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EDICION ESPAÑOLA “Anatomía humana – Atlas interactivo multimedia” está hecho de un apoyo en papel y de una plataforma web (Virtual Campus) combinando una estrategia de aprendizaje innovadora para la anatomía, basada en una representación precisa de las estructuras anatómicas y espacios virtuales en el ser humano vivo. El Atlas en su versión de papel sigue un enfoque regional y topográfico. Cada capítulo está ilustrado con la ayuda de: • Dibujos anatómicos hechos magistralmente con un alto impacto educativo • Secuencias estratográficas para reconstruir la organización de las regiones anatómicas • Imágenes 2D y 3D (volumen renderizato) obtenidas de imágenes TC o RM correspondientes • Fotografías de exámenes laparoscópicos y endoscópicos • Leyendas claras, tablas y cuadros sinópticos para facilitar el aprendizaje • Iconos de llamada a los contenidos de la plataforma web. La plataforma web “Virtual Campus”, tan innovadora para el mundo del Atlas de Anatomía, muestra una colección rica de contenido de materiales didácticos: • Un curso completo de anatomía topográfica ofrece los recorridos guiados a través de recursos específicos • Reconstrucciones de órganos y regiones anatómicas 3D interactivas obtenidas de imágenes TC o MR correspondientes • Videos comentados de laparoscopias y endoscopias • Filmes comentados de disecciones • Animaciones estratográficas interactivas Plan de la obra Volumen 2 Cabeza Cuello Tórax

ENGLISH EDITION “Human Anatomy - Multimedial Interactive Atlas” is made of a paper Atlas and a web platform (Virtual Campus), combining an innovative learning strategy for anatomy, based on a precise representation of the anatomical structures and virtual spaces in the living human. The Paper Atlas follows a regional and a topographic approach. Each illustrated chapter provides: • Anatomical drawings masterfully made with a high educational impact • Stratigraphic sequences to reconstruct the organization of anatomical regions • Realistic 2D and 3D renderings from CT or MR imaging exams • Photographs of laparoscopic and endoscopic exams • Clear legends, summary tables and schemes to facilitate learning • Recall icons to web platform contents. The web platform Virtual Campus, so innovative in the Anatomy Atlas world, displays a rich collection of didactic material content: • a complete course of Topographic Anatomy offers guided tracks through specific resources; • 3D interactive reconstructions of organs or anatomical regions from CT or MRI exams; • commented laparoscopy and endoscopy videos; • commented dissection movies; • interactive stratigraphic animations.

“Human Anatomy - Multimedial Interactive Atlas” is made of a paper Atlas and a web platform, combining an innovative learning strategy for anatomy, based on a precise representation of the anatomical structures and virtual spaces in the living human. The Paper Atlas follows a regional and a topographic approach. Each illustrated chapter provides: Anatomical drawings masterfully made with a high educational impact; Stratigraphic sequences to reconstruct the organization of anatomical regions; Realistic 2D and 3D renderings from CT or MR imaging exams; Photographs of laparoscopic and endoscopic exams; Clear legends, summary tables and schemes to facilitate learning; Recall icons to web platform contents. The web platform, so innovative in the Anatomy Atlas world, displays a rich collection of didactic material content: a complete course of Topographic Anatomy offers guided tracks through specific resources; 3D interactive reconstructions of organs or anatomical regions from CT or MRI exams; commented laparoscopy and endoscopy videos; commented dissection movies; interactive stratigraphic animations. Volume 3: Abdomen; Pelvis; Perineum; Upper and Lower limbs; Nervous system

Long-term exposure to cigarette smoke induces severe injuries to respiratory system through several mechanisms, some of them are well defined, but many others are not yet elucidated. Beside its classical role in nervous system, we have previously shown that Nerve Growth Factor (NGF) and its receptors have a crucial role in airway inflammatory diseases, such as Chronic Obstructive Pulmonary Disease. To expand our knowledge about the relevance of NGF and its receptors in airway diseases induced by cigarette smoking, we exposed for 16 weeks the bronchial epithelial cell line BEAS-2B to sub-toxic concentrations of whole cigarette smoke extract or pure nicotine. Viability, cell cycle gene expression, cell morphology and migration ability were tested and compared to NGF release and gene expression. Modulation of its receptors TrKA and p75NTR was also analyzed. The present study shows that long term exposure of BEAS-2B cells to cigarette smoke extract or nicotine induces: (A) differences: in cell viability, in the expression of cell cycle-related genes, in NGF release and in gene expression of NGF and its receptors; (B) similarities: in morphology and migration ability. Taken together, our data provide new insights about the biological role of NGF and its receptors in airway diseases induced by long-term cigarette smoking and, finally, our data evidence the opportunity not to use nicotine lozenges or e-cigarettes as anti smoking replacement therapy in patients with a previous airway disease according to the ability of nicotine to increase the amount of the pro-inflammatory cytokine NGF into the bronchial environment.

