Immunohistochemical detection of p53 and pp53 Ser392 in canine hemangiomas and hemangiosarcomas located in the skin.

BACKGROUND: p53 protein is essential for the regulation of cell proliferation. Aberrant accumulation of it usually occurs in cutaneous malignancies. Mutant p53 is detected by immunohistochemistry because it is more stable than the wild-type p53. However, post-translational modifications of p53 in response to ultraviolet radiation are important mechanisms of wild-type p53 stabilization, leading to positive staining in the absence of mutation. The aims were: 1) to analyze the immunohistochemical expression of p53 and phospho-p53 Serine392 in canine skin endothelial tumours; and 2) to determine if any relationship exists between p53 and phospho-p53 Serine392 overexpression and cell proliferation. RESULTS: p53 and phospho-p53 Serine392 immunolabeling was examined in 40 canine cutaneous endothelial tumours (13 hemangiomas and 27 hemangiosarcomas). Their expression was associated with tumour size, hemangiosarcoma stage (dermal versus hypodermal), histological diagnosis and proliferative activity (mitotic count and Ki-67 index). Statistical analysis revealed a significant increase of p53 immunoreactivity in hemangiosarcomas (median, 74.61%; interquartile range [IQR], 66.97-82.98%) versus hemangiomas (median, 0%; IQR, 0-20.91%) (p < .001) and in well-differentiated hemangiosarcomas (median, 82.40%; IQR, 66.49-83.17%) versus hemangiomas (p = .002). Phospho-p53 Serine392 immunoreactivity was significantly higher in hemangiosarcomas (median, 53.80%; IQR, 0-69.50%) than in hemangiomas (median, 0%; IQR, 0.0%) (p < .001). Positive correlation of the overexpression of p53 and phospho-p53 Serine392 with mitotic count and Ki-67 index was found in the cutaneous vascular tumours (p < .001). The Ki-67 index of the hemangiomas (median, 0.50%; IQR, 0-2.80%) was significantly lower than that of the hemangiosarcomas (median, 34.85%; IQR, 23.88-42.33%) (p < .001), and that specifically of well-differentiated hemangiosarcomas (median, 24.60%; IQR, 15.45-39.35%) (p = .001). Immunolabeling of 18 visceral hemangiosarcomas showed that the p53 (median, 41.59%; IQR, 26.89-64.87%) and phospho-p53 Serine392 (median, 0%; IQR, 0-22.53%) indexes were significantly lower than those of skin (p = .001; p = .006, respectively). CONCLUSIONS: The p53 and phospho-p53 Serine392overexpression together with high proliferative activity in hemangiosarcomas versus hemangiomas indicated that p53 might play a role in the acquisition of malignant phenotypes in cutaneous endothelial neoplasms in dogs. The Ki-67 index may be useful in distinguishing canine well-differentiated hemangiosarcomas from hemangiomas.

Correction to: Mixed-method tutoring support improves learning outcomes of veterinary students in basic subjects.

The original article [1] contains an error whereby Fig. 2a and b are mistakenly swapped with each other, and thus do not correspond to their correct respective sub-headings in the caption.

Mixed-method tutoring support improves learning outcomes of veterinary students in basic subjects.

BACKGROUND: Tutoring is a useful tool in the university teaching-learning binomial, although its development is impaired in large classes. Recent improvements in information and communication technologies have made tutoring possible via the Internet. The aim of this study was to evaluate the efficacy of mixed-method academic tutoring in two basic subjects in Veterinary Science studies at the University of León (Spain) to optimize the usefulness of tutoring support in the college environment. This quasi-experimental study was firstly carried out as a pilot study in a small group of tutored students of "Cytology and Histology" (CH) (47/186; 25.3%) and "Veterinary Pharmacology" (VP) (33/141; 23.4%) subjects, and was implemented in a large class of CH the next academic year (150 students) while comparing the results with those obtained in a previous tutorless course (162 students). Tutored students were given access to online questionnaires with electronic feedback on each subject. In addition to traditional tutoring carried out in both tutored and tutorless students, the pilot study included three sessions of face-to-face tutoring in order to monitor the progress of students. Its efficacy was assessed by monitoring students' examination scores and attendance as well as a satisfaction survey. RESULTS: Although the examination attendance rate in the pilot study was not significantly different between tutored and tutorless groups in both subjects, an increase for numerical scores in tutored groups was observed, with a significant higher final score in VP (p = 0.001) and in the CH practice exams (first term, p = 0.009; final, p = 0.023). Good and merit scores were also better in tutored students with significant differences in VP (p = 0.005). Students felt comfortable with the tutoring service (100% in CH; 91.7% in VP). Implementation of this additional support in CH also resulted in a significant increase of attendance at the final exam in tutored courses (87.3% versus 77.2%; p = 0.026), scaled (p = 0.001) and numerical scores (final score, p = 0.001). CONCLUSIONS: Online tutoring support, together with conventional teaching methods, may be a useful method to incorporate student-centered learning in basic subjects in Veterinary Science.

