“New insights in the etiopathogenesis of muscle inflammation and aging in animals”

Muscle pathology has unique relevance in many different fields of veterinary research, ranging from the diagnostics, to the comparative medicine involving animal models of human diseases, to the animal based food safety. The Laboratory of Comparative of Neuromuscular Disorders was established decades ago at the Department Veterinary Medicine of Naples in order to contribute to the knowledge of muscle pathology in domestic animals using modern techniques and undertaking international scientific collaborations. In this context, the project of this doctoral thesis had the main purpose to produce new scientific data in at least two interest area: the etiopathogenesis of inflammatory myopathies and skeletal muscle aging. Thus, the experimental results of four research project conducted in four different species are presented, including: 1) Inflammatory myopathy in horses affected by chronic piroplasmosis; 2) Lymphoplasmacytic myositis and expression of Major Histocompatibility Complex class I and II in ovine muscular sarcocystosis; 3) Age related skeletal muscle atrophy and upregulation of autophagy in dogs; 4) Expression and biochemical properties of cellular Prion protein in skeletal muscle of aged cows. In the first and second chapters of the thesis, two inflammatory myopathies associated with parasites having or not having a specific tropism for skeletal muscle (Sarcocystis tenella and Theileria equi -Babesia caballi, respectively) are explored. In the first study, an inflammatory myopathy characterized by a mainly CD8-CD4+ lymphocytic population and fiber degeneration is described in 16 horses serologically positive for equine piroplasms with clinical signs and serum biochemical evidence of a myopathy. To test the hypothesis of circulating autoantibodies directed against an unknown muscular antigen, the serum from affected horses was tested by indirect immunofluorescence to sections of normal equine muscle. In all cases, distinct sarcolemmal staining was detected in sections incubated with serum from affected horses, in contrast to sections incubated with phosphate-buffered saline or equine control sera. Furthermore, a significant increase of interferon-γ, interleukin-12, and tumor necrosis factor-α gene expression by compared to healthy controls was revealed by Reverse transcription polymerase chain reaction (RT-PCR). Thus, we concluded that inflammatory myopathy associated with equine piroplasmosis may involve an autoimmune pathogenesis with upregulation of inflammatory cytokines that may cause myofiber atrophy and degeneration. The aim of the second chapter, leading with muscle inflammation in ovine muscular sarcosytosis, was to investigate if parasitized muscle fibers could play a role in immune-stimulation, as sporadically described in accidental muscular sarcocystosis in definitive hosts. Skeletal muscle samples from 78 sheep presenting muscular sarcocysts revealed inflammatory changes in 69% of cases, consisting of an endomysial lymphoplasmacellular infiltrate, with attendant myofiber degeneration and necrosis. The predominant T cell populations were CD3+, CD8+ with lesser numbers of CD4+ and CD79α+ cells. Eosinophils were constantly absent. Notably, moderate to strong sarcolemmal labeling to Major histocompatibility complex (MHC) I and II was found both in biopsies with evident inflammatory infiltrate and in cases without inflammation. The wall of the cysts resulted strongly positive to MHC Iand II, and occasionally co-localized with the membrane associated protein Dystrophin. Our data suggest that muscle fibers respond to the presence of cysts by expression of MHC I and II that can play a role in stimulating and maintaining the lymphoplasmacellular inflammation. The possible role of MHC I on the cyst wall is discussed, but basically more research is needed in order to evaluate its possible involvement in inflammation. Moreover we speculated about the possibility that the abnormal expression of MHC I and II on muscle fibers is not only typical of myopathies having an immune-mediated pathogenesis but also of myositis associated with muscular parasites. The last two chapters of the thesis are dedicated to muscle aging, and in particular to the role of autophagy in canine sarcopenia and of cellular prion protein in bovine muscle aging. Sarcopenia, the age related loss of muscle mass and strength, is a multifactorial condition that occurs in a variety of species and represents a major healthcare concern in human medicine. We evaluated the expression of three markers of autophagy, Beclin 1, LC3 and p62, in muscle wasting of 25 geriatric dogs, to establish whether the levels of autophagy change with increasing age. Our results indicated a marked intracytoplasmic staining for Beclin 1 and LC3 in 80% of the muscle samples from the older dogs and a significantly greater expression of LC3 II and Beclin 1, by Western blotting. The results of the study suggest that enhanced autophagy might be one of the factors underlying muscle atrophy in canine age related muscle wasting. Finally, we explored the expression and biochemical properties of cellular prion protein (PrPC) in skeletal muscle of cows as well as their possible changes in geriatric animals. PrPC over expression has been described in many different human neuromuscular disorders but little is known about PrPC in health and disease in bovine muscle tissue. Skeletal muscle and brain samples of 12 aged and 8 young Podolica cows were collected and compared by histomorphological techniques as well as several experiments involving immunoblot and specifically analyzing the amount of PrPC, its glycosylation profile, proteinase resistance, solubility levels and tendency to aggregate. Histologically, muscle biopsies of aged cows showed typical myopathic features already described in bovine sarcopenia. Immunohistochemistry for PrP revealed a distinct granular positivity in intramuscular nerve branches and muscle spindles in all cases. Membrane immunopositivity was more prominent on angular atrophic fibers. Increased levels of positivity were found in degenerated fibers (desmin depleted) with a positive labeling of rimmed vacuoles. Prion protein positivity levels was also significantly increased in cases displaying inflammatory changes. No differences between muscles and brains of young and old animals were detected by quantitative immunoblot and Proteinase K titration. The glycoform profile and the molecular mass of PrPC in muscle samples appeared slightly different compared to the brain. Sucrose gradient velocity sedimentation gave variable results, with a generally lower amount of PrP in high density sucrose fractions in the muscle compared to the brain. After high speed centrifugation the majority of muscle PrPC was found in the soluble fraction, without significant differences between young and old animals. Our preliminary data suggest that: 1) the amount and the main biochemical properties of prion protein do not change with age, 2) PrPC is overexpressed in degenerated, vacuolated and angular muscle fibers; 3) The glycoform profile of PrPC differs between brain and muscle tissue. In conclusion, the main innovative aspects of this thesis are: 1) The first description in the literature of a likely immune-mediated myositis occurring in chronic equine piroplasmosis 2) The demonstration of high prevalence of lymphoplasmacellular myositis in ovine muscular sarcocystosis implying the overexpression of MHC I on muscle fibers as well as on the cyst wall. 3) The report of enhanced autophagy in age-related muscle atrophy in dogs. 4) The first detailed description of PrPC localization and biochemical properties in bovine muscle tissue and its possible involvement in neurogenic atrophy, muscle degeneration and chronic inflammation. Collectively, these results enhance the relevance of morphological studies as starting point to investigate the pathogenesis of muscle diseases in veterinary and comparative pathology