EDICION ESPAÑOLA “Anatomía humana – Atlas interactivo multimedia” está hecho de un apoyo en papel y de una plataforma web (Virtual Campus) combinando una estrategia de aprendizaje innovadora para la anatomía, basada en una representación precisa de las estructuras anatómicas y espacios virtuales en el ser humano vivo. El Atlas en su versión de papel sigue un enfoque regional y topográfico. Cada capítulo está ilustrado con la ayuda de: • Dibujos anatómicos hechos magistralmente con un alto impacto educativo • Secuencias estratográficas para reconstruir la organización de las regiones anatómicas • Imágenes 2D y 3D (volumen renderizato) obtenidas de imágenes TC o RM correspondientes • Fotografías de exámenes laparoscópicos y endoscópicos • Leyendas claras, tablas y cuadros sinópticos para facilitar el aprendizaje • Iconos de llamada a los contenidos de la plataforma web. La plataforma web “Virtual Campus”, tan innovadora para el mundo del Atlas de Anatomía, muestra una colección rica de contenido de materiales didácticos: • Un curso completo de anatomía topográfica ofrece los recorridos guiados a través de recursos específicos • Reconstrucciones de órganos y regiones anatómicas 3D interactivas obtenidas de imágenes TC o MR correspondientes • Videos comentados de laparoscopias y endoscopias • Filmes comentados de disecciones • Animaciones estratográficas interactivas Plan de la obra Volumen 3 Abdomen Pelvis Periné Miembro superior Miembro inferior Sistema nervioso

Melanoma is a severe form of cancer, resistant to conventional therapies. According to in vitro studies, sulforaphane, a dietary component, has been considered a promising antineoplastic candidate. The present study analyzes the in vitro biological effects of sulforaphane in A375 melanoma cell line with or without the addition of Nerve Growth Factor. For the first time, our results show that a supplementation of Nerve Growth Factor partially reverses the sulforaphane-induced: i) inhibition of cell migration, ii) pro apoptotic changes in cell cycle and iii) modulation of active caspase-3. Furthermore, we report the sulforaphane-induced modulation in the expression of Nerve Growth Factor receptors TrKA and p75NTR, shifting their ratio from pro survival to pro apoptotic. In conclusion, the present study evidences that in vivo the antineoplastic effects of sulforaphane may be reduced by the contemporaneous presence of other biological elements such as Nerve Growth Factor and it contributes to a better definition of the real in vivo potentiality of sulforaphane as antineoplastic candidate.

Purpose Human melanoma is a highly aggressive incurable cancer due to intrinsic cellular resistance to apoptosis, reprogramming, proliferation and survival during tumour progression. Sulforaphane (SFN), an isothiocyanate found in cruciferous vegetables, plays a role in carcinogenesis in many cancer types. However, the cytotoxic molecular mechanisms and gene expression profiles promoted by SFN in human melanoma remain unknown. Methods Three different cell lines were used: two human melanoma A375 and 501MEL and human epidermal melanocytes (HEMa). Cell viability and proliferation, cell cycle analysis, cell migration and invasion and protein expression and phosphorylation status of Akt and p53 upon SFN treatment were determined. RNA-seq of A375 was performed at different time points after SFN treatment. Results We demonstrated that SFN strongly decreased cell viability and proliferation, induced G2/M cell cycle arrest, promoted apoptosis through the activation of caspases 3, 8, 9 and hampered migration and invasion abilities in the melanoma cell lines. Remarkably, HEMa cells were not affected by SFN treatment. Transcriptomic analysis revealed regulation of genes involved in response to stress, apoptosis/cell death and metabolic processes. SFN upregulated the expression of pro-apoptotic genes, such as p53, BAX, PUMA, FAS and MDM2; promoted cell cycle inhibition and growth arrest by upregulating EGR1, GADD45B, ATF3 and CDKN1A; and simultaneously acted as a potent inhibitor of genotoxicity by launching the stress-inducible protein network (HMOX1, HSPA1A, HSPA6, SOD1). Conclusion Overall, the data show that SFN cytotoxicity in melanoma derives from complex and concurrent mechanisms during carcinogenesis, which makes it a promising cancer prevention agent. Electronic supplementary material The online version of this article (doi:10.1007/s00394-017-1527-7) contains supplementary material, which is available to authorized users.

Nerve growth factor (NGF) receptors, TrKA and p75(NTR), are being investigated in cancer therapy. Our previous data show that, in HTB114 uterine leiomyosarcoma cells, p75(NTR)-dependent apoptosis is inducible by cytotoxic drugs and can suppress nerve growth factor-dependent growth. Although amitriptyline can kill cancer cells and bind TrKA/B, its effects on p75-dependent apoptosis are unknown. The aim of this paper was to evaluate the antineoplastic potential of amitriptyline, and the role of p75(NTR)-dependent apoptosis in the chemoresistant uterine HTB114 leiomyosarcoma. Using proliferation assays and fluorescence-activated cell sorting analysis, we found that amitriptyline caused a marked reduction in HTB114 cell viability, associated with the parallel upregulation of p75(NTR) expression. This converted the TrKA⁺-proliferating cells into TrKA⁺/p75(NTR⁺), leading to downregulation of TrKA-prosurvival signaling (AKT) and activation of p75(NTR)-dependent apoptosis (through caspase-3). Overall, we provide novel evidence that HTB114 uterine leiomyosarcoma cells are highly sensitive to amitriptyline, supporting the role of p75(NTR)-dependent apoptosis as a novel cytotoxic mechanism of this drug and of p75(NTR) as an inducible stress receptor and a novel target in clinical oncology.

