancreatitis and groove pancreatitis.

[Intraductal papillary mucinous tumours of the pancreas: imaging features]. / Aspect radiologique des tumeurs intracanalaires pancréatiques mucineuses et papillaires.

IPMTP is a pancreatic duct disease that can better be diagnosed due to advances in imaging techniques. This probably explains the recent increased frequency of this disease. Enlargement of the main pancreatic duct and/or branch ducts is a typical feature. CT and MRI with MRCP are useful for diagnosis. Features of malignant degeneration are better known. Preoperative staging is performed at CT. Differential diagnosis includes main pancreatic duct dilatation and pancreatic cysts. Recent papers indicate that isolated side branch IPMTP is less frequently malignant. Surgery is indicated in the presence of acute pancreatitis or suspicion of malignant degeneration. Imaging is useful for the follow up of patients with isolated side branch IPMTP. In this paper, the diagnostic, staging and malignant features of IPMTP will be reviewed.

Genetic control of autoantibody expression in autoimmune myasthenia gravis: role of the self-antigen and of HLA-linked loci.

Autoantibodies against the muscle acetylcholine receptor (AChR) play an essential role in the pathophysiology of autoimmune myasthenia gravis (MG). Their serum titers, however, vary considerably among patients. Our aim was to investigate whether their variation might be explained by genetic factors. Using different methods, we have obtained strong evidence for a three-locus association influencing autoantibody titers in MG patients with thymus hyperplasia or with a normal thymus. Two of the loci, one encoding the AChR alpha-subunit, the other encoding the alpha-chain of the class II antigen-presentation molecule, HLA-DQ, demonstrated interaction to determine high autoantibody titers. The third locus was associated with the 8.1 ancestral HLA haplotype. It exerted an additive effect and it is postulated to have a nonantigen specific immunoregulatory function. Our study demonstrates for the first time that polymorphism of an autoantigen gene may quantitatively modify the immune response against it. Altogether, the data lend support to a three-gene model to explain autoantibody expression in a subset of MG patients.

Association of the gene encoding the delta-subunit of the muscle acetylcholine receptor (CHRND) with acquired autoimmune myasthenia gravis.

The muscle acetylcholine receptor (AChR) is the main target self-antigen in acquired autoimmune myasthenia gravis (MG). Here, we investigated an association of MG with the CHRND gene encoding the delta-subunit of the AChR. Using a microsatellite repeat located in the second intron of the gene, we observed a preferential transmission of the allele 268 in 114 one-generation families with one myasthenic child (Pc=0.0154). This allele was also over-represented in a group of 350 unrelated nonthymoma MG patients (OR=1.78, P=0.038), but not in 84 thymoma patients, compared to 168 healthy controls. Moreover, among nonthymoma patients, those lacking serum anti-titin antibodies appeared to be best associated (OR=2.07, P=0.017). In contrast, there was no distortion in the transmission of a single-nucleotide substitution polymorphisms (SNPs) in the 3' untranslated region of CHRND nor in that of two SNPs located in the closely linked CHRNG gene, 4.5 kb telomeric to CHRND. The data warrant a detailed investigation of CHRND polymorphism in MG patients.

Linkage of HLA to myasthenia gravis and genetic heterogeneity depending on anti-titin antibodies.

BACKGROUND: MG is an autoimmune disease of the neuromuscular junction. MG with thymus hyperplasia has been associated with, but not genetically linked to, the HLA-DR3 haplotype. OBJECTIVE: To re-evaluate the association of HLA with MG in 656 patients with generalized disease and to test linkage of HLA to MG with thymus hyperplasia. METHOD: Patients were genotyped for HLA-DRB1. Data analysis included case-control comparisons after subgrouping patients by thymus histopathology. The transmission of parental alleles to MG offspring with thymus hyperplasia was studied in simplex families using the transmission/disequilibrium test (TDT) as a test of linkage. RESULTS: MG with thymus hyperplasia was positively associated with DR3 (OR = 4.5, p = 1 x 10(-6)) and negatively associated with DR7 (OR = 0.28, p = 1 x 10(-6)), based on both case-control comparisons and TDT. No association was detected with thymomas. Conversely, patients who lacked thymus anomalies but expressed anti-titin antibodies (ATA) had an increase of DR7 (OR = 2.08, p = 4 x 10(-3)) and a decrease of DR3 (OR = 0.33, p = 9 x 10(-3)). CONCLUSIONS: The authors established linkage of HLA to MG and thymus hyperplasia, defining the MYAS1 locus. Moreover, DR3 and DR7, or closely linked genes, have opposing effects on MG phenotypes. Nonthymomatous patients with ATA may be a pathogenetically distinct subset of MG patients.