Magnetic resonance imaging and immunohistochemistry of primary vertebral hemangiosarcoma in a dog and implications for diagnosis and therapy.

A vertebral mass in a dog with an acute onset paraparesis was identified by magnetic resonance imaging. A poorly differentiated hemangiosarcoma was diagnosed by histopathology and immunohistochemistry. Endothelial nitric oxide synthase could be a new differential marker for poorly differentiated hemangiosarcoma in dogs. Immunohistochemical detection of p53 phosphorylated at Serine392, p53, CD117, and CD44 suggest targets for design of therapeutic strategies.

Imagerie par résonance magnétique et immunistochimie d'un hémangiosarcome vertébral primaire chez un chien et répercussions pour le diagnostic et le traitement. Une masse vertébrale chez un chien atteint d'une manière soudaine d'une paraparésie a été identifiée à l'aide d'imagerie par résonance magnétique. Un hémangiosarcome mal différencié a été diagnostiqué par histopathologie et immunohistochimie. La synthase à l'oxyde nitrique endothélial pourrait être un nouveau marqueur différentiel pour l'hémangiosarcome mal différencié chez les chiens. La détection immunohistochimique de p53 phosphorylé à la sérine392, p53, CD117 et CD44 suggère des cibles pour la conception de stratégies thérapeutiques.(Traduit par Isabelle Vallières).

In vivo long-term effects of retinoic acid exposure in utero on induced tumours in adult mouse skin.

BACKGROUND: Retinoic acid (RA) and its analogues (retinoids) are promising agents in skin cancer prevention following either topical application or oral administration. However, long-term in vivo effects of RA on chemically induced hyperplastic epidermal foci in adult mouse skin have also been described, casting some doubt with regard to its chemopreventive activity. HYPOTHESIS/OBJECTIVES: To characterize chemically induced skin tumours and to investigate the in vivo long-term action and preventive effect of RA on adult mouse skin carcinogenesis. ANIMALS: Fifty-six adult Naval Medical Research Institute mice, exposed (n = 28) or not exposed (n = 28) to RA in utero. METHODS: Mice were treated with a standard two-stage skin carcinogenesis protocol, which included an initiating application of 7,12-dimethylbenz(a)anthracene followed by promotion with 12-O-tetradecanoylphorbol 13-acetate. RESULTS: Retinoic acid administered to pregnant mice showed a long-term inhibitory action on cell differentiation and development of chemically induced tumours on the adult skin of their offspring, as well as a stimulatory effect on cell proliferation and expression of an early marker of malignant progression (keratin 13). CONCLUSIONS AND CLINICAL IMPORTANCE: The results suggest that RA exposure in utero confers long-lasting effects on adult mouse skin carcinogenesis. These include chemopreventive activity (reduced number of tumours), as well as enhancement of squamous papilloma progression, which appears to be due to enhanced keratinocyte proliferation and suppression of epidermal maturation. The clinical significance of these findings is not known for other routes of RA administration at this time.

Evaluation of the COVID-19 Lockdown-Adapted Online Methodology for the Cytology and Histology Course as Part of the Degree in Veterinary Medicine.

