Coronary arterial compression testing by simultaneous balloon valvuloplasty and coronary angiography in an English bulldog with pulmonary valve stenosis.

A 4-year-old male neutered English bulldog presented for heart murmur evaluation. Echocardiography identified severe pulmonic stenosis (an echocardiography-derived transpulmonary pressure gradient of 100 mmHg), and computed tomography confirmed the presence of an anomalous coronary artery with a prepulmonic course of the left coronary artery arising from the right coronary ostium. Before artificial pulmonic valve implantation, a coronary compression test was performed. A simultaneous aortic root angiogram and pulmonic balloon valvuloplasty revealed complete occlusion of the circumflex branch. Artificial valve implantation was aborted with concern for fatal coronary compression after implantation. Coronary compression testing is a critical component of the evaluation before catheter-based implantation of conduits across the pulmonic valve.

Production of fructooligosaccharides by Bacillus subtilis natto CCT7712 and their antiproliferative potential.

AIMS: Fructooligosaccharides (FOSs) known for their health properties and ß-(2→6)-levan-type FOSs have shown prebiotic and immunomodulatory activities that overcome those of commercial ß-(2→1)-FOSs, but costs do not favour their use. Moreover, FOSs can reach the bloodstream through the diet, and little is known about their direct effect on cells. The aim of this work was to produce high-content FOSs by Bacillus subtilis natto CCT7712 in a bioreactor using commercial sucrose and to evaluate their antiproliferative effects in OVCAR-3 cells. METHODS AND RESULTS: FOS production reached 173·60 g l-1 , 0·2 vvm aeration and uncontrolled pH. Levan-type FOSs, composed of ß-(2 â†’ 6) links and mainly GF3 (6-nystose), were identified using RMN spectroscopy, FT-IR and ESI-MS. FOSs decreased the viability and proliferation of OVCAR-3 cells, and the effects were associated with an increased pro-inflammatory response by the induction of IL-8 and TNF-α, and the repression of ER-ß genes. The metabolic profiles showed disruption of cellular homeostasis that can be associated with a decrease in proliferation. CONCLUSIONS: The high production of levan-type FOSs from B. subtilis natto CCT7712 in a bioreactor was achieved, and they showed antiproliferative potential in OVCAR-3 cells. SIGNIFICANCE AND IMPACT OF THE STUDY: FOS could be a good target for future therapeutic studies and commercial use.

Jararhagin disruption of endothelial cell anchorage is enhanced in collagen enriched matrices.

Hemorrhage is one of the most striking effects of bites by viper snakes resulting in fast bleeding and ischemia in affected tissues. Snake venom metalloproteinases (SVMPs) are responsible for hemorrhagic activity, but the mechanisms involved in SVMP-induced hemorrhage are not entirely understood and the study of such mechanisms greatly depends on in vivo experiments. In vivo, hemorrhagic SVMPs accumulate on basement membrane (BM) of venules and capillary vessels allowing the hydrolysis of collagen IV with consequent weakness and rupture of capillary walls. These effects are not reproducible in vitro with conventional endothelial cell cultures. In this study we used two-dimension (2D) or three-dimension (3D) cultures of HUVECs on matrigel and observed the same characteristics as in ex vivo experiments: only the hemorrhagic toxin was able to localize on surfaces or internalize endothelial cells in 2D cultures or in the surface of tubules formed on 3D cultures. The contribution of matrigel, fibronectin and collagen matrices in jararhagin-induced endothelial cell damage was then analyzed. Collagen and matrigel substrates enhanced the endothelial cell damage induced by jararhagin allowing toxin binding to focal adhesions, disruption of stress fibers, detachment and apoptosis. The higher affinity of jararhagin to collagen than to fibronectin explains the localization of the toxin within BM. Moreover, once located in BM, interactions of jararhagin with α2ß1 integrin would favor its localization on focal adhesions, as observed in our study. The accumulation of toxin in focal adhesions, observed only in cells grown in collagen matrices, would explain the enhancement of cell damage in these matrices and reflects the actual interaction among toxin, endothelial cells and BM components that occurs in vivo and results in the hemorrhagic lesions induced by viper venoms.

First-trimester euploid miscarriages analysed by array-CGH.