Il Testo Atlante di Anatomia – Prometheus rappresenta un atlante anatomico di moderna impostazione e di raffinata realizzazione; non solo strumento utile per gli studenti, in grado di fornire nozioni anatomiche precise, dettagliate e selezionate nell’ottica della professione medica e quindi estremamente valido durante l’apprendimento dell’anatomia, ma anche preziosa opera di aggiornamento, verifica e consultazione nella professione quotidiana per i Medici, per gli Specialisti e, in generale, per tutti i Professionisti Sanitari. I contenuti trattati sono stati scelti in base a criteri di "completezza", ma anche in base alla valutazione dell'importanza di un determinato argomento per la comprensione di base dell'Anatomia o per creare collegamenti logici con l'attività clinica del medico. Le migliaia di illustrazioni, create appositamente per quest'opera, forniscono una visione incredibilmente chiara delle strutture del corpo umano e sono corredate da un ampio testo che serve a renderle più comprensibili mediante spiegazioni, indicazioni didattiche, riferimenti anatomo- funzionali ed anatomo-clinici. Questa nuova edizione include anche ossa, legamenti, articolazioni e organi del collo con i relativi vasi e nervi; alcuni argomenti di Neuroanatomia sono stati completamente rielaborati per correlarli meglio alla conoscenza delle funzioni connesse alla Neurofisiologia, agli approfondimenti clinici e ad esami diagnostici. Gli argomenti che sono stati profondamente rivisti includono, per esempio, la formazione reticolare, la terminologia di lemnischi e nuclei olivari e la classificazione dei gangli autonomi e sensitivi dei nervi cranici. Il Capitolo “Testa e Collo” è stato ampliato introducendo lo sviluppo dei denti, la radiologia diagnostica e l’anestesia; inoltre nuove immagini ricostruiscono rigorosamente la struttura di denti e gengive rendendo l’intero capitolo adatto anche agli studenti di Odontoiatria

Osteoarthritis (OA) of the knee is the most common form of non-traumatic joint disease. Previous studies have shown the involvement of β-NGF and its receptors TrKA and p75NTR in OA-related pain, but their role in its pathogenesis is still unclear. The aim of our study was to investigate the amount of β-NGF and the expression levels of its receptors on cells isolated from synovial fluid and blood from OA patients who had undergone total knee arthroplasty, in order to check any possible correlation with the disease staging. Our results show a progressive stage-related increase of β-NGF and its receptors both in serum and synovial fluid. Furthermore, with respect to control subjects, OA patients show an increased amount of inflammatory monocytes along with an increased expression of β-NGF, TrKA and p75NTR. In conclusion, our study suggests a stage-related modulation of β-NGF and its receptors in the inflammatory process of OA.

Background Biomechanical and histological properties of stapled bronchi with and without bovine pericardial matrix plus collagen reinforcement are analyzed. Materials and methods Pneumonectomy with mechanical bronchial suture was performed in the swine model. Pigs were randomly assigned to three groups: three-row staplers without reinforcement (Traditional), with reinforcement (Buttressed) and control “wild type”, non-resected normal bronchus (Normal). Intraoperative test was carried for air leaks at 20/30/40 mm Hg endobronchial pressure. After 60 d, tracheobronchial specimen was harvested, stocked, and analyzed. Tensile test was performed using INSTRON 5965 loading frame machine. Maximal strain resistance value, length of elongation at rupture parameter, and stiffness coefficient (K) were evaluated. Histological analysis was performed. Sample size calculation was assessed (four per group), and the Student t-test was used to statistically evaluate differences in biomechanical variables. Results No fistula occurred. Biomechanical analysis showed that maximal strain resistance is 41.22 ± 2.11 N (Traditional), 24.53 ± 3.47 N (Buttressed), and 30.91 ± 0.29 N (Normal); elongation at rupture is 16.01 ± 1.82 mm (Traditional), 12.89 ± 0.48 mm (Buttressed), and 9.32 ± 0.11 mm (Normal). Finally, K is 2.59 ± 0.16 N/mm (Traditional), 1.91 ± 0.33 N/mm (Buttressed), and 3.32 ± 0.01 N/mm (Normal). Stumps without reinforcement proved higher resistance and length of elongation than reinforced ones. Normal bronchial tissue shows the highest stiffness coefficient. Statistical analysis produced significant values for each biomechanical feature. Group Buttressed stumps show greater thickness and a substantial inflammatory reaction with granulation tissue along the whole scar and around areas of discontinuity within the scar, not yet healed. Conclusions Reinforcing the mechanical suture line of bronchial stump with bovine pericardial matrix plus collagen shows suboptimal biomechanical and histological characteristics compared to using the stapler alone.

BACKGROUND The localization of low affinity nerve growth factor receptor (p75) in prostate carcinogenesis is still unclear. Our aim was to reinvestigate the localization of p75 in normal and pathological prostate and to check a possible correlation to neoplastic grading. METHODS Specimens from 33 prostate cancers and from normal prostatic tissue were analyzed for p75 expression at light and ultrastructural levels. RESULTS In normal tissue p75-immunoreactivity was restricted to basal cells in the epithelial compartment and to nerves and blood vessel in stroma. During carcinogenesis, p75-immunoreactivity progressively decreased at the periphery of the foci according to the increase in malignancy. No p75-immunoreactivity was detected inside of the foci. On the contrary, in stroma we found a dramatic increase in p75-immunoreactivity correlated to an increase in malignancy. In this compartment, for the first time ultrastructural analysis identified p75-immunoreactivity in smooth muscle cells (SMC) that are p75-negative in normal conditions. CONCLUSION The present study confirms at ultrastructural level a malignant-dependent p75 decrease in basal cells of neoplastic foci. Furthermore, we show a novel, malignant-dependent localization of p75 in SMC in the stroma around the neoplastic foci. Since p75 expression is present in muscle cells only during the earliest stages of differentiation and mature muscle cells lose this expression, we hypothesize that p75 re-expression in stromal SMC is a further mechanism related to the general de-differentiation of the stroma connected to the neoplastic invasion. According to this hypothesis, our results suggest that p75 analysis could be a novel prognostic marker for prostate cancer. Prostate 70: 555–561, 2010. © 2009 Wiley-Liss, Inc.

