Sebbene il sistema nervoso enterico (“enteric nervous system”, ENS) svolga un ruolo cruciale nella patogenesi della Scrapie ovina, non esistono tuttavia in letteratura dati sulle popolazioni cellulari progressivamente coinvolte nel corso dell’infezione, né sugli eventuali danni morfo-funzionali da esse subiti. Il presente studio è stato condotto sui plessi mienterici e sottomucosi dell’ileo di 46 pecore di razza Sarda, recanti diversi polimorfismi del gene Prnp (ARQ/ARQ, ARQ/AHQ, ARQ/ARR, ARR/ARR). I suddetti animali, infettati per os all’età di 8 mesi con un ceppo di Scrapie precedentemente caratterizzato nel topo, sono stati sacrificati mediante eutanasia a determinati intervalli di tempo post-infezione (p.i.). E’ stata quindi valutata, tramite immunoistochimica ed immunofluorescenza indiretta su sezioni tissutali e su preparati “wholemount”, l’immunoreattività (IR) nei confronti della PrPSc, del “marker” panneuronale Hu C/D, dell’ossido-nitrico sintetasi (nNOS), della calbindina (CALB) e della proteina fibrillare acida gliale (GFAP). In 8 pecore con genotipo ARQ/ARQ, clinicamente sane e sacrificate a 12-24 mesi p.i., nonché in 5 ovini clinicamente affetti (2 con genotipo ARQ/ARQ, 3 con genotipo ARQ/AHQ), questi ultimi sacrificati rispettivamente a 24, 36 e 40 mesi p.i., le indagini immunoistochimiche hanno consentito di dimostrare la presenza di PrPSc a livello sia dell’encefalo (obex), sia dell’ENS, in particolar modo nei plessi mienterici. In tali distretti il deposito della PrPSc risultava pienamente compatibile con un interessamento delle cellule enterogliali (“enteroglial cells”, EGCs), mentre occasionalmente si notava un contestuale coinvolgimento della componente neuronale ivi residente. In conclusione, i dati della presente indagine consentono di ipotizzare un verosimile coinvolgimento delle EGCs e dei neuroni residenti a livello dei plessi dell’ENS nella patogenesi della Scrapie sperimentale realizzata per os in ovini di razza Sarda. / The enteric nervous system (ENS) is believed to play a key role in the pathogenesis of sheep Scrapie infection, although no information exists on the ENS cytotypes which are progressively involved, nor on the morpho-functional damage of such cells in the time course of infection. We investigated the ileal myenteric (MPs) and submucosal plexuses (SMPs) of 46 Sarda breed sheep carrying different PrP genotypes (ARQ/ARQ, ARQ/AHQ, ARQ/ARR, ARR/ARR), which had been orally dosed with Scrapie at 8 months of age and euthanized at definite time intervals post-infection (p.i.). PrPSc immunoreactivity (IR), along with pan-neuronal marker Hu C/D, nitric oxide synthase (nNOS), calbindin (CALB) and glial fibrillary acidic protein (GFAP)-IR, were evaluated by means of immunohistochemistry (IHC) and indirect immunofluorescence (IF) on paraffin-embedded sections and wholemount preparations. Eight clinically-healthy ARQ/ARQ sheep euthanized at 12-24 months p.i., along with 2 ARQ/ARQ (euthanized at 24 months p.i.) and 3 ARQ/AHQ (euthanized at 36 and 40 months p.i., respectively) clinicallyaffected sheep, showed IHC evidence of PrPSc in both their brain (obex) and ENS, especially in MPs. PrPSc deposition was fully compatible with an involvement of enteroglial cells (EGCs) and, occasionally, also with an involvement of neurons residing within ileal ENS plexuses. In conclusion, EGCs and neurons residing within ileal ENS plexuses may be likely involved in the pathogenesis of oral experimental Scrapie infection in Sarda breed sheep.