It is estimated that 10-15 % of all clinically recognised pregnancies results in a miscarriage, most of which occur during the first trimester. Large-scale chromosomal abnormalities have been found in up to 50 % of first-trimester spontaneous abortions and, for several decades, standard cytogenetic analysis has been used for their identification. Recent studies have proven that array comparative genomic hybridisation (array-CGH) is a useful tool for the detection of genome imbalances in miscarriages, showing a higher resolution, a significantly higher detection rate and overcoming problems of culture failures, maternal contamination and poor chromosome morphology. In this study, we investigated the possibility that submicroscopic chromosomal changes, not detectable by conventional cytogenetic analysis, exist in euploid miscarriages and could be causative for the spontaneous abortion. We analysed with array-CGH technology 40 foetal tissue samples derived by first-trimester miscarriages with a normal karyotype. A whole-genome microarray with a 100-Kb resolution was used for the analysis. Forty-five copy number variants (CNVs), ranging in size between 120 Kb and 4.3 Mb, were identified in 31 samples (24 gains and 21 losses). Ten samples (10/31, 32 %) have more than one CNV. Thirty-one CNVs (68 %) were defined as common CNVs and 14 were classified as unique. Six genes and five microRNAs contained within these CNVs will be discussed. This study shows that array-CGH is useful for detecting submicroscopic CNVs and identifying candidate genes which could account for euploid miscarriages.

Trisomic dose of several chromosome 21 genes perturbs haematopoietic stem and progenitor cell differentiation in Down's syndrome.

Children with Down's syndrome (DS) have 20-50-fold higher incidence of all leukaemias (lymphoid and myeloid), for reasons not understood. As incidence of many solid tumours is much lower in DS, we speculated that disturbed early haematopoietic differentiation could be the cause of increased leukaemia risk. If a common mechanism is behind the risk of both major leukaemia types, it would have to arise before the bifurcation to myeloid and lymphoid lineages. Using the transchromosomic system (mouse embryonic stem cells (ESCs)) bearing an extra human chromosome 21 (HSA21)) we analyzed the early stages of haematopoietic commitment (mesodermal colony formation) in vitro. We observed that trisomy 21 (T21) causes increased production of haemogenic endothelial cells, haematopoietic stem cell precursors and increased colony forming potential, with significantly increased immature progenitors. Transchromosomic colonies showed increased expression of Gata-2, c-Kit and Tie-2. A panel of partial T21 ESCs allowed us to assign these effects to HSA21 sub-regions, mapped by 3.5 kbp-resolution tiling arrays. The Gata-2 increase on one side, and c-Kit and Tie-2 increases on the other, could be attributed to two different, non-overlapping HSA21 regions. Using human-specific small interfering RNA silencing, we could demonstrate that an extra copy of RUNX1, but not ETS-2 or ERG, causes an increase in Tie-2/c-Kit levels. Finally, we detected significantly increased levels of RUNX1, C-KIT and PU.1 in human foetal livers with T21. We conclude that overdose of more than one HSA21 gene contributes to the disturbance of early haematopoiesis in DS, and that one of the contributors is RUNX1. As the observed T21-driven hyperproduction of multipotential immature precursors precedes the bifurcation to lymphoid and myeloid lineages, we speculate that this could create conditions of increased chance for acquisition of pre-leukaemogenic rearrangements/mutations in both lymphoid and myeloid lineages during foetal haematopoiesis, contributing to the increased risk of both leukaemia types in DS.

Extracellular alkalinization induces endothelium-derived nitric oxide dependent relaxation in rat thoracic aorta.

