Inducers of salmon innate immunity: An in vitro and in vivo approach.

Maintaining fish health is one of the most important aims in aquaculture. Prevention of fish diseases therefore is crucial and can be achieved by various different strategies, including most often a combination of different methods such as optimal feed and fish density, as well as strengthening the immune system. Understanding the fish innate immune system and developing methods to activate it, in an effort to prevent infections in the first place, has been a goal in recent years. In this study we choose different inducers of the innate immune system and examined their effects in vitro on the salmon cell line CHSE-214. We found that the butyrate derivatives 4-phenyl butyrate (PBA) and ß-hydroxy-ß-methyl butyrate (HMB) induce the expression of various innate immune genes differentially over 24-72 h. Similarly, lipids generated from fish oils were found to have an effect on the expression of the antimicrobial peptides cathelicidin and hepcidin, as well as iNOS and the viral receptor RIG-1. Interestingly we found that vitamin D3, similar as in mammals, was able to increase cathelicidin expression in fish cells. The observed induction of these different innate immune factors correlated with antibacterial activity against Aeromonas salmonicida and antiviral activity against IPNV and ISAV in vitro. To relate this data to the in vivo situation we examined cathelicidin expression in juvenile salmon and found that salmon families vary greatly in their basal cathelicidin levels. Examining cathelicidin levels in families known to be resistant to IPNV showed that these QTL-families had lower basal levels of cathelicidin in gills, than non QTL-families. Feeding fish with HMB caused a robust increase in cathelicidin expression in gills, but not skin and this was independent of the fish being resistant to IPNV. These findings support the use of fish cell lines as a tool to develop new inducers of the fish innate immune system, but also highlight the importance of the tissue studied in vivo. Understanding the response of the innate immune system in different tissues and what effect this might have on infections and downstream cellular pathways is an interesting research topic for the future.

Prognostic Value of the Leuko-Glycaemic Index in the Postoperative Period of Coronary Artery Bypass Grafting.

INTRODUCTION: High leuko-glycaemic index (LGI) (> 2000) has been associated with poor prognosis in many critical care settings. However, there is no evidence of LGI's prognostic value in the postoperative period of coronary artery bypass grafting (CABG). This study aims to analyze the prognostic value of LGI in the postoperative period of CABG. METHODS: Single-center retrospective analysis of prospectively collected data was performed. Consecutive adult patients undergoing CABG between 2007 and 2019 were included. Blood glucose levels and white blood cells count were evaluated in the immediate postoperative period. LGI was calculated by multiplying both values and dividing them by 1,000 and analyzed in quartiles. Receiver operating characteristic curve was used to determine the best cutoff value. The primary combined endpoint was in-hospital mortality, low cardiac output (LCO), or acute kidney injury (AKI). Secondary endpoints included in-hospital death, AKI, atrial fibrillation, and LCO. RESULTS: The study evaluated 3,813 patients undergoing CABG (88.5% male, 89.8% off-pump surgery, aged 64.6 years [standard deviation 9.6]). The median of LGI was 2,035. Presence of primary endpoint significantly increased per LGI quartile (9.2%, 9.7%, 11.8%, and 15%; P<0.001). High LGI was associated with increased occurrence of in-hospital mortality, LCO, AKI, and atrial fibrillation. The best prognostic cutoff value for primary endpoint was 2,000. In a multivariate logistic regression model, high LGI was independently associated with in-hospital death, LCO, or AKI. CONCLUSION: High LGI was an independent predictor of inhospital mortality, LCO, or AKI in postoperative period of CABG. It was also associated with higher in-hospital death.

Off-pump bilateral internal thoracic artery grafting - surgical technique.

Numerous coronary revascularization studies have documented superior results associated with bilateral internal thoracic artery grafting compared with single internal thoracic artery grafting. However, concerns about perioperative complications and the technical challenges inherent in bilateral grafting limit its broad utilization. In this video tutorial we show our routine technique for off-pump bilateral internal thoracic artery grafting, and also discuss the experience of our department and the evolving process of how we have performed myocardial revascularization over the past two decades.

Is sex a risk factor for death in patients with bilateral internal thoracic artery grafts?

