Gabriella Guelfi

Expression of type I GNRH receptor and in vivo and in vitro GNRH-I effects in corpora lutea of pseudopregnant rabbits

The expression of type I GNRH receptor (GNRHR-I) and the direct role of GNRH-I on corpora lutea (CL) function were studied in the pseudopregnant rabbit model. Immunohistochemistry evidenced GNRHR-I and GNRH-I in luteal cells at early (day 4 pseudopregnancy)-, mid (day 9)-, and late (day 13)-luteal stages. Real-time RT-PCR and western blotting revealed GNRHR-I mRNA and protein at the three luteal stages. Buserelin in vivo treatment at days 9 and 13 decreased plasma progesterone levels for 48 and 24  h respectively. In in vitro cultured CL, buserelin reduced progesterone secretion, increased prostaglandin F(2α) (PGF(2α)) secretion and cyclo-oxygenase-2 (COX-2) and nitric oxide synthase (NOS) activities at days 9 and 13, and decreased PGE₂ at day 13. Co-incubation with antagonists for GNRH-I (antide), inositol 1,4,5-trisphosphate (IP₃, 2-amino-ethoxydiphenylborate), and diacylglycerol (DAG, 1-hexadecyl-2-acetyl glycerol) or inhibitors for phospholipase C (PLC, compound 48/80), and protein kinase C (PKC, staurosporine) counteracted the buserelin effects. Buserelin co-incubated with COX inhibitor (acetylsalicylic acid) increased progesterone and decreased PGF(2α) and NOS activity at days 9 and 13, whereas co-incubation with NOS inhibitor (N-nitro-l-arginine methyl ester) increased progesterone at the same luteal stages. These results suggest that GNRHR-I is constitutively expressed in rabbit CL independently of luteal stage, whereas GNRH-I down-regulates directly CL progesterone production via PGF(2α) at mid- and late-luteal stages of pseudopregnancy, utilizing its cognate type I receptor with a post-receptorial mechanism that involves PLC, IP₃, DAG, PKC, COX-2, and NOS.

Different levels of serum microRNAs in prostate cancer and benign prostatic hyperplasia: evaluation of potential diagnostic and prognostic role

Introduction Diagnosis of prostate cancer (PCa) is based on prostate biopsy that is performed when prostate specific antigen (PSA) is persistently altered over time and/or abnormal digital rectal examination is found. Serum PSA levels increase in both PCa and benign prostatic hyperplasia, leading to an increased number of unnecessary biopsies. There is an urgent need to unravel PCa-specific molecular signatures. Patients and methods This study aimed at characterizing a panel of circulating micro RNAs (miRNAs) that could distinguish PCa from benign prostatic hyperplasia in a population of age-matched patients with increased PSA levels. Both miRNAs targeting genes involved in PCa onset and miRNAs whose role in PCa has been highlighted in other studies were included. For this purpose, let-7c, let-7e, let-7i, miR-26a-5p, miR-26b-5p, miR-24-3p, miR-23b-3p, miR-27-b-3p, miR-106a-5p, miR-20b-5p, miR-18b-5p, miR-19b-2-5p, miR-363-3p, miR-497, miR-195, miR-25-3p, miR-30c-5p, miR-622, miR-874-3p, miR-346 and miR-940 were assayed through real-time PCR in 64 patients with PCa and compared with 60 patients with benign prostatic hyperplasia. The ability of miRNAs to predict the stage of disease was also analyzed. Results Let-7c, let-7e, let-7i, miR-26a-5p, miR-26b-5p, miR-18b-5p and miR-25-3p were able to discriminate patients with PCa from those harboring benign prostatic hyperplasia, both presenting altered PSA levels (>3 ng/mL). MiR-25-3p and miR-18b-5p showed the highest sensitivity and specificity to predict PCa, respectively. The combination of these two miRNAs improved the overall sensitivity. A correlation between pathological Gleason score and miRNA expression levels was reported; miR-363-3p, miR-26a-5p, miR-26b-5p, miR-106a-5p, miR-18b-5p, miR-25-3p and let-7i decreased in expression concomitantly with an increase in malignancy. Conclusion This study confirms serum miRNAs to be reliable candidates for the development of minimally invasive biomarkers for the diagnosis and prognosis of PCa, particularly in those cases where PSA acts as a flawed marker.