Tumor residual macroscòpic persisteix després de la quimioteràpia neoadjuvant (QTN) ( 70% de pacients) tenen un risc de metàstasis del 35% (3 anys). Hem trobat una signatura predictiva de bona resposta de QTN (coeficient de determinació 55,6%; Odds-ratio 26,6; p=0,00004). Els tumores receptors hormonals (RH) negatius presenten Ciclina D1, Aldehíd deshidrogenasa-1, HER4, p-HER3 i p-AKt com a marcadors predictius de bona resposta a QTN. Els RH positius presenten Bcl-2 com a marcador de resistència. Els RH negatius presenten major expressió de p-AKt, EGFR1, HER2 i p-HER3 i menor expressió de p27 i Bcl-2 que els RH positius. Els tumors Triples negatius presenten menor expressió de 53BP1 i major expressió Captesina L nuclear. La majòria dels Triples Negatius són BRCA1 deficients i susceptibles a inhibidors de PARP. Els tumors BRCA1 deficients, poden restablir la recombinació homòloga i ser resistents al tratament, quan 53BP1 és degradada per Catepsina L nuclear, davant baixos nivells del receptor de la vitamina D. / Tumor residual macroscópico tras quimioterapia neoadyuvante (QTN) ( 70% de pacientes) tiene riesgo de metástasis del 35% (3 años). Hemos encontrado una firma predictiva de buena respuesta de QTN (coeficiente de determinación 55,6%, Odds-ratio 26,6; p=0,00004). Los tumores receptores hormonales (RH) negativos presentan Ciclina D1, Aldehído deshidrogenasa-1, HER4, p-HER3 y p-AKt como marcadores predictivos de buena respuesta a QTN. Los RH positivos presentan Bcl-2 como un marcador de resistencia. Los RH negativos presentan mayor expresión de p-AKt, EGFR1, HER2 y p-HER3 y menor expresión de p27 y Bcl-2 que lo RH positivos. Los tumores Triples Negativos presentan la menor expresión de 53BP1 y la mayor expresión Captesina L nuclear. La mayoría de los TNBC son BRCA1 deficientes y susceptibles a inhibidores de PARP. Los tumores BRCA1 deficientes, pueden restablecer la recombinación homóloga y ser resistentes al tratamiento, cuando 53BP1 es degradado por Catepsina L nuclear ante bajos níveles del receptor de la vitamina D. / Large (>1 cm) residual tumours are present after neoadjuvant chemotherapy (NCT) (70% from patients) will relapse 35% of patients (3 years). We have found a predictive signature of good response to NCT (determination coefficient 55.6%; Odds-ratio 26.6; p=0.00004). In tumors lacking hormone receptor (HR), CyclinD1, Aldehyde dehydrogenase isoform 1, HER4, p-HER3 and p-AKt are markers of good response. In tumors expressing HR, Bcl-2 is a marker of resistance. Tumors lacking HR expression have an increase of p-AKt, EGFR1, HER2 and p-HER3 and a decrease of p27 and Bcl-2 compared in HR positive tumors. TNBC tumors express the highest nuclear Cathepsin L causing the lowest 53BP1. The majority of TNBC are BRCA1 deficient and likely to receive treatment with PARP inhibitors. In tumors without BRCA1 and with 53BP1 out of the way due to high nuclear CTSL, the cells are able to resume homologous recombination and become resistant to treatment. The activity of nuclear Cathepsin L is blocked by high levels of vitamin D receptor.

El propòsit del nostre treball és valorar morfomètricament les diferències existents entre la Neoplàsia Intraepitelial Cervical (CIN) grau 2 i 3, així com el tipus de Virus del Papil·loma Humà (VPH) present, estudiat mitjançant Hibridació in situ.Hem estudiat 66 peces quirúrgiques d'exèresi del coll uterí de pacients amb diagnòstic histològic de CIN 2 i 82 de CIN 3. Hem demostrat amb el nostre estudi que la superfície afectada en el coll cervical per CIN 3 és significativament més gran que la de CIN 2, a la vegada que són lesions longitudinalment majors, amb major afectació glandular, major profunditat en l'afectació glandular i major índex mitòtic. També hem pogut demostrar que VPH 16/18 predomina en CIN 3. No hem trobat diferències significatives en quant a la presència de papil·les vasculars en ambdues lesions i tampoc hem trobat relació entre tipus de VPH i àrea afectada per CIN, així com entre tipus de VPH i edat de la pacient. / El propósito de nuestro trabajo es valorar morfométricamente las diferencias existentes entre la Neoplasia Intraepitelial Cervical (CIN) grado 2 y 3, así como el tipo de Virus del Papiloma Humano (VPH) presente, estudiado mediante Hibridación in Situ. Hemos estudiado 66 piezas quirúrgicas de exéresis del cuello cervical de pacientes con diagnóstico histológico de CIN 2 y 82 de CIN 3. Hemos demostrado con nuestro estudio que la superficie afectada en el cuello cervical por CIN 3 es significativamente mayor que la de CIN 2, a la vez que son lesiones longitudinalmente mayores, con mayor afectación glandular, mayor profundidad en la afectación glandular y mayor índice mitótico. También hemos podido demostrar que VPH 16/18 predomina en CIN 3. No hemos encontrado diferencias significativas en cuanto a la presencia de papilas vasculares en las dos lesiones y tampoco hemos encontrado relación entre tipo de VPH y área afectada por CIN, así como entre tipo de VPH y edad de la paciente.