Skype-Hunter: A real-time system for the detection and classification of Skype traffic

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Platelet lysate-derived neuropeptide y influences migration and angiogenesis of human adipose tissue-derived stromal cells

Neuropeptide Y (NPY), a powerful neurotransmitter of the central nervous system, is a key regulator of angiogenesis and biology of adipose depots. Intriguingly, its peripheral vascular and angiogenic powerful activity is strictly associated to platelets, which are source of clinical hemoderivates, such as platelet lysate (PL), routinely employed in several clinical applications as wound healing, and to preserve ex vivo the progenitor properties of the adipose stromal cells pool. So far, the presence of NPY in PL and its biological effects on the adipose stromal cell fraction (ASCs) have never been investigated. Here, we aimed to identify endogenous sources of NPY such as PL-based preparations and to investigate which biological properties PL-derived NPY is able to exert on ASCs. The results show that PL contains a high amount of NPY, which is in part also excreted by ASCs when stimulated with PL. The protein levels of the three main NPY subtype receptors (Y1, Y2, Y5) are unaltered by stimulation of ASCs with PL, but their inhibition through selective pharmacological antagonists, considerably enhances migration, and a parallel reduction of angiogenic features of ASCs including decrease in VEGF mRNA and intracellular calcium levels, both downstream targets of NPY. The expression of VEGF and NPY is enhanced within the sites of neovascularisation of difficult wounds in patients after treatment with leuco-platelet concentrates. Our data highlight the presence of NPY in PL preparations and its peripheral effects on adipose progenitors