AIM: To investigate the mechanism through which the extracellular alkalinization promotes relaxation in rat thoracic aorta. METHODS: The relaxation response to NaOH-induced extracellular alkalinization (7.4-8.5) was measured in aortic rings pre-contracted with phenylephrine (Phe, 10(-6) M). The vascular reactivity experiments were performed in endothelium-intact and -denuded rings, in the presence or and absence of indomethacin (10(-5) M), NG-nitro-l-arginine methyl ester (L-NAME, 10(-4) M), N-(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide/HCl (W-7, 10(-7) M), 2,5-dimethylbenzimidazole (DMB, 2×10(-5) M) and methyl-ß-cyclodextrin (10(-2) M). In addition, the effects of NaOH-induced extracellular alkalinization (pH 8.0 and 8.5) on the intracellular nitric oxide (NO) concentration was evaluated in isolated endothelial cells loaded with diaminofluorescein-FM diacetate (DAF-FM DA, 5 µM), in the presence and absence of DMB (2×10(-5) M). RESULTS: The extracellular alkalinization failed to induce any change in vascular tone in aortic rings pre-contracted with KCl. In rings pre-contracted with Phe, the extracellular alkalinization caused relaxation in the endothelium-intact rings only, and this relaxation was maintained after cyclooxygenase inhibition; completely abolished by the inhibition of nitric oxide synthase (NOS), Ca(2+)/calmodulin and Na(+)/Ca(2+) exchanger (NCX), and partially blunted by the caveolae disassembly. CONCLUSIONS: These results suggest that, in rat thoracic aorta, that extracellular alkalinization with NaOH activates the NCX reverse mode of endothelial cells in rat thoracic aorta, thereby the intracellular Ca(2+) concentration and activating the Ca(2+)/calmodulin-dependent NOS. In turn, NO is released promoting relaxation.

Action of neuwiedase, a metalloproteinase isolated from bothrops neuwiedi venom, on skeletal muscle: an ultrastructural and immunocytochemistry study

The damaging effects of neuwiedase, a non-hemorrhagic snake venom metalloproteinase from P-I class, on gastrocnemius muscle are studied herein. Following neuwiedase injection, ultrastructural alterations were detected early showing disarrangement of skeletal muscle fibers (characterized by discontinuity of Z lines), mitochondrial swelling, and disruption of plasma membrane and basal lamina. Degradation of skeletal muscle and the appearance of an amorphous substance, primarily composed of cellular debris, were noted after 24 hours. The presence of neuwiedase at the injection site (detected by immunocytochemistry) revealed highly specific labeling of myofibril components of damaged myocytes. In addition, proteolysis of muscle proteins assayed through myofibrils extracted from gastrocnemius muscle indicated that neuwiedase provoked degradation of myofibrils, especially myosin. These results suggest that skeletal muscle damage, induced by neuwiedase, is probably due to its proteolytic action on myofibrils, which are responsible for the maintenance of the cellular architecture.

[Maternally Inherited Diabetes and Deafness]. / Diabetes de herencia materna y sordera.

CASE REPORT: We described the follow up of a patient with diabetes mellitus type 2 who had a macular pattern dystrophy and bilateral neurosensory hearing loss. Electrophysiological studies revealed abnormal pattern electroretinography and impaired electro-oculogram responses. DISCUSSION: Maternally Inherited Diabetes, neurosensory Deafness and generally macular pattern distrophy (MIDD syndrome), is a rare mitochondrial disease, responsible for approximately 0.5 to 2.8% of diabetes mellitus.

Diabetes de herencia materna y sordera / Maternally inherited diabetes and deafness

Caso clínico: Se describe el seguimiento de unpaciente diabético tipo 2 con una degeneraciónmacular en patrón y sordera neurosensorial bilateral.En la electrofisiología mostraba un electrorretinograma(ERG) patrón anormal y un electrooculograma(EOG) disminuido.Discusión: La presencia de diabetes de herenciamaterna y sordera neurosensorial, a los que suelesumarse una distrofia macular en patrón, constituyenel síndrome MIDD (Maternally Inherited Diabetesand Deafness), una rara enfermedad mitocondrialresponsable de un 0,5% a 2,8% de los diabéticos(AU)

Case report: We described the follow up of apatient with diabetes mellitus type 2 who had amacular pattern dystrophy and bilateral neurosensoryhearing loss. Electrophysiological studiesrevealed abnormal pattern electroretinography andimpaired electro-oculogram responses.Discussion: Maternally Inherited Diabetes, neurosensoryDeafness and generally macular patterndistrophy (MIDD syndrome), is a rare mitochondrialdisease, responsible for approximately 0.5 to2.8% of diabetes mellitus(AU)

Effects of acid-base imbalance on vascular reactivity.