The COVID-19 pandemic and lockdown brought numerous teaching challenges requiring innovative approaches to teaching and learning, including novel modes of content delivery, virtual classrooms, and online assessment schemes. The aim of this study is to describe and assess the efficacy of the methods implemented at the University of León (Spain) to adapt to lockdowns in the context of the Cytology and Histology (CH) course for veterinary medicine undergraduate students. To evaluate the success of lockdown-adapted methodologies, we used inferential statistical analysis to compare the academic outcomes of two cohorts: 2018-2019 (traditional face-to-face-presential-learning and evaluation) and 2019-2020 (some face-to-face and some online lockdown-adapted learning and online lockdown-adapted evaluation). This analysis considered scores in both theoretical and practical exams and students' final subject score. We also evaluated the number of logs onto the Moodle platform throughout the 2019-2020 period, as well as performing a student satisfaction survey in both courses. The use of explanatory pre-recorded lectures, continuous online self-assessment tests, and virtual microscopy (VM) may have produced significant improvements in the acquisition of histology competencies among students in the lockdown cohort. However, we need to implement further strategies to improve the assessment of students' true level of knowledge acquisition. According to the student feedback, VM is a well-accepted resource that is perceived as a flexible and enjoyable tool to use. However, while students found that the resource enhances their ability to learn about microscopic structures, they felt that it should not completely replace optical microscopy.

Goats Naturally Infected with the Spanish Goat Encephalitis Virus (SGEV): Pathological Features and An Outbreak.

In autumn 2011, a disease outbreak caused by Spanish goat encephalitis virus (SGEV) was reported in a herd of goats from Asturias (north-western Spain), expanding the known geographic distribution of tick-borne encephalitis in Europe. The virus was classified as a new subtype (subspecies) within the Louping-ill virus species of the mammalian tick-borne flavivirus group. The aims of the present study were to describe the pathology in goats naturally infected with SGEV, as well as discuss the pathogenesis of the disease in that outbreak. A total of 22/85 (25.88%) goats (20 adults and 2 kids) died between October 2011 and June 2012, showing neurological clinical signs. Over three years, the mortality rate in the herd reached 100%. Neuropathological lesions caused by SGEV were severe and widespread throughout the central nervous system but were more severe and numerous in the proximal cervical spinal cord, medulla oblongata, pons and cerebellar cortex. They consisted of neuron necrosis, neuronophagia, mononuclear inflammatory cell perivascular cuffs (lymphocytes, plasma cells and macrophages) and gliosis. The distribution of viral antigens was restricted to the cytoplasm of neurons in several brain areas but not associated with inflammatory foci nor inflammatory cells. SGEV should be considered a significant pathogen of goats that results in severe neurological clinical disease and high mortality.

Coronary microcirculation damage in anthracycline cardiotoxicity.

AIMS: The aim of this study was to study changes in coronary microcirculation status during and after several cycles of anthracycline treatment. METHODS AND RESULTS: Large-white male pigs (n=40) were included in different experimental protocols (ExPr.) according to anthracycline cumulative exposure [0.45 mg/kg intracoronary (IC) doxorubicin per injection] and follow-up: control (no doxorubicin); single injection and sacrifice either at 48 h (ExPr. 1) or 2 weeks (ExPr. 2); 3 injections 2 weeks apart (low cumulative dose) and sacrifice either 2 weeks (ExPr. 3) or 12 weeks (ExPr. 4) after third injection; five injections 2 weeks apart (high cumulative dose) and sacrifice 8 weeks after fifth injection (ExPr. 5). All groups were assessed by serial cardiac magnetic resonance (CMR) to quantify perfusion and invasive measurement of coronary flow reserve (CFR). At the end of each protocol, animals were sacrificed for ex vivo analyses. Vascular function was further evaluated by myography in explanted coronary arteries of pigs undergoing ExPr. 3 and controls. A single doxorubicin injection had no impact on microcirculation status, excluding a direct chemical toxicity. A series of five fortnightly doxorubicin injections (high cumulative dose) triggered a progressive decline in microcirculation status, evidenced by reduced CMR-based myocardial perfusion and CFR-measured impaired functional microcirculation. In the high cumulative dose regime (ExPr. 5), microcirculation changes appeared long before any contractile defect became apparent. Low cumulative doxorubicin dose (three bi-weekly injections) was not associated with any contractile defect across long-term follow-up, but provoked persistent microcirculation damage, evident soon after third dose injection. Histological and myograph evaluations confirmed structural damage to arteries of all calibres even in animals undergoing low cumulative dose regimes. Conversely, arteriole damage and capillary bed alteration occurred only after high cumulative dose regime. CONCLUSION: Serial in vivo evaluations of microcirculation status using state-of-the-art CMR and invasive CFR show that anthracyclines treatment is associated with progressive and irreversible damage to the microcirculation. This long-persisting damage is present even in low cumulative dose regimes, which are not associated with cardiac contractile deficits. Microcirculation damage might explain some of the increased incidence of cardiovascular events in cancer survivors who received anthracyclines without showing cardiac contractile defects.