Anatomia Umana - Atlante si avvale di un supporto cartaceo e di una piattaforma web, combinati in modo innovativo per l’apprendimento dell’anatomia. L’Atlante cartaceo segue un’organizzazione regionale e topografica. Ciascun capitolo è illustrato con l’ausilio di: disegni anatomici magistralmente realizzati e di sicuro effetto didattico; sequenze stratigrafiche per ricostruire l’organizzazione delle regioni anatomiche; realistiche immagini di renderizzazione 2D o 3D a partire da esami TC o RM; fotografie di esami laparoscopici o endoscopici; leggende chiare, tavole riassuntive e schemi che favoriscono l’apprendimento; richiami didascalici alle risorse presenti sulla piattaforma web. La piattaforma web, innovativa nel mondo degli atlanti di anatomia, raccoglie un ricco catalogo di materiale didattico: corso di Anatomia Topografica completo, organizzato in percorsi guidati che conducono lo studente attraverso specifiche risorse della piattaforma web; ricostruzioni 3D interattive di organi o regioni anatomiche, a partire da esami TC o RM; video commentati di laparoscopie ed endoscopie; video commentati di dissezioni anatomiche su cadavere; animazioni stratigrafiche interattive.

Experiments were devised to characterize the expression of nerve growth factor, beta polypeptide (NGF), and its cognate receptors neurotrophic tyrosine kinase receptor type 1 (NTRK1) and nerve growth factor receptor (NGFR) in rabbit male sex organs, as well as the concentrations of NGF in both seminal and blood plasma of sexually mature male rabbits. Immunoreactivity and gene expression for NGF and cognate receptors were detected in testis, prostate gland and seminal vesicle. The highest levels of NGF and NTRK1 transcripts were found in the prostate, while intermediate expressions were found in the testis. NGFR transcripts were expressed at the same levels in both testis and prostate and were more abundant than in seminal vesicles. The widespread distribution of NGF in all prostate glandular cells, together with its relative high mRNA abundance, confirms that the prostate of rabbits is the main source of this neurotrophin. In conclusion, the present data suggest that the NGF system is involved in the testicular development and spermatogenesis of rabbits and that NGF may act as a potential ovulation-inducing factor being abundantly present in the seminal plasma.

Uterine leiomyosarcoma is a severe neoplasia resistant to conventional therapies. In previous studies, we have shown that human SK-UT-1 (ATCC HTB114) uterine leiomyosarcoma cell line secretes nerve growth factor (NGF) and expresses its receptors tyrosine kinase A receptor (TrKA) and low affinity nerve growth factor receptor (p75NTR). Furthermore, we have demonstrated that direct chemical inhibition or IgG neutralization of TrKA receptor induce apoptosis through p75NTR. In the present study, HTB114 cells were exposed to the phosphoinositide 3-kinase (PI3K) inhibitor LY294002 with and without β-NGF: apoptosis, cell cycle, activation of caspase-3 and protein kinase B (AKT) and TrKA/p75NTR phenotypic expression were evaluated. According to the type of exposure, LY294002 not only induced a relevant increase in apoptosis, but also produced a novel and unexpected phenotypic modulation of the NGF receptors with a downregulation of TrKA and an upregulation of p75NTR. This latter increase enhanced HTB114 apoptosis. Our study confirms that the interference on NGF transduction is a promising therapeutical approach in uterine leiomyosarcoma.

The effects of sciatic nerve lesions on the expression of serotonin 5-HT3 receptor (5-HT3R) alpha subunit in motoneurons of the spinal cord was investigated by semi-quantitative immunohistochemistry. Following sciatic nerve crush, a significant reduction in density of staining in motoneurons was observed in longitudinal sections of the ventral horn at 3 and 15 days on the lesioned side when compared to the contralateral side (p

Neurotrophins are expressed in muscle cells both during development and postnatally. Furthermore, during development muscle cells express high levels of the common p75 neurotrophin receptor, which binds all neurotrophins. Only fragmentary and controversial data are available regarding the responsiveness of muscle cells to neurotrophins and the importance of low-affinity p75 receptor in muscle development. The present study investigates in vitro the immunocytochemical expression of p75 in a rat myogenic cell line (L6) at various time points and in response to different coating substrates as a first step in elucidating the regulation of p75 in muscle. We found that in L6 myoblasts, p75 is expressed only at very early stages of maturation and its levels of expression are regulated by the nature of the coating substrates. p75 expression decreases in cells growing on substrates more suitable for myoblast fusion into myotubes. Time course analysis indicates a reverse correlation between myoblast fusion into myotubes and the levels of p75 expression. Myotubes were always p75 negative. Substrates not suitable for the fusion process induced a prolonged presence of p75 in myoblasts with an increase of their apoptosis. We conclude that expression of p75, at least in this in vitro condition, is regulated by the stages of myoblast differentiation and the nature of the coating substrates. According to the observed time- and substrate-related evidences, future studies should investigate in vivo both the regulation of p75 in the myoblast fusion and the effects and the importance of neurotrophins binding during myoblast differentiation.