Angiostrongylus vasorum infection in red foxes (Vulpes vulpes) in southern Italy

Angiostrongylus vasorum (Nematoda: Angiostrongylidae) infection was detected at post-mortem examination in the pulmonary arteries and hearts of 34/102 (33,3%) of red foxes (Vulpes vulpes) from the Campania Region in southern Italy. Pathological changes consisted of granulomatous interstitial pneumonia caused by larvae and intravascular pulmonary adult nematodes. These changes confirm that angiostrongylosis infection in red foxes has a mainly chronic course, in which the infected host may disperse parasite larvae in the environment over its lifetime. Results suggest that the life cycle of A. vasorum is well established in the red fox in the Campania Region representing a potential infection risk for dogs

Edad y crecimiento del pámpano, Trachinotus ovatus, del Estrecho de Messina (Mediterráneo central)

This is the first paper to provide detailed information on the age and growth of Trachinotus ovatus. The size of the 244 individuals collected in the Strait of Messina ranged from 2.7 to 30.4 cm in fork length (LF) and 0.31 to 508.6 g in body mass (M). The relationship between these parameters (M - LF) was investigated and showed a good fit. Age estimation based on vertebrae and otoliths yielded similar results, suggesting a maximum age of five years. However, the precision and accuracy tests, such as percentage of agreement (PA), mean coefficient of variation (ACV) and average percent error (APE) indicated that the otolith readings (97.83% PA, 0.54% ACV and 0.38% APE) were more reliable for age estimation than vertebrae readings (82.17% PA, 5.33% ACV and 3.77% APE). The multi-model inference approach allowed us to compare different non-linear growth models. The von Bertalanffy model (L∞=29.139, k=0.496 and t0=−0.347) fitted the length-at-age data best. This species has a relatively rapid growth and an estimated longevity of five to seven years. This information could be used for management and first stock assessment studies on T. ovatus in the Mediterranean Sea.Este es el primer artículo que proporciona información detallada sobre la edad y el crecimiento de Trachinotus ovatus. El tamaño de los 244 individuos recolectados en el Estrecho de Messina osciló entre 2.7 y 30.4 cm de longitud a la horquilla (LF) y 0.31 a 508.6 g de masa corporal (M). Se investigó la relación entre estos parámetros (M - LF) y mostró un buen ajuste. La estimación de la edad basada en vértebras y otolitos arrojó resultados similares, lo que sugiere una edad máxima de cinco años. Sin embargo, las pruebas de precisión y exactitud, como el porcentaje de concordancia (PA), el coeficiente medio de variación (ACV) y el porcentaje de error medio (APE), indicaron que las lecturas del otolito (97.83% PA, 0.54% ACV y 0.38 % APE) fueron más confiables para la estimación de la edad que las lecturas de vértebras (82.17% PA, 5.33% ACV y 3.77% APE). El enfoque de inferencia de modelos múltiples nos permitió comparar diferentes modelos de crecimiento no lineal. El modelo de von Bertalanffy (L∞=29.139, k=0.496 and t0=−0.347) se ajusta mejor a los datos de talla por edad. Esta especie tiene un crecimiento relativamente rápido y una longevidad estimada de cinco a siete años. Esta información podría utilizarse para la ordenación y los primeros estudios de evaluación de poblaciones de T. ovatus en el mar Mediterráneo

