ABCB1 c.-6-180 T > G polymorphism and clinical risk factors in a multi-breed cohort of dogs with refractory idiopathic epilepsy

Epilepsy is the most common chronic neurological disorder in dogs. Approximately 20-30% of dogs do not achieve satisfactory seizure control with two or more anti-epileptic drugs at appropriate dosages. This condition, defined as refractory epilepsy, is a multifactorial condition involving both acquired and genetic factors. The P glycoprotein might play and important role in the pathophysiological mechanism and it is encoded by the ABCB1 gene. An association between a single nucleotide variation of the ABCB1 gene (c.-6-180 T > G) and phenobarbital resistance has previously been reported in a Border collie population with idiopathic epilepsy. To date, the presence and relevance of this polymorphism has not been assessed in other breeds. A multicentre retrospective, case-control study was conducted to investigate associations between ABCB1 c.-6-180 T > G, clinical variables, and refractoriness in a multi-breed population of dogs with refractory idiopathic epilepsy. A secondary aim was to evaluate the possible involvement of the ABCB1 c.-6-180 T > G single nucleotide variation this population. Fifty-two refractory and 50 responsive dogs with idiopathic epilepsy were enrolled. Of these, 45 refractory and 50 responsive (control) dogs were genotyped. The G allele was found in several breeds, but there was no evidence of association with refractoriness (P = 0.69). The uncertain role of the c.-6-180T>G variation was further suggested by an association between the T/T genotype with both refractoriness and responsiveness in different breeds. Furthermore, high seizure density (cluster seizure) was the main clinical risk factor for refractory idiopathic epilepsy (P = 0.003)

Tracing anti-cancer and cancer-promoting actions of all-trans retinoic acid in breast cancer to a RARα epigenetic mechanism of mammary epithelial cell fate

A hallmark of cancer cells is the ability to evade the growth inhibitory/pro-apoptotic action of physiological all-trans retinoic acid (RA) signal, the bioactive derivative of Vitamin A. However, as we and others reported, RA can also promote cancer cell growth and invasion. Here we show that anticancer and cancer-promoting RA actions in breast cancer have roots in a mechanism of mammary epithelial cell morphogenesis that involves both transcriptional (epigenetic) and non-transcriptional RAR\u3b1 (RARA) functions. We found that the mammary epithelial cell-context specific degree of functionality of the RARA transcriptional (epigenetic) component of this mechanism, by tuning the effects of the non-transcriptional RARA component, determines different cell fate decisions during mammary morphogenesis. Indeed, factors that hamper the RARA epigenetic function make physiological RA drive aberrant morphogenesis via non-transcriptional RARA, thus leading to cell transformation. Remarkably, also the cell context-specific degree of functionality of the RARA epigenetic component retained by breast cancer cells is critical to determine cell fate decisions in response to physiological as well as supraphysiological RA variation. Overall this study supports the proof of principle that the epigenetic functional plasticity of the mammary epithelial cell RARA mechanism, which is essential for normal morphogenetic processes, is necessary to deter breast cancer onset/progression consequent to the insidious action of physiological RA

Identification of an estrogen-regulated circadian mechanism necessary for breast acinar morphogenesis

Altered estrogen receptor \u3b1 (ERA) signaling and altered circadian rhythms are both features of breast cancer. By using a method to entrain circadian oscillations in human cultured cells, we recently reported that the expression of key clock genes oscillates in a circadian fashion in ERA-positive breast epithelial cells but not in breast cancer cells, regardless of their ERA status. Moreover, we reported that ERA mRNA oscillates in a circadian fashion in ERA-positive breast epithelial cells, but not in ERA-positive breast cancer cells. By using ERA-positive HME1 breast epithelial cells, which can be both entrained in vitro and can form mammary gland-like acinar structures in three-dimensional (3D) culture, first we identified a circuit encompassing ERA and an estrogen-regulated loop consisting of two circadian clock genes, PER2 and BMAL1. Further, we demonstrated that this estrogen-regulated circuit is necessary for breast epithelial acinar morphogenesis. Disruption of this circuit due to ERA-knockdown, negatively affects the estrogen-sustained circadian PER2-BMAL1 mechanism as well as the formation of 3D HME1 acini. Conversely, knockdown of either PER2 or BMAL1, by hampering the PER2-BMAL1 loop of the circadian clock, negatively affects ERA circadian oscillations and 3D breast acinar morphogenesis. To our knowledge, this study provides the first evidence of the implication of an ERA-circadian clock mechanism in the breast acinar morphogenetic process