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Akt (also called PKB) is a 63 kDa serine/threonine kinase involved in promotion of cell survival, proliferation a nd metabolic responses downstream the phosphoinositide-3-kinase (PI 3-kinase) signaling pathway. In resting cells, Akt is a predominantly cytosolic enzyme; however generation of PI 3-kinase lipid products recruits Akt to the plasma membrane, resulting in a conformational change which confers full enzymatic activity through the phosphorylation of the membrane-bound protein at two residues, Thr308, and Ser473. Activated Akt redistributes to cytoplasm and nucleus, where phosphorylation of specific substrates occurs. Both the presence and the activity of Akt in the nucleus have been described. An interesting mechanism that mediates nuclear translocation of Akt has been described in human mature T-cell leukemia: the product of TCL1 gene, Tcl1, interacts with the PH domain of phosphorylated Akt, thus driving Akt to the nucleus. In this context, Tcl1 may act as a direct transporter of Akt or may contribute to the formation of a complex that promotes the transport of active Akt to the nucleus, where it can phosphorylate nuclear substrates. A well described nuclear substrate if Foxo. IGF-1 triggers phosphorylation of Foxo by Akt inside the nucleus, where phospho-Foxo associates to 14.3.3 proteins that, in turn, promote its export to the cytoplasm where it is sequestered. Remarkably, Foxo phosphorylation by Akt has been shown to be a crucial event in Akt-dependent myogenesis. However, most Akt nuclear substrates have so far remained elusive, as well as nuclear Akt functions. This lack of information prompted us to undertake a search of substrates of Akt in the nucleus, by the combined use of 2D-separation/mass spectrometry and anti-Akt-phosphosubstrate antibody. This study presents evidence of A-type lamins as novel nuclear substrates of Akt. Lamins are type V intermediate filaments proteins found in the nucleus of higher eukaryotes where, together with lamin-binding proteins, they form the lamina at the nuclear envelope, providing mechanical stability for the nuclear membrane. By coimmunoprecipitation, it is demonstrated here that endogenous lamin A and Akt interact, and that A-type lamins are phosphorylated by Akt both in vitro and in vivo. Moreover, by phosphoaminoacid analysis and mutagenesis, it is further demonstrated that Akt phosphorylates lamin A at Ser404, and, more importantly, that while lamin A/C phosphorylation is stable throughout the cell cycle, phosphorylation of the precursor prelamin A becomes detectable as cells enter the G2 phase, picking at G2/M. This study also shows that lamin phosphorylation by Akt creates a binding site for 14.3.3 adaptors which, in turn, promote prelamin A degradation. While this mechanism is in agreement with a general role of Akt in the regulation of a subset of its substrates, opposite to what has been described, degradation is not mediated through a ubiquitination and proteasomal mechanism but through a lysosomal pathway, as indicated by the reverting action of the lysosomal inhibitor cloroquine. Phosphorylation is a key event in the mitotic breakdown of the nuclear lamina. However, the kinases and the precise sites of phosphorylation are scarcely known. Therefore, these results represent an important breakthrough in this very significant but understudied area. The phosphorylation of the precursor protein prelamin A and its subsequent degradation at G2/M, when both the nuclear envelop and the nuclear lamina disassemble, can be view as part of a mechanism to dispose off the precursor that is not needed in this precise context. The recently reported finding that patients affected by Emery-Dreifuss muscular dystrophy carry a mutation at Arg 401, in the Akt phosphorylation motif, open new perspective that warrant further investigation in this very important field.

Background. A new classification system of human breast tumours based on the immunohistochemical characterization has been applied to mammary tumours of the female dog with the aim to verify its association with invasion and grade, and prognostic aid in veterinary medicine. Methods. Forty-five canine mammary carcinomas with a two-year post-mastectomy follow-up were selected from our database, and the following antibodies were applied: anti-cytokeratines 14, 5/6, oestrogen receptor (ER), progesterone receptor (PR), and ERB-B2. . The tumours were grouped for phenotype as: luminal-like (ER+ and/or PR+, CK14-, CK5/6-) type A (ERB-B2-), and B (ERB-B2+); basal-like (ER-, PR-, CK14+ and/or CK5/6+, ERB-B2-); ERB-B2 (ER-, PR-, CK14-, CK5/6-, ERB-B2+). Association with invasion, grade and histotypes were evaluated and Kaplan-Meier survival curves estimated, then compared by survival analysis. Results. Thirty-five cases with luminal pattern (ER+ and PR+) were subgrouped into 13 A type and 22 B type, if ERB-B2 positive or negative . Most luminal-like A and basal-like cases were grade 1 carcinomas, while the percentage of luminal B cases was higher in grade 2 and 3 (Pearson Chi-square P=0.009). No difference in the percentage of molecular subtypes was evidenced between simple and complex/mixed carcinomas (Pearson Chi-square P=0.47). No significant results were obtained by survival analysis, even if basal-like had a more favourable prognosis than luminal-like. Conclusion. The panel of antibodies identified only 3 groups (luminal-like A and B, and basal-like) in the dog. Even though canine mammary tumours may be a model of human breast cancer, the existence of the same types of carcinoma as in the woman need to be confirmed. Canine mammary carcinomas show high molecular heterogeneity, which would benefit from a classification based on molecular differences. However, by multivariate analysis, the molecular classification appears a variable with a dependent value if compared to invasion and grade that are independent, suggesting that, at present, caution should be used in the application of such a classification to the dog, in which invasion and grade supply the most important prognostic information.

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