Miguel Cerdá

Effect of simultaneous inhibition of TNF-α production and xanthine oxidase in experimental acute pancreatitis: The role of mitogen activated protein kinases

Objective:To assess the effects of inhibiting both tumor necrosis factor (TNF)-α production and xanthine oxidase activity on the inflammatory response, mitogen-activated protein kinase (MAPK) activation and mortality in necrotizing acute pancreatitis in rats. Summary Background Data:Pancreatic injury triggers 2 major pathways involved in the systemic effects of severe acute pancreatitis: pro-inflammatory cytokines and oxidative stress. Methods:Pancreatitis was induced by intraductal infusion of 3.5% sodium taurocholate. We examined whether treatment with oxypurinol, a specific inhibitor of xanthine oxidase, and/or pentoxifylline, an inhibitor of TNF-α production, affects pancreatic damage, ascites, lung inflammation, and MAPK phosphorylation. Results:Oxypurinol prevented p38 phosphorylation in the pancreas and partially avoided the rise in lung myeloperoxidase activity. Pentoxifylline prevented erk 1/2 and JNK phosphorylation in the pancreas, and it partially reduced ascites and the rise in lung myeloperoxidase activity. Combined treatment with oxypurinol and pentoxifylline almost completely abolished ascites, MAPK phosphorylation in the pancreas, and the increase in lung myeloperoxidase activity. Histology revealed a reduction in pancreatic and lung damage. These changes were associated with a significant improvement of survival. Conclusions:Simultaneous inhibition of TNF-α production and xanthine oxidase activity greatly reduced local and systemic inflammatory response in acute pancreatitis and decreased mortality rate. These effects were associated with blockade of the 3 major MAPKs.

Inhibition of xanthine oxidase by allopurinol prevents skeletal muscle atrophy: role of p38 MAPKinase and E3 ubiquitin ligases.

Alterations in muscle play an important role in common diseases and conditions. Reactive oxygen species (ROS) are generated during hindlimb unloading due, at least in part, to the activation of xanthine oxidase (XO). The major aim of this study was to determine the mechanism by which XO activation causes unloading-induced muscle atrophy in rats, and its possible prevention by allopurinol, a well-known inhibitor of this enzyme. For this purpose we studied one of the main redox sensitive signalling cascades involved in skeletal muscle atrophy i.e. p38 MAPKinase, and the expression of two well known muscle specific E3 ubiquitin ligases involved in proteolysis, the Muscle atrophy F-Box (MAFbx; also known as atrogin-1) and Muscle RING (Really Interesting New Gene) Finger-1 (MuRF-1). We found that hindlimb unloading induced a significant increase in XO activity and in the protein expression of the antioxidant enzymes CuZnSOD and Catalase in skeletal muscle. The most relevant new fact reported in this paper is that inhibition of XO with allopurinol, a drug widely used in clinical practice, prevents soleus muscle atrophy by ∼20% after hindlimb unloading. This was associated with the inhibition of the p38 MAPK-MAFbx pathway. Our data suggest that XO was involved in the loss of muscle mass via the activation of the p38MAPK-MAFbx pathway in unloaded muscle atrophy. Thus, allopurinol may have clinical benefits to combat skeletal muscle atrophy in bedridden, astronauts, sarcopenic, and cachexic patients.

Rolipram inhibits leukocyte-endothelial cell interactions in vivo through P- and E-selectin downregulation

Rolipram, a selective phosphodiesterase (PDE) type 4 inhibitor, was used to characterize leukocyte recruitment mechanisms in models of acute and subacute inflammation. Intravital microscopy within the rat mesenteric microcirculation was employed. Mesentery superfusion with PAF (0.1 μM) induced a significant increase in leukocyte rolling flux, adhesion and emigration at 60 min. Rolipram pretreatment, markedly inhibited these parameters by 100, 95 and 95% respectively. Similar effects were observed when the mesentery was superfused with LPS (1 μg ml−1) for the same time period and these leukocyte parameters were nearly abrogated by rolipram pretreatment. LPS exposure of the mesentery for 4 h caused a greater increase in leukocyte rolling flux, adhesion and emigration which were inhibited by rolipram administration by 51, 71 and 81% respectively. Immunohistochemistry revealed a significant increase in P‐selectin expression after 60 min superfusion with PAF which was attenuated by rolipram. LPS exposure of the mesentery for 4 h caused a significant increase in P‐ and E‐selectin, intercellular adhesion molecule‐1 (ICAM‐1) and vascular cell adhesion molecule‐1 (VCAM‐1) expression. Rolipram pretreatment down‐regulated both P‐ and E‐selectin expression but had no effect on ICAM‐1 and VCAM‐1 expression. Significant increases in plasma cyclic AMP levels were detected at 4.5 h after rolipram administration. In conclusion, we have demonstrated that rolipram is a potent in vivo inhibitor of leukocyte‐endothelial cell interactions. The effects observed are mediated through endothelial P‐ and E‐selectin downregulation. Therefore, selective PDE‐4 inhibitors may be useful in the control of different inflammatory disorders.

