Claudia Pérez-Martínez

Endogenous cardiac stem cell activation by insulin-like growth factor-1/hepatocyte growth factor intracoronary injection fosters survival and regeneration of the infarcted pig heart.

OBJECTIVES The purpose of this study was to test the ability of insulin-like growth factor (IGF)-1/hepatocyte growth factor (HGF) to activate resident endogenous porcine cardiac stem/progenitor cells (epCSCs) and to promote myocardial repair through a clinically applicable intracoronary injection protocol in a pig model of myocardial infarction (MI) relevant to human disease. BACKGROUND In rodents, cardiac stem/progenitor cell (CSC) transplantation as well as in situ activation through intramyocardial injection of specific growth factors has been shown to result in myocardial regeneration after acute myocardial infarction (AMI). METHODS Acute MI was induced in pigs by a 60-min percutaneous transluminal coronary angiography left anterior descending artery occlusion. The IGF-1 and HGF were co-administered through the infarct-related artery in a single dose (ranging from 0.5 to 2 μg HGF and 2 to 8 μg IGF-1) 30 min after coronary reperfusion. Pigs were sacrificed 21 days later for dose-response relationship evaluation by immunohistopathology or 2 months later for cardiac function evaluation by cardiac magnetic resonance imaging. RESULTS The IGF-1/HGF activated c-kit positive-CD45 negative epCSCs and increased their myogenic differentiation in vitro. The IGF-1/HGF, in a dose-dependent manner, improved cardiomyocyte survival, and reduced fibrosis and cardiomyocyte reactive hypertrophy. It significantly increased c-kit positive-CD45 negative epCSC number and fostered the generation of new myocardium (myocytes and microvasculature) in infarcted and peri-infarct/border regions at 21 and 60 days after AMI. The IGF-1/HGF reduced infarct size and improved left ventricular function at 2 months after AMI. CONCLUSIONS In an animal model of AMI relevant to the human disease, intracoronary administration of IGF-1/HGF is a practical and effective strategy to reduce pathological cardiac remodeling, induce myocardial regeneration, and improve ventricular function.

Development and Characterization of Protective Haemophilus parasuis Subunit Vaccines Based on Native Proteins with Affinity to Porcine Transferrin and Comparison with Other Subunit and Commercial Vaccines

ABSTRACT Haemophilus parasuis is the agent responsible for causing Glässers disease, which is characterized by fibrinous polyserositis, polyarthritis, and meningitis in pigs. In this study, we have characterized native outer membrane proteins with affinity to porcine transferrin (NPAPT) from H. parasuis serovar 5, Nagasaki strain. This pool of proteins was used as antigen to developed two vaccine formulations: one was adjuvanted with a mineral oil (Montanide IMS 2215 VG PR), while the other was potentiated with a bacterial neuraminidase from Clostridium perfringens. The potential protective effect conferred by these two vaccines was compared to that afforded by two other vaccines, consisting of recombinant transferrin-binding protein (rTbp) A or B fragments from H. parasuis, Nagasaki strain, and by a commercially available inactivated vaccine. Five groups of colostrum-deprived piglets immunized with the vaccines described above, one group per each vaccine, and a group of nonvaccinated control animals were challenged intratracheally with a lethal dose (3 × 108 CFU) of H. parasuis, Nagasaki strain. The two vaccines containing rTbps yielded similar results with minimal protection against death, clinical signs, gross and microscopic lesions, and H. parasuis invasion. In contrast, the two vaccines composed of NPAPT antigen and commercial bacterin resulted in a strong protection against challenge (without deaths and clinical signs), mild histopathological changes, and no recovery of H. parasuis, thus suggesting their effectiveness in preventing Glässers disease outbreaks caused by serovar 5.

Closed-chest experimental porcine model of acute myocardial infarction-reperfusion