A side-reaction in the SPPS of Trp-containing peptides.

Syntheses of several Trp-containing peptides on a Wang solid support afforded significant amounts of a side-product. 1H-NMR and MS data showed that an unexpected alkylation by the linker has occurred on the indole nucleus. This was observed whatever the scavenger used, and whatever the position of the Trp residue in the sequence, unless it was in the C-terminal position.

Structure of ubiquitin-conjugating enzyme 9 displays significant differences with other ubiquitin-conjugating enzymes which may reflect its specificity for sumo rather than ubiquitin.

The three-dimensional structure of ubiquitin-conjugating enzyme 9 (Ubc9) has been obtained to a resolution of 2.8 A by molecular replacement followed by a combination of automated refinement and graphical intervention. Diffraction data were recorded on a single crystal in space group P43 with cell dimensions a = b = 73.9, c = 42. 9 A. The final model has an R factor of 21.3% for all data to 2.8 A. Only the N-terminal methionine, a two-residue N-terminal extension and a four-residue loop are not located by the final electron-density map. Ubc9 is now known to be the first sumo, a new ubiquitin-like protein, conjugating enzyme and does not conjugate ubiquitin. The structure of Ubc9 shows important differences compared with the structures of known ubiquitin-conjugating enzymes. At the N-terminal helix, the structural and sequence alignments are out of register by one amino acid giving Ubc9 a different recognition surface compared to ubiquitin-conjugating enzymes. This is coupled to a profound change in the electrostatic surface of the molecular face remote from the catalytic site. These differences may be important in recognition of other proteins in the Sumo conjugation pathway. The catalytic cysteine in Ubc9 has a positively charged lip and a negatively charged ridge nearby. Both these features seem confined to sumo-conjugating enzymes, and a sequence alignment of sumo and ubiquitin suggests how these might play a role in sumo/ubiquitin discrimination.

The absence of the mitochondrial ATP synthase delta subunit promotes a slow growth phenotype of rho- yeast cells by a lack of assembly of the catalytic sector F1.

In the yeast Saccharomyces cerevisiae, inactivation of the gene encoding the delta subunit of the ATP synthase led to a lack of assembly of the catalytic sector. In addition a slow-growth phenotype was observed on fermentable medium. This alteration appears in strains lacking intact mitochondrial DNA and showing a defect in the assembly of the catalytic sector, such as the yeast strain inactivated in the gene encoding the epsilon subunit. In rho mitochondria having an intact F1, the ion movement resulting from the exchange of ADP formed in the organelle and ATP entering the mitochondrial compartment led to a mitochondrial transmembranous potential delta psi that was sensitive to carboxyactractyloside. This ion movement was dramatically decreased in rho mitochondria lacking the delta subunit and thus the F1 sector, whereas a cell devoid of delta subunit and complemented with a plasmid harboring the ATPdelta gene displayed an assembled F1, a normal generation time and a fully restored mitochondrial potential. This result could be linked to the involvement of the membrane potential delta psi which is indispensible for mitochondrial biogenesis.

N-conjugates of 2,5-disubstituted pyrrole and glutathione. Evaluation of their potency as antioxidants against photosensitization of NCTC 2544 keratinocytes by excess endogenous protoporphyrin IX.

A novel glutathione compound in which the amino group has been derivatized by a 2,5-dimethyl pyrrole is shown to be very effective against cell photosensitization in vitro. Protoporphyrin IX either added to the medium or produced endogenously by incubation of NCTC 2544 keratinocytes with 5-aminolevulinic acid has been chosen as the photosensitizer. The antioxidant effectiveness of glutathione-pyrrole derivatives against protoporphyrin photosensitization depends critically on the type of 2,5 substitution on the pyrrole ring. This structure-function relationship may be attributed to the difference in compartmentation and/or uptake of the various glutathione-pyrrole derivatives under study. The 2,5-dimethyl pyrrole derivative is much more effective than glutathione as a protective agent against phototoxic reactions induced by protoporphyrin IX.

[Criteria for quantification and characterization of aneurysms of the abdominal aorta using ultrasonography. The AFFCA study. French Association of Continuous Education in Angiology]. / Critères de quantification et de caractérisation des anévrysmes de l'aorte abdominale par l'échographie. Etude AFFCA. Association Française de Formation Continue en Angiologie.