Ductal Carcinoma In Situ of the Breast: A Comparative Analysis of Histology, Nuclear Area, Ploidy, and Neovascularization Provides Differentiation Between Low- and High-Grade Tumors

Ductal carcinoma in situ (DCIS) is a heterogeneous group of lesions that has been subdivided into three types: well differentiated (grade I), moderately differentiated (grade II), and poorly differentiated (grade III). Forty‐five cases of DCIS were analyzed for image analysis: nuclear area, DNA ploidy, and vascularization in order to establish a more precise correlation between the histologic grade and these morphometric parameters. Our results confirm that the mean nuclear area, DNA ploidy, and microvessel density (MVD) progressively increased from DCIS grade I to DCIS grade III. The analysis of the nuclear area in relationship to DCIS grading demonstrated a progressive increase of values between grades I/II to grade III, but these data have no statistical significance. An analysis of DNA ploidy demonstrated significant differences between grades I/III (p < 0.05), but there was no statistical significance between grades I/II, grades II/III, or both (p > 0.005). The analysis of MVD was extremely significant between grades I/III (p < 0.001) and grades II/III (p < 0.001), but between grades I/II, these values showed no significant differences (p > 0.05). Based on this study, it can be concluded that image analysis techniques confirm how DCIS presents morphometric values that increase from DCIS grade I to DCIS grade III and that within this spectrum, DCIS grade III can be identified as a group of tumors presenting a large nuclear area, aneuploid DNA, and abundant vascular neogenesis, confirming that this neoplasm displays more aggressive patterns than the other two types. These criteria should justify a higher rate of tumor progression to DCIS grade III.

Closed Chest Radiofrequency Ablation of the Sinoatrial Node in Dogs

Transcatheter ablation of the sinoatrial node with radiofrequency energy (0.6 MHZ, 2.5–5 watts) was performed in 10 dogs under fluoroscopic monitoring and autonomic blockade. Sinus function was previously studied in terms of cycle length, recovery time and atrial activation pattern by catheter mapping. Several discharges (8–22) were applied for variable periods of time (maximum 1 minute). Sinus tachycardia and/or sinus arrest during ablation confirmed correct catheter position. Sinus rhythm was abolished in eight dogs. The ectopic rhythm was atrial in six and AV nodal in two dogs. Ectopic atrial cycle length and recovery time were longer than the baseline sinus values: 724 ± 321 versus 509 ± 147, P < 0.05; 1103 ± 775 versus 638 ± 151, P < 0.05 (values in msec). The study was repeated 10–14 days later in six dogs; three maintained the same atrial rhythm, one persisted in sinus rhythm, and one dog changed from atrial to sinus rhythm, whereas another changed from sinus to atrial rhythm. Gross findings revealed transmural lesions in all dogs, without perforation. Histology in chronic dogs showed sinus cell necrosis and its replacement by granulation tissue. In conclusion: sinus function may be abolished by closed chest radiofrequency ablation.