BACKGROUND: Mortality after coronary artery bypass grafting (CABG) has been reported to be higher in women. The aim of this study was to evaluate whether bilateral internal thoracic artery (BITA) grafting in women has a long-term survival benefit over single internal thoracic artery grafting, possibly equivalent to the male population. METHODS: A retrospective review was undertaken of our prospectively collected database. We included 4406 consecutive patients who underwent isolated CABG, who received their operation between January 2000 and April 2017. From the entire series, 2979 patients (67.6%) received exclusively BITA grafts; 299 (10.1%) were female. The primary end point was follow-up mortality, independently from cause. In-hospital mortality and during follow-up were analyzed. Substratification according to age was performed to answer whether it has an effect. Multivariable Cox proportional hazard analyses was performed to investigate the significant predictors of late mortality. RESULTS: The median follow-up was 5.1 ± 3.9 years. Female BITA patients were older (P < .001), had nonelective surgery (P < .001), more on-pump CABG (P = .015), fewer number of grafts (P < .001) versus male BITA patients. BITA grafting in women had a long-term survival equivalent to that of men (P = .784). In a Cox proportional hazard model, female sex was not an independent risk factor for late death (B, -0.303; hazard ratio, 0.739; 95% confidence interval, 0.470-1.16; P = .189). The stratification analysis showed that the beneficial effect of BITA remained similar among sexes and was not modified by age even after adjusting for confounders. In a risk-adjusted sample, patients older than 65 years with BITA grafting showed superior long-term survival than those with single internal thoracic artery grafting (P = .019). CONCLUSIONS: Although there are some differences between sexes, BITA grafting in women was associated with similar 10-year survival compared with men, and female sex was not an independent risk factor for late death. Among women, the BITA group had better survival, especially those older than 65 years.

Surgical Treatment of Dysphagia Lusoria Caused by Right-Aortic Arch with Kommerell Diverticulum: Left Heart Bypass without Subclavian Revascularization.

The authors present the case of a 26-year-old patient suffering from dysphagia because of compression by a Kommerell diverticulum in right aortic arch anomaly. Open surgical arch and descending aorta replacement with left heart bypass without left subclavian artery reimplantation was performed.

Cirugía de preservación valvular como alternativa en enfermedad de la raíz aórtica / Aortic Valve Sparing Surgery as Alternative in Aortic Root Disease

RESUMEN Introducción: Desde 1968, la enfermedad aneurismática de la raíz aórtica ha sido tratada mediante el remplazo con tubo valvulado. En las últimas décadas la cirugía de preservación valvular surgió y evolucionó como una opción al remplazo protésico. Objetivo: Reportar la experiencia institucional en la técnica de preservación valvular y sus resultados a largo plazo. Material y métodos: Revisión de 116 casos consecutivos con criterios de reparabilidad, intervenidos entre 2005 y 2019. Previo ecocardiograma transesofágico (ETE) y angiotomografía (AngioTC), se procedió quirúrgicamente acorde a la clasificación anatomofuncional, con la combinación de técnicas. Se realizó control intraoperatorio y conversión a remplazo según el criterio del cirujano interviniente. Se reportan las variables intraoperatorias, la morbimortalidad intrahospitalaria y la mortalidad, la libertad de insuficiencia valvular significativa y la reoperación en el seguimiento clínico y ecocardiográfico. Resultados: La edad media era 56 ± 15,6 años, varones 73%, 59% asintomáticos, intervenidos por diámetro aórtico (52 ± 11,7 mm) o progresión de valvulopatía. En el posprocedimiento, 4% de los casos resultó con insuficiencia leve o nula y 2 conversiones (1,7%); mortalidad hospitalaria 0,9%. A 10 años de seguimiento, sobrevida actuarial del 88% y libertad de insuficiencia significativa (moderada/grave) 79%. Se reintervinieron 5 casos, a un intervalo promedio de 9,1 años, libertad de reoperación de 90% a 10 años. No se registraron eventos tromboembólicos ni hemorrágicos mayores. Conclusión: las técnicas de preservación valvular aórtica, en contexto de enfermedad de la raíz, resultan una opción factible, segura y estable en el tiempo.