Estudios preliminares sobre compuestos antioxidantes en materiales clonales del programa de mejoramiento genético de sauce del INTA

En la Estación Experimental Agropecuaria (EEA) INTA Delta del Paraná se desarrolla un programa de mejoramiento clásico de sauces, en el marco del Proyecto Nacional PNFOR- 1104062 y del Programa de Domesticación y Mejoramiento de Especies Forestales Nativas e Introducidas para Usos de Alto Valor (PROMEF). El objetivo es generar materiales clonales con ventajas productivas y de adaptabilidad. En el corriente año, seis nuevos clones mejorados fueron inscriptos en el Registro Nacional de Cultivares del INASE: `Agronales INTA-CIEF´, `Los Arroyos INTA-CIEF´, `Géminis INTA-CIEF´, `Lezama INTA-CIEF´, `Ibicuy INTA-CIEF´ y `Yaguareté INTA-CIEF´. Desde hace unos años, docentes investigadores de la Facultad de Agronomía de la UBA colaboran a través de un trabajo interdisciplinario con el Programa, analizando el material clonal a fin de evaluar características químicas que contribuyan a explicar los mecanismos implicados en ciertos comportamientos frente a situaciones de estrés biótico y abiótico, a los cuales naturalmente se ven sometidos. Las investigaciones se realizan en el marco de los proyectos UBACyT (2012-2015) 20020110200266 y UBACyT (2011-2014) 20020100100229. El presente trabajo comprende un análisis preliminar de los compuestos antioxidantes presentes en individuos de Salix spp. Los derivados fenólicos son metabolitos secundarios de amplia distribución en las plantas superiores, y particularmente los sauces se caracterizan por la presencia de glicósidos fenólicos y salicilatos los cuales constituyen una verdadera barrera de defensa y resistencia a adversidades. En esta primera etapa exploratoria se trabajó con clones de las especies Salix nigra, S. alba, S. humboldtiana, S. viminalis y S. matsudana, de los cuales se tomaron tres repeticiones y se analizaron, mediante métodos espectrofotométricos, su capacidad antioxidante (CA) y el contenido de fenoles totales (FT) expresados como moles equivalentes de ácido ascórbico y moles equivalentes de ácido clorogénico /g de hojas frescas, respectivamente. Los resultados de este trabajo exploratorio muestran que los clones recientemente registrados poseen diferencias contrastantes en cuanto a contenidos de CA y FT. Los valores de CA, en orden decreciente, corresponden a: Lezama > Yaguareté, Geminis > Ibicuy, Agronales y Los Arroyos. Respecto al contenido de FT, se observa el mismo orden decreciente presentando Lezama un valor de 186 moles eq ác. clorogénico /g de hojas frescas. Mediante HPLC-C18 se compararon los perfiles cromatográficos entre clones de una misma especie y especies diferentes observándose importantes variaciones entre los materiales. Estas observaciones preliminares aportan conocimientos que podrían ayudar a comprender aspectos de la susceptibilidad de los clones a las adversidades y los mecanismos implicados.EEA Delta del ParanáFil: Pagano, Natalia. Universidad de Buenos Aires. Facultad de Agronomía. Cátedra de Química de Biomoléculas; ArgentinaFil: Cerrillo, Teresa. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Delta del Paraná; ArgentinaFil: Leicach, Silvia Rosa. Universidad de Buenos Aires. Facultad de Agronomía. Cátedra de Química de Biomoléculas; ArgentinaFil: Chludil, Hugo Daniel. Universidad de Buenos Aires. Facultad de Agronomía. Cátedra de Química de Biomoléculas; Argentin

Extracorporeal shock waves alone or combined with raloxifene promote bone formation and suppress resorption in ovariectomized rats