Blood stage malaria parasites causing a mild and self limited infection in mice have been obtained with either radiation or chemical mutagenesis showing the possibility of developing an attenuated malaria vaccine. Targeted disruption of plasmepsin-4 (pm4) or the merozoite surface protein-7 (msp7) genes also induces a virulence-attenuated phenotype in terms of absence of experimental cerebral malaria (ECM), delayed increase of parasitemia and reduced mortality rate. The decrease in virulence in parasites lacking either pm4 or msp7 is however incomplete and dependent on the parasite and mouse strain combination. The sequential disruption of both genes induced remarkable virulence-attenuated blood-stage parasites characterized by a self-resolving infection with low levels of parasitemia and no ECM. Furthermore, convalescent mice were protected against the challenge with P. berghei or P. yoelii parasites for several months. These observations provide a proof-of-concept step for the development of human malaria vaccines based on genetically attenuated blood-stage parasites.

Spinal and brainstem motoneurons of the adult rat reexpress low-affinity nerve growth factor receptor (LNGFR) and its mRNA after axotomy. We have previously reported the time courses of this reexpression after cut (no regeneration) or crush (followed by regeneration) of the sciatic nerve. We have shown that the length of the different phases of this reexpression (appearance, maintenance and disappearance) can vary according to the type of axotomy. With the present study we expand our previous data and describe and analyze the modulation the LNGFR expression in adult spinal cord motoneurons following different lesion paradigms. In one approach we have imposed three traumatic injuries that still allow regeneration of the sciatic nerve but with a different time course with respect to the crush injury (application of a silicone regeneration chamber, multiple crushes and delayed repair of ligated nerves). In a second approach, we have determined the capability of three toxic or metabolic injuries to induce LNGFR expression without any direct trauma of the nerve (experimental diabetogenesis, botulinum and alpha-bungarotoxin intoxication and 2,5-hexanedione intoxication). In a third approach, we have investigated the effect of the block of the axoplasmic transport on the LNGFR expression following different topical applications of vincristine combined with a nerve crush. The results we present are consistent with the idea that: (1) LNGFR immunoreactivity in adult motoneurons is expressed by motoneurons that are attending to an axonal outgrowth and not a generic signal of cellular damage or impairment of the motor function; (2) LNGFR expression in these motoneurons is related to and parallels the outgrowth process time frame, and (3) the signal/s that trigger and sustain this reexpression may be retrogradely transported from the periphery. © 1993 Wiley-Liss, Inc.

The axon regeneration following a peripheral nerve injury often fails to restore a complete functional recovery. One of the causes of this unsatisfactory result has been attributed to regrowth of regenerating fibers to inappropriate peripheral targets. The accuracy of reinnervation by axons regenerating across a 10-mm gap within an impermeable chamber has been studied by using a sequential retrograde double-labeling technique. Despite the long gap between the nerve stumps, at 4 weeks a mean of 30.5% of the regenerating axons can reinnervate the original muscular area. These data confirm previous studies in which a preferential reinnervation is reported not to be absolutely dependent on the axon's mechanical alignment.

Neurotrophins, originally identified as neuronal survival and differentiation factors, exert their actions through tyrosine kinase receptors such as TrKA, in the case of the nerve growth factor. Neurotrophins also interact with p75, a common receptor devoid of kinase activity and connected to apoptosis. Here we show that nerve growth factor, TrKA and p75 are expressed in cell lines of human cancers of various non-neuronal lineages, including a panel of muscular sarcomas, and we show that all cell lines investigated actively release nerve growth factor into the medium. Treatment by AG879 (a tyrosine kinase inhibitor that inhibits TrKA phosphorylation, but not TrKB and TrKC) or by neutralizing antibodies anti-nerve growth factor and anti-TrKA dramatically decreases their proliferation with a variable increase in apoptosis. Similarly, p75 transfection induced a significant increase in apoptosis. Furthermore, for the first time we have determined by high-performance liquid chromatography the pharmacokinetic profile of a novel preparation of AG879 and we have established an optimal plasmatic concentration for in-vivo administration. Treatment with AG879 in immunodepressed mice grafted with leiomyosarcoma or promyelocytic leukemia cells resulted in dramatic reductions in tumor sizes. In conclusion, our data have a novel preclinical potential for revealing a possible therapeutical utility in targeting in-vivo nerve growth factor/TrKA by AG879 or neutralizing antibody anti-TrKA in cancer proliferation and in muscle sarcomas, in particular.