Glutathione and Bcl-2 targeting facilitates elimination by chemoradiotherapy of human A375 melanoma xenografts overexpressing bcl-xl, bcl-2, and mcl-1

BackgroundBcl-2 is believed to contribute to melanoma chemoresistance. However, expression of Bcl-2 proteins may be different among melanomas. Thus correlations among expression of Bcl-2-related proteins and in vivo melanoma progression, and resistance to combination therapies, was investigated.MethodsHuman A375 melanoma was injected s.c. into immunodeficient nude mice. Protein expression was studied in tumor samples obtained by laser microdisection. Transfection of siRNA or ectopic overexpression were applied to manipulate proteins which are up- or down-regulated, preferentially, during melanoma progression. Anti-bcl-2 antisense oligonucleotides and chemoradiotherapy (glutathione-depleting agents, paclitaxel protein-binding particles, daunorubicin, X rays) were administered in combination.ResultsIn vivo A375 cells down-regulated pro-apoptotic bax expression; and up-regulated anti-apoptotic bcl-2, bcl-xl, and mcl-1, however only Bcl-2 appeared critical for long-term tumor cell survival and progression in vivo. Reduction of Bcl-2, combined with partial therapies, decreased melanoma growth. But only Bcl-2 targeting plus the full combination of chemoradiotherapy eradicated A375 melanoma, and led to long-term survival (> 120 days) without recurrence in 80% of mice. Tumor regression was not due to immune stimulation. Hematology and clinical chemistry data were within accepted clinical toxicities.ConclusionStrategies to target Bcl-2, may increase the effectiveness of antitumor therapies against melanomas overexpressing Bcl-2 and likely other Bcl-2-related antiapoptotic proteins.

Mapping of atrial activation patterns after inducing contiguous radiofrequency lesions: an experimental study.

CHORRO, F.J., et al.: Mapping of Atrial Activation Patterns After Inducing Contiguous Radiofrequency Lesions: An Experimental Study. High resolution mapping techniques are used to analyze the changes in atrial activation patterns produced by contiguous RF induced lesions. In 12 Langendorff‐perfused rabbit hearts, left atrial activation maps were obtained before and after RF induction of epicardial lesions following a triple‐phase sequential protocol: (phase 1) three separate lesions positioned vertically in the central zone of the left atrial wall; (phase 2) the addition of two lesions located between the central lesion and the upper and lower lesions; and (phase 3) the placement of four additional lesions between those induced in the previous phases. In six additional experiments a pathological analysis of the individual RF lesions was performed. In phase 1 (lesion diameter = 2.8 ± 0.2 mm, gap between lesions = 3 ± 0.8 mm), the activation process bordered the lesions line in two (2.0‐ms cycles) and four experiments (1.0‐ms cycles). In phase 2, activation bordered the lesions line in eight (2.0‐ms cycles, P < 0.01 vs control) and nine experiments (1.0‐ms cycles, P < 0.001), and in phase 3 this occurred in all experiments except one (both cycles, P < 0.001 vs control). In the experiments with conduction block, the increment of the interval between activation times proximal and distal to the lesions showed a significant correlation to the length of the lesions (r = 0.68, P < 0.05, 100‐ms cycle). In two (17%) experiments, sustained regular tachycardias were induced with reentrant activation patterns around the lesions line. In conclusion, in this acute model, atrial RF lesions with intact tissue gaps of 3 mm between them interrupt conduction occasionally, and conduction block may be frequency dependent. Lesion overlap is required to achieve complete conduction block lines. Tachycardias with reentrant activation patterns around a lesions line may be induced.

Acute Effects of Radiofrequency Ablation upon Atrial Conduction in Proximity to the Lesion Site