A Retrospective Investigation on Canine Papillomavirus 1 (CPV1) in Oral Oncogenesis Reveals Dogs Are Not a Suitable Animal Model for High-Risk HPV-Induced Oral Cancer

CPV1 (also called COPV) is a papillomavirus responsible for oral papillomatosis in young dogs. The involvement of this viral type in oral oncogenesis has been hypothesized in oral squamous cell carcinomas (SCCs), but has never been investigated in other neoplastic and hyperplastic oral lesions of dogs. Aim of this study was to investigate the presence of CPV1 in different neoplastic and hyperplastic lesions in order to assess its role in canine oral oncogenesis; according to the results obtained, a second aim of the study was to define if the dog can be considered a valid animal model for oral high risk HPV-induced tumors. Eighty-eight formalin-fixed, paraffin-embedded (FFPE) canine oral lesions including 78 oral tumors (papillomas, SCCs, melanomas, ameloblastomas, oral adenocarcinomas) and 10 hyperplastic lesions (gingival hyperplasia) were investigated with immunohistochemistry for the presence of papillomavirus L1 protein and with Real-Time PCR for CPV1 DNA. RT-PCR for RNA was performed on selected samples. All viral papillomas tested were positive for immunohistochemistry and Real-time PCR. In 3/33 (10%) SCCs, viral DNA was demonstrated but no viral RNA could be found. No positivity was observed both with immunohistochemistry and Real-Time PCR in the other hyperplastic and neoplastic lesions of the oral cavity of dogs. Even though the finding of CPV1 DNA in few SCCs in face of a negative immunohistochemistry could support the hypothesis of an abortive infection in the development of these lesions, the absence of viral RNA points out that CPV1 more likely represents an innocent bystander in SCC oncogenesis. The study demonstrates a strong association between CPV1 and oral viral papillomas whereas viral contribution to the pathogenesis of other oral lesions seems unlikely. Moreover, it suggests that a canine model of CPV1 infection for HPV-induced oncogenesis could be inappropriate.

Direct actions of ACTH on ovarian function of pseudopregnant rabbits.

The present study sought to assess whether the receptors for adrenocorticotropic hormone (ACTH), MC2R, and for glucocorticoid (GR) are expressed in corpora lutea (CL) of pseudopregnant rabbits and whether ACTH and cortisol exert any direct action on luteal function. By immunohistochemistry, positive reaction for MC2R and GR was detectable within luteal cells of CL. The MC2R mRNA levels were five-fold less abundant in day 9 than in day 4 CL (P<0.01). At both stages, ACTH agonist (ACTH 1-24) increased progesterone and prostaglandin (PG) E(2) (PGE(2)) (P<0.01), but reduced PGF(2α) releases (P<0.01) in vitro. ACTH 1-24 injection increased plasma cortisol levels within 4h (P<0.01), but decreased (P<0.01) progesterone 24h later and for the following two days. ACTH administration to estrous rabbits caused a transitory increase in blood progesterone concentrations (P<0.01). Daily injections of ACTH did not modify progesterone profile following ovulation. In conclusion, ACTH directly up-regulates CL progesterone production in vitro via MC2R, but indirectly hampers luteal function via cortisol-GR associated mechanism.

Circulating microRNAs and kallikreins before and after radical prostatectomy: are they really prostate cancer markers?