It is known that gamma-tocopherol inhibits human prostate cancer cell proliferation via down-regulation of cyclin-related signalling but tocopherol and tocotrienol metabolites with a shortened phytyl chain, carboxyethyl hydroxychromans, were not previously investigated as anti-proliferative agents. In this study, the effect of the two main tocopherols, namely, alpha-tocopherol and gamma-tocopherol, and their corresponding metabolites (alpha- and gamma-carboxyethyl hydroxychromans) was studied on proliferation and cyclin D1 expression of the prostate cancer cell line PC-3. The hydrosoluble vitamin E analogues Trolox and alpha-tocopherol succinate were also tested. The most effective inhibitors of PC-3 proliferation were gamma-tocopherol and gamma-carboxyethyl hydroxychroman. Their effect was discernable at 1 microM and reached a plateau at concentrations > or = 10 microM with maximal inhibition values ranging between 70 and 82%. alpha-Tocopherol, alpha-carboxyethyl hydroxychroman, and the analogue Trolox were much less effective; a weak effect was observed for concentrations < or = 10 microM and a maximal inhibition of less than 45% was found at 50 microM concentration. PC-3 cells showed higher inhibition, particularly by the gamma derivatives, than HTB-82 and HECV cells. Tocopherols and carboxyethyl hydroxychromans exerted an inhibitory effect on cyclin D1 expression parallel to the retardation of cell growth. gamma-Carboxyethyl hydroxychroman and gamma-tocopherol showed effects also upstream of the cyclin modulation. Furthermore, the inhibition of cyclin D1 expression by gamma-carboxyethyl hydroxychroman was competed for by alpha-carboxyethyl hydroxychroman. In conclusion, this study shows that carboxyethyl hydroxychroman metabolites are as effective as their vitamin precursors to inhibit PC-3 growth by specific down-regulation of cyclin expression, with the gamma forms being the most effective ones. Although the inhibition of PC-3 cell growth and diminution of cyclin expression are clearly visible, more subtle mechanistic effects of tocopherols and their corresponding carboxyethyl hydroxychroman metabolites deserve further investigations.

Prosaposin is the precursor of the saposins and has both neurotrophic and myelinotrophic activity in vitro and in vivo. Using an antibody specific for the holoprotein, an immunocytochemical survey demonstrated intense staining of adult rat skeletal, cardiac, and smooth muscle cells. Prosaposin immunoreactivity in muscle appears dependent on innervation, as denervated adult rat skeletal muscles showed decreased immunostaining that returned to normal levels after reinnervation. TX14(A), a peptide derived from the neurotrophic sequence of prosaposin, attenuated the decline in muscle mass loss following nerve injury induced by a constricting ligature. In vitro, both L6 myoblasts and primary chick-embryo myoblasts showed similar prosaposin immunopositivity, mainly in myotubes. TX14(A) induced a threefold increase in L6 myoblast fusion during early stages of differentiation without affecting cell proliferation. The fusion process was decreased in vitro in a dose-dependent fashion by addition of a neutralizing anti-prosaposin antibody. These data suggest that, in addition to neurotrophic and myelinotrophic activities, prosaposin has myotrophic properties.

Classic studies have established that muscle cells exert trophic actions on neurons of the developing peripheral nervous system through the production of neurotrophins. For this reason neurotrophins are also known as ‘target-derived factors’. During differentiation, muscle cells also express some neurotrophin receptors, such as the low-affinity p75 neurotrophin receptor, which binds all neurotrophins, and the high affinity tyrosine kinase receptor TrKA, nerve growth factor (NGF) transducing receptor. The functional roles of these receptors in muscle cells are still unclear and only fragmentary and controversial data are available regarding the responsiveness of muscle cells to NGF. The aim of the present study is to investigate the effects of NGF on cells of myogenic lineage. The rat myogenic cell line L6, primary cultures of adult human myoblasts, and the human rhabdomyosarcoma cell line TE-671 were used in this study. As expected, all the three cell types expressed NGF, p75 and TrKA. NGF was expressed by L6 and primary myoblasts following differentiation, but it was constitutively expressed at high levels in the TE-671 rhabdomyosarcoma cells. In L6 myoblasts, p75 receptor was expressed in myoblasts but not in myotubes early after plating; while some primary human myoblasts expressed it at all the time-points tested. Some fusiform cells of the TE-671 rhabdomyosarcoma cell line also expressed p75. TrKA was constitutively immunodetected in all the three cell lines, suggesting that these cells may respond to NGF. Addition of exogenous NGF increased the fusion rate of both primary and L6 myoblasts; as well as the proliferation of the slowly dividing primary myoblasts. Consistently, blocking the action of endogenously produced NGF with a specific neutralizing antibody decreased the percentage of fusion in both primary and L6 myoblasts. On the contrary, blocking the binding of NGF to p75 did not affect the percentage of fusion. Furthermore, neither exogenous NGF nor NGF- or p75-neutralizing antibodies appeared to affect the rhabdomyosarcoma cells, which have a high proliferation rate and do not fuse. Pharmacological inhibition of TrKA signal transduction with K252a (in the nM range) and tyrphostin AG879 (in the low μM range) resulted in a dramatic dose-dependent decrease in proliferation of all of the myogenic cell lines tested. Interestingly, this was especially evident in the rapidly dividing rhabdomyosarcoma cell line. The TrKA inhibitors also blocked fusion of L6 and primary myoblasts and induced morphological changes characterized by the flattening of the cells and a ‘spider-like’ rearrangement of the intermediate filaments in all three cell lines with some minor differences. A transfection study showed that p75-overexpressing L6 cells do not fuse and present changes in their morphology similar to the TrKA-inhibitors treated L6 cells. These data support the notion that NGF expression in skeletal muscle is not only associated with a classical target-derived neurotrophic function for peripheral nervous system neurons, but also with an autocrine action which affects the proliferation, fusion into myotubes, and cell morphology of developing myoblasts. The present data also suggest that these effects of NGF are mediated by TrKA receptors and that a sustained presence of NGF is needed for increase fusion into myotubes. Lastly, the dramatic anti-proliferative effect of TrKA inhibitors on myogenic cells, and especially on the TE-671 rhabdomyosarcoma cell line, suggests that pharmacological interference with NGF signal transduction could be effective in the control of these malignancies.