ABSTRACT Background: Since 1968, ascending aorta replacement with a valved conduit has been the standard practice for aortic root aneurysm. By the end of the 20th century, aortic valve sparing operation emerged and evolved as an alternative to aortic valve replacement. Objective: The aim of this study was to report our experience with aortic valve sparing technique and its long -term outcomes. Methods: A total of 116 consecutive cases with criteria of repairability operated on between 2005 and 2019 were analyzed. Preopera- tive transesophageal echocardiography (TEE) and computed tomography angiography (CTA) were used in combination to determine the aortic phenotype based on a previous anatomical and functional classification. Perioperative control was performed and conver- sion to aortic valve replacement was left to the discretion of the attending surgeon. Intraoperative variables, in-hospital morbidity and mortality, freedom from significant aortic regurgitation (AR) and reoperation in the clinical and echocardiographic follow-up were reported. Results: Mean age was 56±15.6 years and 73% were men; 59% were asymptomatic, and the reason for the intervention was the aortic diameter (52±11.7 mm) or progression of AR. After the procedure, 4% of the cases presented mild or trivial AR and 2 patients required conversion to aortic valve replacement (1.7%). In hospital mortality was 0.9%. Actuarial survival was 88% at 10 years, and 79% were free from significant (moderate/severe) AR. Five cases underwent reoperation after a mean interval of 9.1 years and free- dom from reoperation at 10 years was 90%. There were no major thromboembolic or bleeding events. Conclusion: Aortic valve sparing technique in the setting of aortic root disease is a feasible and safe option, and stable over time.

Prognostic Value of the Leuko-Glycaemic Index in the Postoperative Period of Coronary Artery Bypass Grafting

Abstract Introduction: High leuko-glycaemic index (LGI) (> 2000) has been associated with poor prognosis in many critical care settings. However, there is no evidence of LGI's prognostic value in the postoperative period of coronary artery bypass grafting (CABG). This study aims to analyze the prognostic value of LGI in the postoperative period of CABG. Methods: Single-center retrospective analysis of prospectively collected data was performed. Consecutive adult patients undergoing CABG between 2007 and 2019 were included. Blood glucose levels and white blood cells count were evaluated in the immediate postoperative period. LGI was calculated by multiplying both values and dividing them by 1,000 and analyzed in quartiles. Receiver operating characteristic curve was used to determine the best cutoff value. The primary combined endpoint was in-hospital mortality, low cardiac output (LCO), or acute kidney injury (AKI). Secondary endpoints included in-hospital death, AKI, atrial fibrillation, and LCO. Results: The study evaluated 3,813 patients undergoing CABG (88.5% male, 89.8% off-pump surgery, aged 64.6 years [standard deviation 9.6]). The median of LGI was 2,035. Presence of primary endpoint significantly increased per LGI quartile (9.2%, 9.7%, 11.8%, and 15%; P<0.001). High LGI was associated with increased occurrence of in-hospital mortality, LCO, AKI, and atrial fibrillation. The best prognostic cutoff value for primary endpoint was 2,000. In a multivariate logistic regression model, high LGI was independently associated with in-hospital death, LCO, or AKI. Conclusion: High LGI was an independent predictor of in-hospital mortality, LCO, or AKI in postoperative period of CABG. It was also associated with higher in-hospital death.

Necrosis of infectious pancreatic necrosis virus (IPNV) infected cells rarely is preceded by apoptosis.

Infectious pancreatic necrosis virus (IPNV)-infected cells were labeled with Annexin V and propidium iodide in order to determine the proportion of cells, which developed necrosis and/or apoptosis during the time course of infection. Contrasting with earlier reports, we found that at any time during IPNV multiplication cycle, the percentage of apoptotic cells never exceeded the 12% of the whole population of the infected cells. In addition, the percentage of necrotic cells increased continuously until reaching the 75% of the infected cells at 15 h post infection. Apoptotic cells were also identified by in situ terminal deoxynucleotidyl transferase-mediated BrdUTP nick end labeling (TUNEL). Our results are in accordance with the idea that apoptosis rarely precedes necrosis.

Visualization of infectious pancreatic necrosis virus (IPNV) particles labeled with fluorescent probes.

Infectious pancreatic necrosis virus (IPNV) particles were labeled with SYBR Green I or a monoclonal antibody and FITC-conjugated secondary antibody and examined in a fluorescence microscope. Labeled viral particles were visualized in a narrow range of pixels. Comparing IPNV particles with fluorescent phage T4 virions, the former, as expected, were seen smaller in size. The method allows the rapid and accurate counting of viral particles both on filters and bound to the cell surface. In addition, IPNV particles can be specifically enumerated in the presence of other virions and the ratio between physical particles and virus infectivity can be easily calculated as well.