The electrophysiological effects of RF ablation upon the areas in proximity to the lesioned zones have not yet been well characterized. An experimental model is used to investigate atrial conduction in the boundaries of RF damaged zones. In 11 isolated and perfused rabbit hearts, endocardial atrial electrograms were recorded using an 80‐Iead multiple electrode positioned in the left atrium. Both before and after the RF application (5 W, 8 s, 1‐mm diameter unipolar epicardial electrode) in the mid‐portion of the free left atrial wall, measurements were made of conduction time from the pacing zone (posterior wall of the left atrium) to three points between 7.5 and 7.9 mm distal to the damaged zone. Conduction velocity and the direction of the activation propagation vector were determined in ten groups of four electrodes positioned around the damaged zone, and at the left atrial appendage. The mean diameter (± SEM) of the transmural lesions produced by RF ablation and defined by macroscopic examination was 4.2 ± 0.2 mm. The conduction times to the three points distal to the lesion site were significantly prolonged as a result of RF ablation: 7.6 ± 0.4, 7.4 ± 0.5, and 6.9 ± 1.0 ms (control); and 11.3 ± 1.0 (P ≤ 0.01), 11.1 ± 1.3 (P < 0.01), 10.6 ± 1.4 ms (P < 0.05) (post‐RF). The differences between the conduction velocities determined in the areas surrounding the lesion, before and after RF application, failed to reach statistical significance: 86.2 ± 6.5 cm/s (control) versus 75.5 ± 5.7 cm/s (post‐RF) (NS). After RF, significant variations were only observed in the direction of impulse propagation in the proximal‐inferior quadrant adjacent to the lesion site, the difference being ‐61°± 18° (P < 0.02). In 2 of 4 experiments in which the lesion size was increased by a second RF application (5 W, 16 s), tachycardias with activation sequence around the lesion could be induced, with cycle lengths of 56 and 50 ms, respectively. In the atrial wall, the conduction times to the regions distal to the RF lesion are significantly prolonged. No significant changes are observed in conduction velocity in the areas in proximity to the lesion. Prolonged conduction to the areas distal to the ablation site is due to the lengthened pathway traveled by the impulses in reaching these areas. Tachycardias with activation patterns that suggest reentry around the RF damaged zone may be induced.

Co-administration of pentoxifylline and thiopental causes death by acute pulmonary oedema in rats.

Pentoxifylline exhibits rheological properties that improve microvascular flow and it is widely used in vascular perfusion disorders. It also exhibits marked anti‐inflammatory properties by inhibiting tumour necrosis factor α production. Thiopental is one of the most widely used drugs for rapid induction of anaesthesia. During experimental studies on the treatment of acute pancreatitis, we observed that when pentoxifylline was administered after anaesthesia with thiopental, most of the rats exhibited dyspnea, signs of pulmonary oedema and died. The aim of the work described here was to investigate the cause of the unexpected toxic effect of the combined treatment with thiopental and pentoxifylline.

Características de los electrogramas auriculares registrados en las líneas de bloqueo producidas con radiofrecuencia en un modelo experimental

Objetivos Analizar y cuantificar las modificaciones de los electrogramas auriculares tras la realizacion de lesiones lineales en la pared auricular utilizando procedimientos de ablacion con radiofrecuencia. Metodos En 12 preparaciones de corazon aislado de conejo segun la tecnica de Langendorff se ha utilizado un electrodo multiple epicardico (221 electrodos unipolares) para analizar la activacion auricular antes y despues de la realizacion de una lesion lineal en la pared auricular izquierda mediante aplicaciones sucesivas de radiofrecuencia. Tras comprobar la existencia de bloqueo de la conduccion en la zona lesionada mediante cartografia epicardica y analisis de los vectores de propagacion, en cada experimento se han seleccionado seis electrodos en la zona lesionada y otros seis en la no lesionada. En ambas zonas se ha comparado la amplitud, la maxima pendiente negativa y la morfologia de los electrogramas antes (control) y despues de las aplicaciones de radiofrecuencia. Resultados El analisis de la reproducibilidad de las mediciones en dos ciclos consecutivos ha puesto de manifiesto una variacion en la amplitud de un 1 ± 5% (NS) y en la pendiente de un 1 ± 9% (NS). En la zona no lesionada, la amplitud (105 ± 22%) y la pendiente (92 ± 16%) (valores normalizados con respecto a los obtenidos en el control) no han variado significativamente tras las aplicaciones de radiofrecuencia, y los registros de electrogramas simples han sido los mas frecuentes (el 82 frente al 83% en el control; NS). En la zona lesionada, la amplitud (19 ± 7%, p Conclusiones Los electrogramas obtenidos directamente sobre lineas de bloqueo originadas con radiofrecuencia se caracterizan por presentar una reduccion significativa de la amplitud y de la maxima pendiente negativa. En estos registros predominan los electrogramas dobles cuyos dos componentes representan la activacion a ambos lados de la lesion. En la linea de bloqueo pueden registrarse tambien en un porcentaje reducido de casos electrogramas simples y multiples. Palabras clave: Ablacion con radiofrecuencia Lesiones lineales. Activacion auricular. Cartografia. Electrofisiologia cardiaca. Modelos experimentales.