Osteoporosis is a metabolic skeletal disease characterized by an imbalance between osteoclast-mediated bone resorption and osteoblast-mediated bone formation. We examined the beneficial effect of shock waves (SW) alone or in combination with raloxifene (RAL) on bone loss in ovariectomized rats (OVX). Sixteen weeks after surgery, OVX were treated for five weeks with SW at the antero-lateral side of the right hind leg, one session weekly, at 3 Hz (EFD of 0.33 mJ/mm2), or with RAL (5 mg/kg/die, per os) or with SW+RAL. Sera, femurs, tibiae and vertebrae were sampled for following biochemical and histological analysis. SW, alone or combined with RAL, prevented femur weight reduction and the deterioration of trabecular microarchitecture both in femur and vertebrae. All treatments increased Speed of Sound (SoS) values, improving bone mineral density, altered by OVX. Serum parameters involved in bone remodeling (alkaline phosphatase, receptor activator of nuclear factor kappa-B ligand, osteoprotegerin) and osteoblast proliferation (PTH), altered by ovariectomy, were restored by SW and RAL alone or in combination. In tibiae, SW+RAL significantly reduced cathepsin k and TNF-α levels, indicating the inhibition of osteoclast activity, while all treatments significantly increased runt-related transcription factor 2 and bone morphogenetic-2 expression, suggesting an increase in osteoblastogenic activity. Finally, in bone marrow from tibiae, SW or RAL reduced PPARγ and adiponectin transcription, indicating a shift of mesenchymal cells toward osteoblastogenesis, without showing a synergistic effect. Our data indicate SW therapy, alone and in combination with raloxifene, as an innovative strategy to limit the hypoestrogenic bone loss, restoring the balance between bone formation and resorption

When opportunistic predators interact with swordfish harpoon fishing activities: shark depredation over catches in the Strait of Messina (central Mediterranean Sea)

We describe the interaction between harpoon fishing activity and sharks, which opportunistically depredated harpoon catches in the Strait of Messina. Shark bite marks were observed on harpooned swordfish during the period 2014–2020, with different damages to the catches. Most of the depredation events have focused on large swordfish, generally weighing more than 60 kg. Data on direct observations were implemented by interviews and questionnaires to fishermen aimed to recover the information on their local fishing and ecological knowledge. Fishermen provided additional data on shark-harpoon fishing interactions also supplying information on by-catch species (i.e., bluefin tuna). Therefore, these results suggest that sharks migrating through the Strait of Messina are occasionally attracted by injured prey, due to their ability to detect chemical cues, fish distress stimuli and body fluids (i.e. blood) in the water. In addition, our investigations showed an increase in shark attacks on harpooned fish over time, likely due to an increase in harpoon swordfish catches. This may be related to the effects of the driftnets' ban enforced by European Regulations in the last decades

Self-determination in people with intellectual disability: The mediating role of opportunities

Abstract: The Convention on the Rights of Persons with Disabilities have proclaimed the basic right of people to make one?s own choices, have an e ective participation and inclusion. Research in the field of disability have stressed self-determination as a key construct because of its impact on their quality of life and the achievement of desired educational and adulthood related outcomes. Self-determination development must be promoted through specific strategies and especially, by providing tailored opportunities to practice those skills. Providing these opportunities across environments could be especially relevant as a facilitator of self-determination development. This manuscript aims to ascertain if opportunities at home and in the community to engage in self-determined actions are mediating the relationship between people intellectual disability level and their self-determination. Results have confirmed direct e ects of intellectual disability level on self-determination scores. Indirect e ects also predicted self-determination and almost all its related components (self-initiation, self-direction, self-regulation, self-realization, and empowerment) through opportunities in the community and at home. Autonomy was predicted by the intellectual disability level through an indirect e ect of opportunities at home, but not in the community. These results highlight the need for further research to better operationalize and promote contextually rooted opportunities for people with intellectual disability to become more self-determined.This research was funded by the Spanish Ministry of Economy and Competitiveness, grant number PSI2016-75826-P. AEI/FEDER, UE