INTRODUCTION Progress in cardiovascular regenerative medicine research requires the availability of appropriate experimental animal models that are as close to humans as feasible. Our objective was to assess the validity of a porcine endovascular model of myocardial infarction and reperfusion. METHODS Fifteen domestic pigs (Large White race) were anesthetized and pre-medicated with amiodarone. Endovascular fluoroscopy-guided coronary procedures were performed to occlude the mid-left anterior descending artery using a coronary angioplasty balloon. Occlusion was confirmed by angiography and electrocardiography. After 75 min the balloon catheter system was withdrawn and the presence of reperfusion flow was verified. The animals were sacrificed after 1 and 2 weeks of follow-up, the hearts were explanted, and the extent of myocardial infarction with respect to the left ventricle was quantified. RESULTS Overall survival rate was 67%. Five animals died prematurely: 3 showing signs of heart failure, 1 had reperfusion failure (final TIMI flow grade 1) and 1 succumbed to acute stress. The most common adverse event was ventricular fibrillation (87% of the animals) and defibrillation was effective in all affected animals. The extent of myocardial infarct in the animals followed-up for 1 and 2 weeks was similar (20.4+/-4.3% vs. 20.9+/-2.8%, respectively; p=0.8) but was significantly greater in the animals that died prematurely (29.5+/-3.6%, p=0.02). CONCLUSIONS The endovascular porcine model we have explored constitutes a feasible and reproducible alternative for the evaluation of human myocardial infarction and reperfusion.

La reestenosis en el stent depende del daño vascular inducido. ¿Son válidos los modelos experimentales actuales de análisis de los stents farmacoactivos?

INTRODUCTION AND OBJECTIVES Drug-eluting stents are useful for preventing restenosis, but the patho-physiological processes involved in the proliferative response after implantation are still not known in detail. The aim of this study is to compare the coronary vascular histomorphometry after implanting drug-eluting stents and bare metal stents in a swine model. METHODS Sixty stents were randomly implanted in 20 Large White female pigs with a ratio of baremetal/drug-eluting stents of 1:2. After 28 days, euthanasia and histomorphometry were performed. We defined the vessel injury score in accordance to whether the internal elastic lamina was intact or ruptured. RESULTS There were no differences between drug-eluting stents and bare metal stents in the intact internal elastic lamina group regarding neointimal area or % restenosis (1.3 [1.1-2.2]) vs 2.0 [1.3-2.5] mm²; P=.6; and 14.0 [12.1-20.8] vs 22.2 [14.1-23.3] %; P=.5). We assessed statistically significant differences for the ruptured internal elastic lamina group, (neointimal area 1.2 [0.8-2.0] vs 2.9 [2.3-3.7] mm²; P=.001 and % restenosis 16.63 [11.2-23.5] vs 30.4 [26.4-45.7] %; P=.001). CONCLUSIONS In our swine model, we did not find any differences between proliferative response of drug-eluting stents and bare metal stents when the internal elastic lamina is intact; differences are only found when vascular injury is deeper.

Preclinical Evaluation of Coronary Stents: Focus on Safety Issues

In recent years, we have witnessed a revolution in the treatment of coronary artery disease. The development and improvement of drug eluting stents (DES) have lowered the incidence of restenosis to one-digit figures. In the search for a superior efficacy, animal models have played a key role. The classical swine model of coronary stenting remains the preferred model to measure restenosis, although the rabbit iliac artery stenting has become an accepted alternative. After widespread clinical use of DES, an unforeseen complication arose: late stent thrombosis. In a back-to-bench step, some data from animal models helped to explain the phenomenon. A delayed and incomplete vascular healing was detected. Toxic and hypersensitivity reactions to polymers and/or drugs seem to be the underlying causes. So, translational research focused on the safety aspect of these devices: development of better drug carriers as absorbable polymers or fully bioresorbable scaffolds, selection of different drugs and assessment of the re-endothelialization process. We review and evaluate the efficacy and safety of coronary stents in different animal models. Further improvements in this field such as, the selection of better animal models (e.g. hyperlipidemic, diabetic, atherosclerotic) that closely mimic the clinical setting and longer follow-up periods to detect late complications are also discussed.

Epidemic infection caused by Citrobacter rodentium in a gerbil colony

Non-motile, Gram-negative rods, isolated from the intestinal tract and kidney of several dead animals in a gerbil colony, were identified as Citrobacterrodentium (formerly included in C freundii species) on the basis of 31 biochemical tests. The isolates were tested against 40 antimicrobial agents and were all susceptible to ticarcillin plus clavulanate, ceftazidime and most of the quinolones studied, but were all resistant to most of the penicillins and aminoglycosides tested, and to fosfomycin, metronidazole and tiamulin. This bacterial species has been primarily associated with transmissible murine colonic hyperplasia, and this appears to be the first report of an epidemic infection in a gerbil colony with a fatal outcome in most of the animals affected.