Acid-base homeostasis maintains systemic arterial pH within a narrow range. Whereas the normal range of pH for clinical laboratories is 7.35-7.45, in vivo pH is maintained within a much narrower range. In clinical and experimental settings, blood pH can vary in response to respiratory or renal impairment. This altered pH promotes changes in vascular smooth muscle tone with impact on circulation and blood pressure control. Changes in pH can be divided into those occurring in the extracellular space (pHo) and those occurring within the intracellular space (pHi), although, extracellular and intracellular compartments influence each other. Consistent with the multiple events involved in the changes in tone produced by altered pHo, including type of vascular bed, several factors and mechanisms, in addition to hydrogen ion concentration, have been suggested to be involved. The scientific literature has many reports concerning acid-base balance and endothelium function, but these concepts are not clear about acid-base disorders and their relations with the three known mechanisms of endothelium-dependent vascular reactivity: nitric oxide (NO/cGMP-dependent), prostacyclin (PGI2/cAMP-dependent) and hyperpolarization. During the last decades, many studies have been published and have given rise to confronting data on acid-base disorder and endothelial function. Therefore, the main proposal of this review is to provide a critical analysis of the state of art and incentivate researchers to develop more studies about these issues.

Effects of acid-base imbalance on vascular reactivity

Acid-base homeostasis maintains systemic arterial pH within a narrow range. Whereas the normal range of pH for clinical laboratories is 7.35-7.45, in vivo pH is maintained within a much narrower range. In clinical and experimental settings, blood pH can vary in response to respiratory or renal impairment. This altered pH promotes changes in vascular smooth muscle tone with impact on circulation and blood pressure control. Changes in pH can be divided into those occurring in the extracellular space (pHo) and those occurring within the intracellular space (pHi), although, extracellular and intracellular compartments influence each other. Consistent with the multiple events involved in the changes in tone produced by altered pHo, including type of vascular bed, several factors and mechanisms, in addition to hydrogen ion concentration, have been suggested to be involved. The scientific literature has many reports concerning acid-base balance and endothelium function, but these concepts are not clear about acid-base disorders and their relations with the three known mechanisms of endothelium-dependent vascular reactivity: nitric oxide (NO/cGMP-dependent), prostacyclin (PGI2/cAMP-dependent) and hyperpolarization. During the last decades, many studies have been published and have given rise to confronting data on acid-base disorder and endothelial function. Therefore, the main proposal of this review is to provide a critical analysis of the state of art and incentivate researchers to develop more studies about these issues.

BnP1, a novel P-I metalloproteinase from Bothrops neuwiedi venom: biological effects benchmarking relatively to jararhagin, a P-III SVMP.

Snake venom metalloproteinases (SVMPs) have been extensively studied and their effects associated with the local bleeding observed in human accidents by viper snakes. Representatives of P-I and P-III classes of SVMPs similarly hydrolyze extracellular matrix proteins or coagulation factors while only P-III SVMPs induce significant hemorrhage in experimental models. In this work, the effects of P-I and P-III SVMPs on plasma proteins and cultures of muscle and endothelial cells were compared in order to enlighten the mechanisms involved in venom-induced hemorrhage. To reach this comparison, BnP1 was isolated from B. neuwiedi venom and used as a weakly hemorrhagic P-I SVMPs and jararhagin was used as a model of potently hemorrhagic P-III SVMP. BnP1 was isolated by size exclusion and anion-exchange chromatographies, showing apparent molecular mass of approximately 24kDa and sequence similarity with other members of SVMPs, which allowed its classification as a group P-I SVMP. The comparison of local effects induced by SVMPs showed that BnP1 was devoid of significant myotoxic and hemorrhagic activities and jararhagin presented only hemorrhagic activity. BnP1 and jararhagin were able to hydrolyze fibrinogen and fibrin, although the latter displayed higher activity in both systems. Using HUVEC primary cultures, we observed that BnP1 induced cell detachment and a decrease in the number of viable endothelial cells in levels comparable to those observed by treatment with jararhagin. Moreover, both BnP1 and jararhagin induced apoptosis in HUVECs while only a small increase in LDH supernatant levels was observed after treatment with jararhagin, suggesting that the major mechanism involved in endothelial cell death is apoptosis. Jararhagin and BnP1 induced little effects on C2C12 muscle cell cultures, characterized by a partial detachment 24h after treatment and a mild necrotic effect as evidenced by a small increase in the supernatants LDH levels. Taken together, our data show that P-I and P-III SVMPs presented comparable effects except for the hemorrhagic activity, suggesting that hydrolysis of coagulation factors or damage to endothelial cells are not sufficient for induction of local bleeding.