OBJECTIVE: To validate ultrasonographic quantification and characterization criteria for aneurysms of the abdominal aorta (AAA) by comparing computed tomography data and surgical findings in order to standardize diagnosis and follow-up of AAA. PATIENTS AND METHODS: A multicentric prospective study included 80 patients with AAA (January to August 1996). Standardized data on quantitative (diameter, surface area) and qualitative (topography, form, wall, thrombus) data were obtained at each ultrasound examination and at surgery. Ultrasound (US) and computed tomography (CT) explorations were performed and interpreted without prior knowledge by independent operators. US findings versus CT reference were compared in 77 cases, US findings versus surgical reference in 31, and CT findings versus surgery reference in 28. RESULTS: Compared with CT-scan, B mode ultrasound (n = 77) underestimated the anteroposterior diameter of the aneurysm (mean difference -2.16 mm; p < 0.001), the anteroposterior diameters of the flow channel lumen (-5.54 mm; p < 0.001) and upper neck of the aneurysm (-2.74 mm; p < 0.001). Surface area measurements were not significantly different both the aneurysmal sac (p = 0.3) and the flow channel lumen (p = 1). Compared with surgical findings, US (n = 30) underestimated the transverse diameter (mean difference (-4.29 mm; p = 0.0037). Compared with surgery, US findings (n = 28) were not significantly different for the anteroposterior ans transverse diameters. Analyzing the form and wall of the AAA, US/CT-scan performance was good for symmetrical fusiform aneurysms (sensitivity 77%, specificity 67%). US and CT-scan were reliable for detecting wall bugles (same sensitivity, 29%). The angle formed by the thrombus with the wall (expressed in degrees) was not significantly different (p = 0.9). When the lower pole of the aneurysm was situated above the aortic bifurcation, US (sensitivity 75%) was more reliable than the CT-scan (sensitivity 50%) compared with surgical findings. CONCLUSION: Ultrasonography should not be limited to the simple positive diagnosis of AAA. A precise analysis of the diameters and surface areas of the aneurysm should be performed.

Laryngotracheal stenosis after intubation or tracheostomy in patients with neurological disease.

OBJECTIVE: This retrospective study evaluated the incidence of airway complications in neurological patients following translaryngeal intubation, tracheostomy, or both. DESIGN: The medical records of 315 consecutive patients (200 with traumatic brain injuries, 31 traumatic tetraplegics, and 84 with other neurological disorders) were reviewed. The type of artificial airway, duration of intubation, and use of nocturnal ventilation were recorded. Eighty-six percent of the patients underwent some combination of tracheal tomograms, flow-volume loop analysis, and fiberoptic tracheolaryngoscopy. Stenosis was classified as severe if it required surgery, if it required maintaining the tracheostomy, or was lethal. It was classified as benign if it was successfully treated by medical or local means. RESULTS: Fifty-five percent of the patients were intubated translaryngeally only (mean = 17 days). Three percent underwent tracheostomy only, and 42% underwent tracheostomy after intubation for a mean of 13 days. The overall incidence of airway stenosis was 20%, 1/4 of which was severe. Fifteen percent of these patients died as a result of tracheal complications. The incidence of stenosis was higher following tracheostomy than following intubation only (29% vs 13%, p < .01). The incidence of severe stenosis in intubated-only patients was low (1%) compared with that following tracheostomy (10%, p < .01). No significant relationship was found between the length of intubation or the timing of tracheostomy. CONCLUSION: Fewer complications are associated with transtracheal intubation than with tracheostomy. The data suggest that longer periods of intubation be used when attempting ventilator weaning before restoring to tracheostomy if weaning fails.

Clinical and molecular heterogeneity of cytochrome c oxidase deficiency in the newborn.

We report 8 cases of severe cytochrome c oxidase deficiency with onset in the neonatal period. Clinical symptoms were heterogeneous: antenatal cerebral malformations, neurological distress with ketoacidosis, severe myopathy, or isolated respiratory control failure. Lactic acid was elevated in blood and/or CSF in 7 cases. Muscle biopsy (7 patients), liver biopsy (4 patients), and cultured skin fibroblasts (7 patients) were used to assess the cytochrome c oxidase deficiency. Among the patients, the enzymatic defect differed in the level of residual activity, expression in different tissues and subunit composition in muscle (as analysed by immunohistochemistry). Southern blot analysis of the mitochondrial DNA was normal in 7 patients. The heterogeneity of cytochrome c oxidase deficiency was therefore demonstrated by these clinical presentations and by the biochemical assessment of the enzyme defect. This reflects, most probably, the diverse nature of the causal mutations.

Morphological studies of skeletal muscle in lactic acidosis.

This paper underscores the contribution of routine morphological examination of skeletal muscle in patients with lactic acidosis. Mitochondrial disorders are by far the most common causes of primary lactic acidosis, in which muscle biopsy analysis helps in diagnosis and in the search for the molecular anomalies. Thus, we focus our attention on one particular point: the contribution of the morphological study of muscle biopsy in primary lactic acidosis due to mitochondrial disorders, especially mitochondrial respiratory-chain diseases.