Entrainment of breast (cancer) epithelial cells detects distinct circadian oscillation patterns for clock and hormone receptor genes

Most physiological and biological processes are regulated by endogenous circadian rhythms under the control of both a master clock, which acts systemically and individual cellular clocks, which act at the single cell level. The cellular clock is based on a network of core clock genes, which drive the circadian expression of non-clock genes involved in many cellular processes. Circadian deregulation of gene expression has emerged to be as important as deregulation of estrogen signaling in breast tumorigenesis. Whether there is a mutual deregulation of circadian and hormone signaling is the question that we address in this study. Here we show that, upon entrainment by serum shock, cultured human mammary epithelial cells maintain an inner circadian oscillator, with key clock genes oscillating in a circadian fashion. In the same cells, the expression of the estrogen receptor \u3b1 (ERA) gene also oscillates in a circadian fashion. In contrast, ER A-positive and -negative breast cancer epithelial cells show disruption of the inner clock. Further, ER A-positive breast cancer cells do not display circadian oscillation of ERA expression. Our findings suggest that estrogen signaling could be affected not only in ER A-negative breast cancer, but also in ER A-positive breast cancer due to lack of circadian availability of ER A. Entrainment of the inner clock of breast epithelial cells, by taking into consideration the biological time component, provides a novel tool to test mechanistically whether defective circadian mechanisms can affect hormone signaling relevant to breast cancer

STUDIO SULLA BIOPSIA TC-GUIDATA DEL POLMONE CON AGO SOTTILE NEL CANE E NEL GATTO

La diagnosi delle patologie polmonari sulla base dell’anamnesi e della visita clinica è spesso difficile. La diagnostica per immagini risulta essere di grande importanza in questo settore. La radiologia è stata considerata per molto tempo la tecnica di elezione per lo studio di queste malattie. Tuttavia spesso non è possibile differenziare una lesione infiammatoria/infettiva da una neoplastica. Risulta quindi necessaria una corretta diagnosi cito-istopatologica per avere una diagnosi accurata, una corretta prognosi ed un preciso piano terapeutico. In medicina umana la TC e la biopsia TC-guidata sono indicate in presenza di lesioni non adeguatamente visualizzate con altre procedure diagnostiche. Nel presente studio sono stati sottoposti a biopsia TC-guidata con ago sottile 30 cani e 9 gatti, di differente sesso, razza e dimensioni. In tutti gli animali sono stati eseguiti esame clinico, esami ematici e le radiografie del torace. Nel presente studio 32 campioni su 39 sono risultati diagnostici. Gli altri 7 casi o a causa di incertezza diagnostica, od in quanto contenevano solo sangue sono stati considerati non diagnostici. Non si sono riscontrate complicazioni gravi, solamente 5 casi di lieve pneumotorace.Diagnosis of pulmonary lesions on the basis of history and physical examination is often challenging. Diagnostic imaging is therefore of paramount importance in this field. Radiology has traditionally been considered the elective diagnostic procedure for these diseases. Nonetheless it is often not possible to differentiate inflammatory/infectious lesions from neoplastic disease. A correct cyto-histopathologic diagnosis is therefore needed for an accurate diagnosis and subsequent prognostic and therapeutic plan. In human medicine TC and TC-guided biopsy are indicated in the presence of lesions which are not adequately imaged with other diagnostic procedures. In the present study 30 dogs and 9 cats of different sex, breed and size underwent TCguided lung fine-needle aspiration. Clinical examination, haematology and chest radiography were performed on all animals. In this study 32 samples out of 39 were diagnostic. Other 7 cases either because of uncertainty or only blood was aspirated, were considered non diagnostic. Only mild pneumothorax was seen in 5 cases. No major complications were encountered