Remote ischaemic preconditioning ameliorates anthracycline-induced cardiotoxicity and preserves mitochondrial integrity.

AIMS: Anthracycline-induced cardiotoxicity (AIC) is a serious adverse effect among cancer patients. A central mechanism of AIC is irreversible mitochondrial damage. Despite major efforts, there are currently no effective therapies able to prevent AIC. METHODS AND RESULTS: Forty Large-White pigs were included. In Study 1, 20 pigs were randomized 1:1 to remote ischaemic preconditioning (RIPC, 3 cycles of 5 min leg ischaemia followed by 5 min reperfusion) or no pretreatment. RIPC was performed immediately before each intracoronary doxorubicin injections (0.45 mg/kg) given at Weeks 0, 2, 4, 6, and 8. A group of 10 pigs with no exposure to doxorubicin served as healthy controls. Pigs underwent serial cardiac magnetic resonance (CMR) exams at baseline and at Weeks 6, 8, 12, and 16, being sacrifice after that. In Study 2, 10 new pigs received 3 doxorubicin injections (with/out preceding RIPC) and were sacrificed at week 6. In Study 1, left ventricular ejection fraction (LVEF) depression was blunted animals receiving RIPC before doxorubicin (RIPC-Doxo), which had a significantly higher LVEF at Week 16 than doxorubicin treated pigs that received no pretreatment (Untreated-Doxo) (41.5 ± 9.1% vs. 32.5 ± 8.7%, P = 0.04). It was mainly due to conserved regional contractile function. In Study 2, transmission electron microscopy (TEM) at Week 6 showed fragmented mitochondria with severe morphological abnormalities in Untreated-Doxo pigs, together with upregulation of fission and autophagy proteins. At the end of the 16-week Study 1 protocol, TEM revealed overt mitochondrial fragmentation with structural fragmentation in Untreated-Doxo pigs, whereas interstitial fibrosis was less severe in RIPC+Doxo pigs. CONCLUSION: In a translatable large-animal model of AIC, RIPC applied immediately before each doxorubicin injection resulted in preserved cardiac contractility with significantly higher long-term LVEF and less cardiac fibrosis. RIPC prevented mitochondrial fragmentation and dysregulated autophagy from AIC early stages. RIPC is a promising intervention for testing in clinical trials in AIC.

Phenotypic Characterization of Encephalitis and Immune Response in the Brains of Lambs Experimentally Infected with Spanish Goat Encephalitis Virus.

Spanish goat encephalitis virus (SGEV), a novel subtype of tick-borne flavivirus closely related to louping ill virus, causes a neurological disease in experimentally infected goats and lambs. Here, the distribution of microglia, T and B lymphocytes, and astrocytes was determined in the encephalon and spinal cord of eight Assaf lambs subcutaneously infected with SGEV. Cells were identified based on immunohistochemical staining against Iba1 (microglia), CD3 (T lymphocytes), CD20 (B lymphocytes), and glial fibrillary acidic protein (astrocytes). In glial foci and perivascular cuffing areas, microglia were the most abundant cell type (45.4% of immunostained cells), followed by T lymphocytes (18.6%) and B lymphocytes (4.4%). Thalamus, hypothalamus, corpus callosum, and medulla oblongata contained the largest areas occupied by glial foci. Reactive astrogliosis occurred to a greater extent in the lumbosacral spinal cord than in other regions of the central nervous system. Lesions were more frequent on the side of the animal experimentally infected with the virus. Lesions were more severe in lambs than in goats, suggesting that lambs may be more susceptible to SGEV, which may be due to species differences or to interindividual differences in the immune response, rather than to differences in the relative proportions of immune cells. Larger studies that monitor natural or experimental infections may help clarify local immune responses to this flavivirus subtype in the central nervous system.