The aim of our study was to monitor serum levels of two miRNAs (miR-21 and miR-141) and three KLKs (hK3/PSA, hK11, and hK13) before and 1, 5, and 30 days after radical prostatectomy, in order to characterize their fluctuations after surgery. 38 patients with prostate cancer were included. miR-21 and miR-141 were quantified through real-time PCR, while ELISA assays were used to quantify hK3 (PSA), hK11, and hK13. Both miR-21 and miR-141 showed a significant increase at the 5th postoperative day, after which a gradual return to the preoperative levels was recorded. These findings suggest that miR-21 and miR-141 could be involved in postsurgical inflammatory processes and that radical prostatectomy does not seem to alter their circulating levels. Postoperative serum kallikreins showed a significant decrease, highlighting the potential usefulness of kallikreins apart from PSA as potential prostate cancer markers.

Stability Assessment of Candidate Reference Genes in Urine Sediment of Prostate Cancer Patients for miRNA Applications

We aimed at assessing the stability of candidate reference genes in urine sediments of men subjected to digital rectal examination for suspected prostate cancer (PCa). Two microRNAs (miR-191 and miR-25) and 1 small nucleolar RNA (SNORD48) were assayed in 35 post-DRE urine sediments of men with PCa and in 26 subjects with histologically confirmed benign prostatic hyperplasia (BPH). The stability of candidate reference genes was assessed through BestKeeper algorithm and equivalence test. miR-200b and miR-452 were used to test for the effect of normalization on target genes. Our results proved miR-191 to be the most stable gene, showing the lowest degree of variation and the highest stability value. miR-25 and SNORD48 values fell beyond the cutoff of acceptability. In conclusion, we recommend the use of miR-191 for normalization purposes in post-DRE urine sediments.

Non-invasive assessment of animal exercise stress: real-time PCR of GLUT4, COX2, SOD1 and HSP70 in avalanche military dog saliva

Exercise has been shown to increase mRNA expression of a growing number of genes. The aim of this study was to assess if mRNA expression of the metabolism- and oxidative stress-related genes GLUT4 (glucose transporter 4), COX2 (cyclooxygenase 2), SOD1 (superoxide dismutase 1) and HSP70 (heat shock protein 70) in saliva changes following acute exercise stress in dogs. For this purpose, 12 avalanche dogs of the Italian Military Force Guardia di Finanza were monitored during simulation of a search for a buried person in an artificial avalanche area. Rectal temperature (RT) and saliva samples were collected the day before the trial (T0), immediately after the descent from a helicopter at the onset of a simulated avalanche search and rescue operation (T1), after the discovery of the buried person (T2) and 2 h later (T3). Expressions of GLUT4, SOD1, COX2 and HSP70 were measured by real-time PCR. The simulated avalanche search and rescue operation was shown to exert a significant effect on RT, as well as on the expression of all metabolism- and oxidative stress-related genes investigated, which peaked at T2. The observed expression patterns indicate an acute exercise stress-induced upregulation, as confirmed by the reductions in expression at T3. Moreover, our findings indicate that saliva is useful for assessing metabolism- and oxidative stress-related genes without the need for restraint, which could affect working dog performance.

Characterization of Kallireins and microRNAs in Urine Sediment for the Discrimination of Prostate Cancer from Benign Prostatic Hyperplasia

Objectives: Prostate Cancer (PCa) and Benign Prostatic Hyperplasia (BPH) are frequently coexisting in elderly men. The measurement of serum PSA together with Digital Rectal Examination (DRE) represents the primary diagnostic tool to suspect PCa, whereas definitive diagnosis is achieved by prostate biopsy. The low specificity of PSA and the modest detection rate of biopsy convict the patient to a quite often unnecessary and uncomfortable clinical itinerary. There is a urgent need for new and more accurate methodologies to diagnosize PCa. In the present study, the expression of 4 mRNAs and 2 miRNAs was evaluated in post DRE urine cell pellets from patients suffering PCa and age-matched subjects affected by BPH with elevated PSA levels. We also evaluated the diagnostic accuracy of markers in predicting PCa. Materials and methods: The expression levels of 4 mRNAs (3 kallikreins - KLK3, KLK11, KLK13 and a prostate cancer antigen - PCA3) and 2 microRNAs (miR-9-3p and miR-19a-3p) were assayed by means of real-time PCR in post DRE urine of 79 men undergoing prostate biopsy for PSA levels > 3 ng/mL. The diagnostic power of tested markers was evaluated through logistic regression analysis. Results: PCA3 was undetectable in 22 out of 38 BPH subjects. KLK3 and KLK11 were significantly upregulated in PCa group (p value < 0.001), while miR-9-3p and miR-19a-3p were up-regulated in BPH group (p value <0.001 and < 0.01, respectively). KLK13 was not differentially expressed between groups. MiR-19a-3p and miR- 9-3p reached the highest specificity (64.29%) and sensitivity (81.08%), respectively. The more accurate bivariate logistic model was obtained combining KLK11 with either miR-9-3p and miR-19a-3p. Conclusions: Our findings demonstrated that selected kallikreins and miRNAs proved to be an accurate diagnostic tool for PCa. Urine cells pellets obtained after DRE represent a reliable biological matrix for minimally invasive gene expression assays.