Análisis de la inflamación luminal inducida por distintos tipos de stent coronario en el modelo coronario animal mediante microscopía electrónica de barrido

There is histological evidence that drug-eluting stents are associated with delayed endothelialization and a persistent inflammatory state. Moreover, clusters of inflammatory cells have been observed on luminal surfaces by scanning electron microscopy. With the aim of quantifying this inflammatory response, we implanted one bare-metal stent and two drug-eluting stents containing different doses of vinblastine embedded in the same polymer into the coronary arteries of 12 domestic pigs. The density of inflammatory cells in a representative area (100 x 100 μm) was quantified at 3 and 7 days. Endothelialization was more complete in bare-metal stents than in drug-eluting stents at both 3 days (P=.016) and 7 days (P=.0001). The degree of inflammation induced by the drug-eluting stents was higher than that induced by the bare-metal stents at both 3 days (11.8±3.5% vs. 4.5±2%; P=.001) and 7 days (26.3±4.4% vs. 1.2±1.5%; P=.0001). In addition, the time sequence was inverted: the inflammatory response increased over time with the drug-eluting stents, while the opposite occurred with the bare-metal stents.

The role of CD44 adhesion factor in canine mammary carcinomas.

CD44 is an adhesion molecule implicated in the progression of human breast cancer. The purpose of this study was to describe CD44 antigen expression in canine mammary carcinomas and to evaluate its prognostic significance in relation to other clinico-pathological variables. A reduction in CD44 expression was significantly related to variables such as tumour size and adherence to underlying tissues but was not related to tumour location or to ulceration of the overlying skin. Complex (grade I) and anaplastic (grade III) carcinomas exhibited more intense expression of this antigen than did some tubulopapillary and most solid carcinomas (grade II). Although reduced CD44 expression was associated with infiltrative growth and vascular invasion in solid carcinomas, intense expression was also observed in anaplastic tumours. Although overall these findings suggest a role for this adhesion factor in canine mammary tumour development and progression, the complexity and apparently paradoxical nature of some of the findings currently limit the use of this immunohistochemical marker as a prognostic indicator.

Spontaneous mouse mammary tumours: incidence and cytokeratin expression.

The purpose of this study was to advance our knowledge of the histogenesis of spontaneous mammary tumours in laboratory mice. Normal mammary tissue and 19 spontaneous mammary tumours from adult female mice were examined using monoclonal and polyclonal antibodies differing in their recognition of various cytokeratin intermediate filament proteins (CKs). All neoplasms were intraductal and were invasive carcinomas with a tubular, papillary, cystic or solid growth pattern. CK8-positive reactions were detected in the normal alveolar and ductal epithelia and CK5- and CK14-positive reactions were seen in myoepithelial cells of nonlactating mammary glands. Positive staining for CK5 and CK8 was detected in all tumours and CK14 was expressed in those with a papillary pattern. Comparisons between non-lactating glands and tumours indicated that the neoplasms were well or moderately differentiated, there was no squamoid differentiation and that they arose from the alveoli and duct system, not the myoepithelial cells.

A vaccine based on a mutant transferrin binding protein B of Haemophilus parasuis induces a strong T-helper 2 response and bacterial clearance after experimental infection.

This study aimed to characterize the type of immune response induced by an experimental vaccine based on a mutant Haemophilus parasuis transferrin binding protein (Tbp) B (Y167A) defective in its ability to bind porcine transferrin. Clinical and pathological signs, bacterial clearance, antibody response and the cytokine profile in alveolar macrophages and spleen after the vaccination and challenge of twenty-two colostrum-deprived pigs with 10(8) CFU of H. parasuis were analysed. Pigs vaccinated with Y167A were compared to those vaccinated with native TbpB (nTbpB), those treated with a commercial bacterin (CB) against Glässers disease, those unvaccinated challenged (CH) and those unvaccinated unchallenged (UNCH) pigs. The rectal temperatures of Y167A pigs resembled those of UNCH pigs and were significantly lower than those of the nTbpB, CB and CH animals. A major reduction in pathological changes of the challenged pigs was observed in the Y167A group. H. parasuis was cleared from 88.9% of the samples from Y167A pigs versus 60.0% and 55.6% from those of the CB and nTbpB groups, respectively. The antibody response elicited by Y167A by ELISA was notably higher than that observed for nTbpB and CB pigs and was capable of preventing the expression and secretion of IL-8. The expression of IL-4 and IL-5, which were associated with the specific antibody levels, suggests that the main mechanism of protection conferred by Y167A vaccine is based on a strong T-helper 2 response.