Rapid simultaneous detection and quantitation of infectious pancreatic necrosis virus (IPNV).

Infectious pancreatic necrosis virus (IPNV) is a pathogen of great concern in the salmon industry as well as in the environment. Taking advantage of the early immunofluorescent visualization of viral proteins in infected cells, a titration method was developed. At 16 h p.i., fluorescent foci were visualized with a monoclonal antibody against VP3-structural protein of the virus. The counting of each fluorescent cell allows the quantitation of infection foci; titres expressed in fluorescent foci/ml were equivalent to plaque forming units (PFU)/ml. With slight modifications, the same method used to detect the virus in field samples, can be applied to estimate virus contents. Some of the samples used during the assays were obtained from routine screening procedures. The titres recorded from positive samples correlated well with the clinical condition of the fish. With this method, rapid diagnosis and quantitation may simultaneously be performed with the same tissue extract.

Aneurisma de aorta ascendente: ¿cuál es el riesgo de reemplazar la raíz? / Ascending Aorta Aneurysm: What is the Risk of Replacing the Aortic Root?

RESUMEN Introducción: El riesgo-beneficio del reemplazo de los senos de Valsalva con el consiguiente reimplante coronario frente a la alternativa de mantenerlos, cuando hay dilataciones moderadas de la raíz, es un tópico que se debe definir. Objetivo: Analizar la morbimortalidad posoperatoria y a largo plazo en pacientes sometidos a reemplazo de la raíz aórtica comparados con aquellos en los que se han respetado los senos de Valsalva. Material y métodos: Entre 2002 y 2016, a 426 pacientes se les realizó reemplazo de aorta ascendente. Tras excluir de esa población las cirugías de urgencia, las aortopatías genéticas (excepto bicúspide), las reoperaciones y las cirugías del arco, se conformó una población de 259 pacientes. En 99 de ellos (38,2%) se reemplazó la aorta ascendente conservando la raíz; estos pacientes fueron comparados con los 160 (61,8%) pacientes restantes, en quienes se reemplazaron los senos de Valsalva. Resultados: El grupo en el que se preservó la raíz fue más añoso, con más mujeres, con un Euroscore mayor, con mayor incidencia de válvula bicúspide y enfermedad coronaria. El tiempo de circulación extracorpórea fue mayor en el grupo en el que se reemplazó la raíz. La mortalidad hospitalaria no fue diferente (1% para la conservación de raíz vs. 3,1% para el reemplazo de los senos de Valsalva (p = 0,272). En el análisis multivariado, el tiempo de circulación extracorpórea fue predictor de mortalidad posoperatoria. La sobrevida a 8 años no mostró diferencias significativas entre grupos. En el seguimiento, ningún paciente requirió reoperación debido a complicaciones de la aorta. En el análisis multivariado, la edad y la presencia de enfermedad valvular mitral fueron predictores de mortalidad alejada. Conclusión: El reemplazo de la aorta ascendente, ya sea reemplazando la raíz o respetando los senos de Valsalva, es una cirugía segura, con baja morbimortalidad hospitalaria. A largo plazo, la preservación de los senos de Valsalva no se asocia con más eventos ni con mayor mortalidad.

ABSTRACT Background: In mildly dilated aortic root, the cost-benefit of replacing of the sinuses of Valsalva with reimplantation of the coronary arteries or preserving them is still a matter of debate. Objective: The goal of this study was to analyze the postoperative and long-term morbidity and mortality of patients undergo-ing aortic root replacement versus aortic root surgery with sinuses of Valsalva preservation. Methods: Between 2002 and 2016, 426 patients underwent replacement of the ascending aorta. After excluding patients under-going urgent procedures, genetic aortic diseases (except for bicuspid aortic valve), reoperations and surgery of the aortic arch, the cohort was made up of 259 patients. In 99 of them (38.2%) the ascending aorta was replaced, preserving the aortic root; these patients were compared with the remaining 160 (61.8%) patients who underwent replacement of the sinuses of Valsalva. Results: Patients undergoing preservation of the aortic root were older, had higher percentage of female sex, higher EuroSCORE and with greater incidence of bicuspid aortic valve and coronary artery disease. Cardiopulmonary bypass time was longer in the group undergoing aortic root replacement. There were no significant differences in in-hospital mortality between both groups (1% in the group with preservation of the aortic root vs. 3.1% for replacement of the sinuses of Valsalva, p=0.272). Multivariate analysis showed that cardiopulmonary bypass was a predictor of in-hospital mortality. Survival at 8 years was similar in both groups. There were no new operations due to complications in the aorta during follow-up. At multivariate analysis, age and mitral valve disease were identified as predictors of long-term mortality. Conclusion: Replacement of the ascending aorta, either replacing the aortic root or preserving the sinuses of Valsalva, is a safe procedure, with low in-hospital mortality. Preservation of the sinuses of Valsalva is not associated with greater rate of events or mortality at the long-term.