My Dog Is Not My Cat: Owner Perception of the Personalities of Dogs and Cats Living in the Same Household

Simple Summary A growing number of dogs and cats live together, sharing both a common home and common owner. Nevertheless, how do owners of both cats and dogs living in the same household perceive their pets’ personalities? We tried to answer this question by using a questionnaire targeted at people who owned both dogs and cats. Sociability, protectiveness, reactivity, neuroticism, and fearfulness were the traits that emerged and that diversified themselves according to species. Moreover, intrinsic animal factors, such as age and gender, demographic characteristics of the owner, and environmental context seem to modulate the perceived personality traits in a species-specific way. As personality might influence pets’ welfare and adaptability to the home environment, owners of multiple pets should know and take into consideration both common and differential aspects of their pet’s personality to optimise cohabitation among dogs and cats. Abstract This study aims to define the personality traits perceived by the owners of multiple pets and to evaluate how they are modulated by experiential-environmental factors. A questionnaire was administered to 1270 owners of multiple pets (dogs and cats) to collect data on the demographics, management, and personality of their pets. Data were analysed by principal component analysis, bivariate, and multivariable models. Five personality traits emerged in dogs and cats: sociability, reactivity, protectiveness, neuroticism, and fearfulness. The owners perceived differences in the personality of their pet: dogs scored higher in sociability, protectiveness, and reactivity, while lower in the neuroticism dimension compared with cats (p < 0.001). Age similarly affected sociability (p < 0.01), and reactivity (p < 0.001) in both dogs and cats, while species-specific gender differences were found as to fearfulness (p < 0.05) and neuroticism (p < 0.001). The age of acquisition modulated several traits in dog personality, while living with conspecifics especially influenced cats. Physiological, behavioural, and evolutionary characteristics could explain species differences. Moreover, intrinsic and extrinsic factors modulated the five dimensions of dogs and cats in a diversified fashion, suggesting complex interactions between species and the environment. However, owners could have had different attitudes with their animals which could have influenced personality perception.

Next Generation Sequencing of urine exfoliated cells: An approach of prostate cancer microRNAs research

There is emerging evidence that microRNAs (miRNAs) dysregulation is involved in the genesis and the progression of Prostate Cancer (PCa), thus potentially increasing their use in urological clinical practice. This is the first pilot study which utilizes Illumina Deep Sequencing to examine the entire miRNAs spectrum existent in urine exfoliated prostate cells (UEPCs) of PCa patients. A total of 11 male patients with histological diagnosis of PCa were enrolled in the present study. First-catch urine (30 mL) was collected following a prostate massage. Total RNA was extracted from urine and sequenced using an HiSeq2500 System (Illumina). QPCR assay was used to validate the highest NGS results in PCA patients and in age-matched, caucasian men. Remarkably, PCA let-7 family was down-regulated (P < 0.01), compared to the controls. The results of our study support the notion of a relatively high diagnostic value of miRNA family for PCa detection, especially in the let-7 family. The present research confirmed the potential use of miRNAs as non-invasive biomarkers in the diagnosis of PCa, potentially reducing the invasiveness of actual clinical strategy.