Immunohistochemical Assessment of Immune Response in the Dermis of Sarcoptes scabiei-Infested Wild Carnivores (Wolf and Fox) and Ruminants (Chamois and Red Deer).

Sarcoptic mange is caused by the mite Sarcoptes scabiei and has been described in several species of domestic and wild mammals. Macroscopic lesions are predominantly hyperkeratotic (type I hypersensitivity) in fox, chamois and deer, but alopecic (type IV hypersensitivity) in wolf and some fox populations. To begin to understand the immune processes underlying these species differences in lesions, we examined skin biopsies from wolves (Canis lupus), foxes (Vulpes vulpes), chamois (Rupicapra rupicapra) and red deer (Cervus elaphus) naturally infested with S. scabiei. Twenty skin samples from five animals per species were used. Sections were immuno-stained with primary antibodies against Iba1 to detect macrophages, lambda chain to detect plasma cells, CD3 to detect T lymphocytes and CD20 to detect B lymphocytes. Skin lesions contained significantly more inflammatory cells in the fox than in the wolf and chamois. Macrophages were the most abundant inflammatory cells in the lesions of all the species studied, suggesting a predominantly innate, non-specific immune response. Lesions from the wolf contained higher proportions of macrophages than the other species, which may reflect a more effective response, leading to alopecic lesions. In red deer, macrophages were significantly more abundant than plasma cells, T lymphocytes and B lymphocytes, which were similarly abundant. The fox proportion of plasma cells was significantly higher than those of T and B lymphocytes. In chamois, T lymphocytes were more abundant than B lymphocytes and plasma cells, although the differences were significant only in the case of macrophages. These results suggest that all the species examined mount a predominantly innate immune response against S. scabiei infestation, while fox and chamois may also mount substantial humoral and cellular immune responses, respectively, with apparently scarce effectiveness that lead to hyperkeratotic lesions.

Phenotypic characterization of encephalitis in the brains of goats experimentally infected with Spanish Goat Encephalitis Virus.

Spanish goat encephalitis virus (SGEV) is a novel tick-borne flavivirus subtype, closely related to the flavivirus louping ill virus (LIV). SGEV caused a severe, acute and mortal neurological disease outbreak in northern Spain in a goat herd. In order to characterize the cell population in lesions and to determine the distribution of the inflammatory cells, central nervous system (CNS) samples of nine female Alpine goats challenged subcutaneously with SGEV over the right thorax behind the elbow were evaluated using immunohistochemistry (microglia-Iba1, T lymphocytes-CD3, B lymphocytes-CD20 and astrocytes-GFAP). The number of microglia (37.8 %) and T lymphocytes (21.5 %) was greater than the number of B lymphocytes (16.8 %). Goats were classified into clusters based on the severity of histological lesions in CNS (A-mild to moderate lesions and B-severe lesions). Microglia was significantly more abundant than T and B lymphocytes in cluster B (severe lesions). The total area occupied by glial foci revealed that medulla oblongata and spinal cord were the most affected tissues. Astrogliosis (GFAP+) was present in the majority of the CNS sections being near to the pial surface. The lesion predominance on the right side of the medulla oblongata, which could be associated to the site of challenge suggestive of neurotropic route was also statistically confirmed. Results suggest that the cellular immune response would be the most important response to the SGEV infection.

Development and Evaluation of a Disease Large Animal Model for Preclinical Assessment of Renal Denervation Therapies.

New-generation catheters-based renal denervation (RDN) is under investigation for the treatment of uncontrolled hypertension (HTN). We assessed the feasibility of a large animal model of HTN to accommodate the human RDN devices. Ten minipigs were instrumented to measure blood pressure (BP) in an awake-state. HTN was induced with subcutaneous 11-deoxycorticosterone (DOCA, 100 mg/kg) implants. Five months after, the surviving animals underwent RDN with the Symplicity® system. Norepinephrine (NE) renal gradients were determined before and 1 month after RDN. Renal arteries were processed for histological (hematoxylin-eosin, Movat pentachrome) and immunohistochemical (S100, tyrosine-hydroxylase) analyses. BP significantly rose after DOCA implants. Six animals died prematurely, mainly from infectious causes. The surviving animals showed stable BP levels after 5 months. One month after RDN, nerve damage was showed in three animals, with impedance drop >10%, NE gradient drop and reduction in BP. The fourth animal showed no nerve damage, impedance drop <10%, NE gradient increase and no change in BP. In conclusion, the minipig model of DOCA-induced HTN is feasible, showing durable effects. High mortality should be addressed in next iterations of this model. RDN may partially offset the DOCA-induced HTN. Impedance drop and NE renal gradient could be markers of RDN success.