Inter-laboratory ring trial to evaluate real-time reverse transcription polymerase chain reaction methods used for detection of infectious pancreatic necrosis virus in Chile

Background: Infectious Pancreatic Necrosis Virus (IPNV) is the etiological agent of a highly contagious disease that affects salmonids. In Chile, the second worldwide salmon producer, IPNV causes great economic loss and is one of the most frequently detected pathogens. Due to its high level of persistence and the lack of information about the efficiency of its diagnostic techniques, the National Reference Laboratory (NRL) for IPNV in Chile performed the first inter-laboratory ring trial, to evaluate the sensitivity, specificity and repeatability of the qRT-PCR detection methods used in the country. Results: Results showed 100% in sensitivity and specificity in most of the laboratories. Only three of the twelve participant laboratories presented problems in sensitivity and one in specificity. Problems in specificity (false positives) were most likely caused by cross contamination of the samples, while errors in sensitivity (false negatives) were due to detection problems of the least concentrated viral sample. Regarding repeatability, many of the laboratories presented great dispersion of the results (Ct values) for replicate samples over the three days of the trial. Moreover, large differences in the Ct values for each sample were detected among all the laboratories. Conclusions: Overall, the ring trial showed high values of sensitivity and specificity, with some problems of repeatability and inter-laboratory variability. This last issue needs to be addressed in order to allow harmonized diagnostic of IPNV within the country. We recommend the use of the NRL methods as validated and reliable qRT-PCR protocols for the detection of IPNV.

Visualization of the infectious pancreatic necrosis virus replication cycle by labeling viral intermediates with a TUNEL assay.

Investigating the capacity of the infectious pancreatic necrosis virus (IPNV) to promote apoptosis with a TUNEL kit, designed to visualize fragmented DNA, a striking labeling pattern was found. In addition to the fluorescence observed in the nucleus of cells with fragmented DNA, the infected cells showed an intense particulate fluorescence in their cytoplasm. The cytoplasmic labeling seems to be concomitant with the timing of virus life cycle because labeled viral proteins coexist with TUNEL cytoplasmic fluorescence at different times during infection. In principle the terminal deoxynucleotidyl transferase (TdT) used in TUNEL assays should not be able to label substrates other than DNA, however, our results are consistent with the idea that viral structures are being labeled. We propose that the TdT enzyme label the 3'-hydroxyl ends of viral RNAs during its polymerization. This kind of reaction could be possible because it has been reported that TdT is an enzyme that can use rNTPs as substrates and the 3'-hydroxyl priming end can be provided by a ribonucleotide as well. Thus, allowing the visualization of virus RNA-containing intermediates during IPNV replication.

Relationship between apoptosis and the BH2 domain sequence of the VP5 peptide of infectious pancreatic necrosis virus / Relación entre apoptosis y secuencia del dominio BH2 del peptido VP5 del virus de la necrosis pancreatica infecciosa

Objective. To determine whether the level of apoptosis induced by infectious pancreatic necrosis virus (IPNV) is related to the amino acid sequence of the BH2 domain of the VP5 protein and the level of infectivity. Materials and methods. Three IPNV strains were used, the VP2 protein gene was amplified for genotyping and the VP5 sequence was also obtained. The infectivity of the strains was calculated using the viral titer obtained at 12, 24, 36 and 45 hpi in CHSE-214 cells. The percentage of apoptosis in infected cells was visualized by TUNEL assay and immunohistochemistry (caspase 3 detection). Results. The V70/06 and V33/98 strains corresponded to genotype Sp, while V112/06 to VR-299; the amino acid analysis of the V70/06 strain allows its classification as middle virulent strain and V33/98 and V112/06 strains as low virulent ones; infection with the V112/06 strain produced a lower viral titer (p<0.05). The VP5 gene of the 3 strains showed four homologous domains to Bcl-2, however, the BH2 domain was truncated in V70/06 and V33/98 (12 kDa), being complete (15kDa) in V112/06, which also showed the Trp155 residue, equivalent to Trp188 considered as a critical factor for the function of Bcl-2. The average apoptosis was below 12%, showing no differences between strains (p>0.05). Conclusions. The results showed that the differences in the BH2 sequence of the VP5 protein, infectivity and the VP2 sequence are not associated with the modulation of apoptosis.