We previously reported that a permanent transection of adult rat sciatic and hypoglossal nerves resulted in distinct changes in the levels of both low-affinity nerve growth factor receptor (p75) and choline acetyltransferase in the corresponding motoneurons as determined by immunoreactivity. Permanent axotomy of hypoglossal motoneurons induced a progressive loss of choline acetyltransferase immunoreactivity and a persistent expression of p75 immunoreactivity, phenomena that were not observed in spinal motoneurons. These observations indicated that spinal and brainstem motoneurons respond to permanent axotomy with a differential immunoreactivity for p75 and choline acetyltransferase. Such differences could be ascribed to specific intrinsic properties of each population of motoneurons or, alternatively, to different factors present in the periphery (nerve stump or target muscle). The aim of the present study was to test these two possibilities by determining if a segment of sciatic nerve transplanted to a transected hypoglossal nerve may counteract or attenuate the loss of choline acetyltransferase immunoreactivity in injured hypoglossal motoneurons. In addition, as further parameter, we analysed the presence of p75 immunoreactivity. Prior to grafting, segments of sciatic nerve were prepared by one of three methods: (i) a fresh piece; (ii) a degenerated piece; and (iii) a heated piece. Seven and 30 days following the placement of grafts, hypoglossal motoneurons were analysed for choline acetyltransferase and p75 immunolabelling. The results revealed that viable sciatic grafts (fresh and degenerated) are able to partially attenuate the loss in the number of choline acetyltransferase-positive injured hypoglossal motoneurons, even if an important decrease in choline acetyltransferase still persists with respect to the contralateral nucleus. In addition, viable sciatic grafts decreased the number of p75 immunoreactive hypoglossal motoneurons both at seven and at 30 days. In conclusion, the effects of viable sciatic grafts on the number of choline acetyltransferase and p75-labelled hypoglossal motoneurons indicate that these adult neurons are able to respond to factors released from the sciatic nerve, and that the number of injured motoneurons positive for choline acetyltransferase and p75 can be influenced by the presence of factors that may reach their proximal stumps. Furthermore, we hypothesize that the differential expression patterns between hypoglossal and sciatic motoneurons may be due, at least in part, to factors released from the nerve trunks themselves.

Adult rat spinal and brainstem motoneurons reexpress low-affinity nerve growth factor receptor (p75) after their axotomy. We have previously reported and quantified the time course of this reexpression in spinal motoneurons following several types of injuries of the sciatic nerve. Other studies reported the reexpression of p75 in axotomized brainstem motoneurons. Results of these previous studies differed regarding the type of the most effective triggering injury for p75 reexpression, the relative duration of this reexpression and the decrease of choline acetyltransferase (ChAT) immunoreactivity (-IR) following a permanent axotomy of spinal or brainatem motoneurons. These differences suggest that these two populations of motoneurons respond to axotomy with a different modulation of p75 and ChAT expression. The aim of the present study was to determine whether differential modulation exists. We have analyzed and quantified the presence of p75- and ChAT-IR motoneurone in the hypoglossal nucleus following the same types of injury and the same time course we previously used for sciatic motoneurons. The results show that a nerve crush is the most effective triggering injury for p75 and that it induces similar temporal patterns of p75 and ChAT expression for sciatic and hypoglossal motoneurons. In contrast, a cut injury of the sciatic and hypoglossal nerves resulted in distinct temporal courses of both p75 and ChAT expression between these two populations of motoneurons. In fact, a permanent axotomy of the hypoglossal motoneurons induced (i) a much longer maintenance phase for p75 than in aciatic motoneurons and (ii) a progressive loss of ChAT-IR with a successive return to normal values in contrast to the modest decrease in the sciatic motoneurons. This evidence indicates that spinal and brainstem motoneurons respond to a permanent axotomy with a different modulation of p75 and ChAT expression. Altogether, the present data and the reported evidence of a differential post- axotomy cell death support the hypothesis that these two populations of motoneurons undergo different dynamic changes after axotomy. © 1995 Wiley-Liss, Inc.

Developing spinal motor neurons (SMN) express low-affinity nerve growth factor receptors (LNGFR) but not high-affinity transducing NGF receptors. Moreover, SMN are not supported by NGF in vitro. In the normal adult rat most SMN are not LNGFR immunoreactive (LNGFR-IR), but they transiently reexpress LNGFR (though not the high-affinity receptor) after peripheral nerve injury. With a cut lesion of the sciatic nerve (when only a neuroma forms), the number of LNGFR-IR SMN at L4-L6 rapidly increases to a maximum between day 1 and 7 and returns to baseline levels by day 30. After a crush lesion (accompanied by regeneration to the muscle), LNGFR-IR SMN appear in about the same numbers, but they start to disappear 1 week later. We speculate that the similar appearance and differential decline of LNGFR-IR seen after the two types of lesions are regulated by the availability of a common signal such as ciliary neurotrophic factor. The adult SMN model provides a good opportunity to investigate the reexpression of LNGFR after peripheral nerve injury, and more generally, the unknown role and regulation of LNGFR.