Kv1.3 blockade inhibits proliferation of vascular smooth muscle cells in vitro and intimal hyperplasia in vivo.

The modulation of voltage-gated K+ (Kv) channels, involved in cell proliferation, arises as a potential therapeutic approach for the prevention of intimal hyperplasia present in in-stent restenosis (ISR) and allograft vasculopathy (AV). We studied the effect of PAP-1, a selective blocker of Kv1.3 channels, on development of intimal hyperplasia in vitro and in vivo in 2 porcine models of vascular injury. In vitro phenotypic modulation of VSMCs was associated to an increased functional expression of Kv1.3 channels, and only selective Kv1.3 channel blockers were able to inhibit porcine VSMC proliferation. The therapeutic potential of PAP-1 was then evaluated in vivo in swine models of ISR and AV. At 15-days follow-up, morphometric analysis demonstrated a substantial reduction of luminal stenosis in the allografts treated with PAP-1 (autograft 2.72 ± 1.79 vs allograft 10.32 ± 1.92 vs allograft + polymer 13.54 ± 8.59 vs allograft + polymer + PAP-1 3.06 ± 1.08 % of luminal stenosis; P = 0.006) in the swine model of femoral artery transplant. In the pig model of coronary ISR, using a prototype of PAP-1-eluting stent, no differences were observed regarding % of stenosis compared to control stents (31 ± 13 % vs 37 ± 18%, respectively; P = 0.372) at 28-days follow-up. PAP-1 treatment was safe and did not impair vascular healing in terms of delayed endothelialization, inflammation or thrombosis. However, an incomplete release of PAP-1 from stents was documented. We conclude that the use of selective Kv1.3 blockers represents a promising therapeutic approach for the prevention of intimal hyperplasia in AV, although further studies to improve their delivery method are needed to elucidate its potential in ISR.

Endothelialization of nonapposed stent struts located over the origin of a side branch: results with different carbofilm-coated stents.

OBJECTIVE: To evaluate the degree of endothelialization of the nonapposed struts located at the ostia of side branches. BACKGROUND: Endothelialization of coronary stents has got considerable relevance because of the phenomenon of late thrombosis. Bifurcation location and incomplete stent apposition have been linked to this complication. METHODS: Domestic pigs (n = 11; weight: 25 +/- 3 kg) were anesthetized and had one stent per coronary artery implanted: one stainless steel (Tecnic), one cobalt-chromium (Chrono), and one tacrolimus-eluting stent (Janus), all of them being Carbofilm-coated (Sorin). One, three, or seven days postprocedure, the pigs were sacrificed, the hearts explanted, and longitudinal sections examined by surface electron microscopy to quantify the percentage of the strut endothelialized over the branches and in the total surface. RESULTS: Forty-four side branches (25 stents) that had stent struts over their origin were evaluated. Different patterns of endothelialization were observed, from the total absence to the complete endothelialization. There were no significant differences in relation to type of stent or to the artery treated. The predictors of higher percentage of endothelialization were the ratio of metal to branch diameter (P = 0.04) and better endothelialization in the rest of the stent (P = 0.0002), only this parameter maintaining significant correlation (P = 0.03) in multivariate analysis. CONCLUSIONS: Carbofilm-coated stent struts located over the origin of side branches follow the pattern of endothelialization for the rest of the stent, even in the case of tacrolimus-eluting stent.

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.

Serial Magnetic Resonance Imaging to Identify Early Stages of Anthracycline-Induced Cardiotoxicity.