Effect of cilostamide treatment on the intercellular coupling, meiotic and embryonic developmental competence in horse oocyte-cumulus cells complexes of different origins

Design of new strategies to improve in vitro embryo production (IVP) efficiency, such as pre-maturation culture with cilostamide (CILO PMC) aimed to prolong oocytes-cumulus cells gap junction-mediated communications (GJC) functionality while delaying meiotic resumption, should consider the high heterogeneity of cumulus-oocyte complexes (COCs) retrieved from ovarian follicles. In the mare, main factors contributing to this heterogeneity are: the follicle diameter, the cumulus oophorus morphology and the reproductive seasonality. Thus, the objectives of this study were: 1) to identify a population of equine oocytes that would benefit from a CILO PMC, based on the assessment the GJC functionality by Lucifer Yellow microinjection, the oocyte chromatin configuration by DNA staining, and the meiotic competence after in vitro maturation (IVM), in COCs of different origins, since these parameters are indicative of the oocyte metabolic state and 2) to assess the effect of CILO PMC, in COCs of different origins, on the GJC functionality, the meiotic and embryonic developmental competence after IVM and intracytoplasmic sperm injection (ICSI). COCs with Compact (Cp) or Expanded (Ex) cumulus oophorus were collected from 2 cm follicles during winter, spring-summer and fall, and cultured with our standard IVM protocol (CTRL) or prematurated in the same medium with 10-4 UI r-hFSH/ml and 10 \u3bcM Cilostamide (CILO PMC) for different times. When meiotic and developmental were assessed, COCs were subjected to CILO PMC for 6 h and than to standard IVM and ICSI. Data were analyzed by Chi square test. Overall, our results suggest that COCs that would more probably benefit from CILO PMC are those with Cp cumuli from <1 and 1-2 cm follicles collected during spring-summer and fall. In fact, they have a lower meiotic competence after 24h IVM (P<0.05), with a mean MII rate of 40%, compared to all the other groups, in which MII rate is around 60%. Moreover, they show a higher % (P<0.05) of COC with open GJC (70%), when compared with both Cp COC in the winter season and Exp COC from all follicular classes in all seasons tested, in which more that 50% of COCs have closed GJC. Independently from the season, chromatin configuration analysis reveals that oocytes with Cp cumuli from 1-2 cm follicles are in a more advanced stage of differentiation since their chromatin is more condensed (P<0.05). Study on GJC indicates that functionality can be maintained up to 10h in Cp COCs from 1-2 cm using CILO PMC when compared to the CTRL group, in which a major GJC drop is detected at 10h. In fact, COCs with open GJC are 74% and 44% in CILO PMC and CTRL groups respectively at 10 h (P<0.05) On the contrary, the exact timing for the treatment of Cp COCs from <1 cm follicles remains to be determined since GJC functionality drops after 16h in the CTRL group, with only 14% of COCs with open GJC, and no differences are observed between groups. Finally, initial evaluation of the effect of CILO PMC for 6h indicates that it does not affect the rate of embryos obtained after ICSI. Evaluation of embryo quality is in progress to assess whether CILO PMC could improve IVP efficiency beyond embryo yield. In conclusion, our data could be useful in setting up IVM strategies to improve horse IVP efficiency as well as the understanding of horse oocyte biology. Funding: Grant n. 26096200 "Ex Ovo Omnia" Regione Sardegna-Lombardia and \u2018Dote Ricerca\u2019 FSE, Regione Lombardia