Objetivo. Determinar si el nivel de apoptosis inducido por cepas del virus de la necrosis pancreática infecciosa (IPNV) tiene relación con la secuencia aminoacídica del dominio BH2 de la proteína VP5 y el nivel de infectividad. Materiales y métodos. Se utilizaron tres cepas de IPNV; el gen de la proteína VP2 fue amplificado para genotipificación y se obtuvo la secuencia de VP5. La infectividad de las cepas se calculó mediante el título viral obtenido a 12, 24, 36 y 45 hpi en células CHSE-214. Los porcentajes de apoptosis en células infectadas se visualizaron mediante ensayo TUNEL e inmuno-histoquímica (detección de caspasa 3). Resultados. Las cepas V70/06 y V33/98 correspondieron a genotipo Sp, mientras que V112/06 a VR-299; el análisis aminoacídico relacionó a V70/06 como cepa de mediana virulencia y a V33/98 y V112/06 de baja virulencia; la infección con V112/06 produjo menor título viral (p<0.05). El gen VP5 de las 3 cepas presentó los cuatro dominios homólogos a Bcl-2; sin embargo, el dominio BH2 fue truncado en V70/06 y V33/98 (12 kDa); siendo completo (15kDa) en V112/06, que además, presentó el residuo Trp155, equivalente a Trp188 considerado factor crítico para la función de Bcl-2. El promedio de apoptosis fue inferior a 12%, no se observaron diferencias entre cepas (p>0.05). Conclusiones. Los resultados mostraron que las diferencias en la secuencia de BH2 de la proteína VP5, la infectividad y en la secuencia de la proteína VP2 no están asociadas con la modulación de apoptosis.

Infectious pancreatic necrosis virus (IPNV) enumeration through epifluorescence microscopy: technical aspects

A method for counting Infectious pancreatic necrosis virus (IPNV) through epifluorescence microscopy was analyzed in detail. Image processing and statistic considerations are included. The particle size of viruses was compared in different experimental conditions such as the staining of the virus with SYBR-Green I or with antibodies for specific fluorescence labeling of viral proteins. The type of surface used as mounting support was assayed as well. The results indicated that the most suitable method involves the mounting of the viral-containing suspension on a membrane filter followed by the staining with a monoclonal antibody specific for a viral protein combined with a FITC (fluorescein isothiocyanate)-conjugated secondary antibody.

Restauraciones clase V sin microfiltración: estudio "in vitro" / Class V restorations without microleakage: an in vitro study

Se estudió la influencia de la contracción de polimerización de la resina compuesta sobre la capacidad de sellado de restauraciones mixtas de clase V. Se prepararon cavidades en bucal y lingual de 21 molares extraídos y se restauraron con Silux-Plus, Scotchbond 2 y Vitrebond. Siete grupos de seis cavidades cada uno fueron preparados. Un grupo fue restaurado sólo con ionómero de vidrio, cuatro grupos recibieron tratamientos diferentes de la superficie del cemento ionomérico. En un grupo la cavidad fue preparada con algunas modificaciones de diseño y finalmente un grupo fue sometido a ciclaje térmico entre 5 y 55 grados. Luego, los dientes fueron sumergidos en una solución de fucsina básica al 2 por ciento por 24 horas, lavados y seccionados para observar el grado de penetración del colorante. Los resultados demostraron que las restauraciones realizadas sólo con Vitrebond no fueron capaces de prevenir la filtración severa en ningún margen. Las restauraciones mixtas fueron superiores en ambos márgenes. La microfiltración en las restauraciones mixtas fue menor en los márgenes de esmalte que de dentina. Los tratamientos realizados sobre la superficie del ionómero, la modificación del diseño cavitario y el ciclaje térmico no modificaron la intensidad de la filtración