Motoneurons of the rat spinal cord express low-affinity nerve growth factor receptor (LNGFR) and corresponding mRNA during development, and re-express it after their axotomy by peripheral nerve injury. The present study establishes the anatomical and quantitative baseline of LNGFR immunoreactive (LNGFR-IR) neurons of the entire normal adult female rat and then investigates the temporal course for the re-expression of LNGFR-IR in lumbar motoneurons after either a crush lesion (which is followed by regeneration and reconnection to the muscle) or a cut lesion with removal of the distal stump (where a neuroma but no reconnection is formed). In the normal adult spinal cord, two types of LNGFR-IR neurons were recognized: (1) small populations of large motoneurons located in the ventral horn mainly in correspondence to the regions of the phrenic, cremasteric and dorsolateral nuclei, and (2) a more numerous and more dorsally located population of small neurons. With a sciatic cut lesion, the number of LNGFR-IR motoneurons at spinal levels L4-L6 rapidly and dramatically increased to a maximum between post-lesion days 1 and 7, apparently involving most axotomized motoneurons of the region, and returned to the baseline level by day 30. With a crush lesion, similar numbers and virtually the same time-course of LNGFR-IR appearance were seen, but the onset of progressive disappearance of LNGFR-IR neurons was delayed by one week, so that at 30 days, the most caudal motoneurons (which are last to reach their target) were still LNGFR-IR. Comparison of these two time courses gives clues to the kind of signals that may be involved in initiating and/or maintaining the LNGFR response.

S-100 is a protein originally believed to be unique to the nervous system. We report here on its immunochemical detection in non-nervous structures of the rabbit eye, namely in the lens, cornea and iris, at a markedly higher concentration in the latter.

The present study reports on the immunohistochemical distribution of S-100 antigen in non-nervous cell types within the human eye at light microscopy. In the cornea the antigen was confined to endothelial cells covering its posterior surface; the lens exhibited immunoreactivity restricted to the epithelial cells located beneath the anterior capsule. In the iris and ciliary body, S-100 was detected in stromal cells and epithelial cells of the pigmented inner layer in the former and inner epithelial cells bounding the posterior chamber in the latter.

In the present study, by the PAP method with an anti S-100, a scanty population of stellate or elongated cells, identifiable as folliculo-stellate cells, was detected in normal human adenohypophysis and in pituitary adenomas, mostly of GH-secreting type. Moreover, in hemangioblastomas, S-100 immunostaining enabled us to detect the presence of stained cells, possibly of a reactive glial nature, while “stromal cells” appeared to be devoid of reaction product.

S-100, a protein originally believed to be unique to the nervous system, has recently been found in extraneural cell types. We report here on the presence of S-100 in the testis, namely in Leydig cells and in lymphatic endothelial cells, using immunohistochemical and immunochemical methods. We show that the protein in the testis is immunologically identical to brain S-100. The S-100-labelled cells in the testis exhibit morphological similarities with other cell types in different tissues known to contain S-100.

The levels and the distribution of S-100 protein were studied in the brain of hypothyroid adult rats. The concentration of the protein was significantly increased in the soluble fraction of hypothyroid rat brain as compared with controls (P less than 0.001). This finding probably reflects the increased number of astroglial cells, which has been widely reported in hypothyroid adult rats. The immunocytochemical ultrastructural distribution of the protein in the cerebellar cortex was not affected by thyroid deficiency. The protein was thus confined to the cytoplasm and the nucleus of the astroglial cells both in treated and untreated rats.

Cellular and subcellular distribution of adenylate cyclase (AC) and guanylate cyclase (GC) activities in crushed peripheral nerves during regeneration were studied at the electron microscope level. In unlesioned nerves, no AC reaction product could be evidenced, whereas GC was detectable on the plasma membranes of Schwann cells, myelinated and nonmyelinated fibers, and within nonmyelinated axons. At 24 hours after the crush, AC reaction product was found within axonal segments proximal to the zone of the crush in association with mitochondria. At this stage, macrophage-like cells, which probably are transformed Schwann cells, polymorphonuclear leucocytes, and endothelial cells displaying an intense AC reaction product could be detected. On the other hand, at 24 hours after the crush, GC was no longer detectable, except on occasional unlesioned nerve fibers. At 48 hours after the lesion, AC reaction product was no longer detectable within axons, and all AC positivity was associated with plasma membranes of non-neuronal cells, including transformed Schwann cells, occasional macrophages, polymorphonuclear leucocytes, fibroblasts, and elongated cells. As to GC, images similar to those obtained at 24 hours were observed until 48 hours after the crush. From the 7th to the 28th postlesion day, AC activity was localized exclusively to the plasma membranes of fibroblasts and elongated cells. Transformed Schwann cells were no longer detectable, whereas normal Schwann cells and regenerating axons could be seen, and these showed no AC reaction product in analogy to the absence of AC reaction product of unlesioned nerves. During the same period, GC again was detectable on regenerating fibers with the same subcellular localization as that of unlesioned nerves. The present results strongly suggest that starting from the second postcrush day, cells invading the lesioned zone and transformed Schwann cells, all taking part in the formation of the new perineurial tissue, display a high AC activity, which should be taken into account when measuring cyclic adenosine monophosphate (cAMP) levels under these conditions. Also, our data suggest that GC is involved primarily in regeneration processes that occur in crushed peripheral nerves. Thus, the pattern of AC distribution in peripheral unlesioned and lesioned nerves appears to be exactly the opposite of the GC localization examined under similar experimental conditions insofar as nervous fibers are concerned.