BACKGROUND: Anthracycline-induced cardiotoxicity is a major clinical problem, and early cardiotoxicity markers are needed. OBJECTIVES: The purpose of this study was to identify early doxorubicin-induced cardiotoxicity by serial multiparametric cardiac magnetic resonance (CMR) and its pathological correlates in a large animal model. METHODS: Twenty pigs were included. Of these, 5 received 5 biweekly intracoronary doxorubicin doses (0.45 mg/kg/injection) and were followed until sacrifice at 16 weeks. Another 5 pigs received 3 biweekly doxorubicin doses and were followed to 16 weeks. A third group was sacrificed after the third dose. All groups underwent weekly CMR examinations including anatomical and T2 and T1 mapping (including extracellular volume [ECV] quantification). A control group was sacrificed after the initial CMR. RESULTS: The earliest doxorubicin-cardiotoxicity CMR parameter was T2 relaxation-time prolongation at week 6 (2 weeks after the third dose). T1 mapping, ECV, and left ventricular (LV) motion were unaffected. At this early time point, isolated T2 prolongation correlated with intracardiomyocyte edema secondary to vacuolization without extracellular space expansion. Subsequent development of T1 mapping and ECV abnormalities coincided with LV motion defects: LV ejection fraction declined from week 10 (2 weeks after the fifth and final doxorubicin dose). Stopping doxorubicin therapy upon detection of T2 prolongation halted progression to LV motion deterioration and resolved intracardiomyocyte vacuolization, demonstrating that early T2 prolongation occurs at a reversible disease stage. CONCLUSIONS: T2 mapping during treatment identifies intracardiomyocyte edema generation as the earliest marker of anthracycline-induced cardiotoxicity, in the absence of T1 mapping, ECV, or LV motion defects. The occurrence of these changes at a reversible disease stage shows the clinical potential of this CMR marker for tailored anthracycline therapy.

Immunogenic characterization of vaccines based on Haemophilus parasuis Nagasaki strain, OmpP2, OmpP5 and OmpD15, in colostrum-deprived pigs experimentally challenged with the same strain.

Three recombinant outer membrane proteins (rOmps) from the Haemophilus parasuis Nagasaki strain (serovar 5 reference strain), rOmpP2, rOmpP5 and rOmpD15, which have previously shown protection against H. parasuis infection in mice, were cloned, expressed and evaluated as vaccine antigens in colostrum-deprived pigs. When these animals were immunized with these rOmps and were later challenged intratracheally with 108 CFUs of the Nagasaki strain, no protection was seen in terms of survival, clinical signs, pathological results and recovery of H. parasuis. We hypothesized that a possible explanation for this lack of protection could be the low number of epitopes accessible to the immune system as a consequence of their poor exposure on the bacterial surface so that the immune response would not be able to protect against experimental infection by H. parasuis when a fully susceptible animal model, such as pigs, was used.

Safety and Efficacy of New Biodegradable Polymer-based Sirolimus-Eluting Stents in a Preclinical Model.

INTRODUCTION AND OBJECTIVES: New drug-eluting stents (DES) designed to overcome the limitations of existing devices should initially be tested in preclinical studies. Our objective was to analyze the safety and efficacy of new biodegradable polymer-based DES compared with bare-metal stents (BMS) and commercially available DES in a model of normal porcine coronary arteries. METHODS: We randomly implanted 101 stents (BMS and biodegradable polymer-based sirolimus-eluting stents: 3 test stent iterations [BD1, BD2, and BD3], Orsiro, Biomime and Biomatrix) in the coronary arteries of 34 domestic pigs. Angiographic and histomorphometric studies were conducted 1 month (n = 83) and 3 months (n = 18) later. RESULTS: The stents were implanted at a stent/artery ratio of 1.31 ± 0.21, with no significant differences between groups. At 1 month, the new test stents (BD1, BD2 and BD3) showed less late loss and angiographic restenosis, as well as lower histologic restenosis and neointimal area (P < .0005), than the BMS. There were no differences in endothelialization, vascular injury, or inflammation between the new test stents and BMS, although the new stents showed higher fibrin deposition (P = .0006). At 3 months, all these differences disappeared, except for a lower neointimal area with the new BD1 stent (P = .027). No differences at any time point were observed between the new test stents and commercially available controls. CONCLUSIONS: In this preclinical model, the new biodegradable polymer-based DES studied showed less restenosis than BMS and no significant differences in safety or efficacy vs commercially available DES.

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 Glässer's 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.