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E-cadherin, a glycoprotein involved in cell-cell adhesion, has a pivotal role in epithelial-mesenchymal transition, a process through which neoplastic epithelial cells develop an invasive phenotype. In human cutaneous melanomas, decreased E-cadherin expression is associated with shorter survival and increased Breslow thickness, whereas in the dog its role is poorly understood. Tumor thickness and modified Clark level were recently proposed as useful features to assess canine melanocytic tumors, but no studies investigated their association with E-cadherin expression. We performed immunohistochemistry on 77 formalin-fixed, paraffin-embedded primary canine melanocytic tumors. A 3-tier and a 2-tier classification system for assessing E-cadherin expression were tested, with the latter being more informative for the assessment of canine melanocytic tumors. E-cadherin expression was lower in cutaneous melanomas than melanocytomas, as well as in amelanotic tumors compared to pigmented tumors. In amelanotic melanomas, absent E-cadherin expression was associated with an unfavorable outcome, suggesting a potential use of this marker in defining the prognosis of amelanotic melanomas. E-cadherin expression was lower in tumors with greater tumor thickness and modified Clark level ≥IV, suggesting its possible utility in identifying the most invasive tumors. The expression of E-cadherin in oral melanomas was heterogeneous, but was associated with pigmentation and clinical outcome; thus, E-cadherin evaluation could be advantageous to detect the most aggressive neoplasms. However, cutaneous melanomas without E-cadherin expression frequently had a favorable clinical outcome. Hence, its importance as prognostic factor should be carefully considered depending on the tumor origin.

The aim of this research was to evaluate the inflammatory and/or oxidative damage related to silver nanoparticles (AgNPs), which are responsible for negative effects on sperm physiology and metabolism. Thirty New Zealand White rabbit bucks were divided into 5 experimental groups (6 animals/group): Control, treated with 0.9% NaCl; AgNP, treated with a 5 mM AgNP solution; LPS, treated with 50 g/kg b.w. E. coli LPS; AgNPs + NSAID, treated with an anti-inflammatory drug at 0.2 mg/kg b.w. and 5 mM AgNPs; and AgNPs + Vit E, treated with 0.18 mg/kg b.w. vitamin E and 5 mM AgNPs. Sperm quality and oxidative and inflammatory status were assessed at different times (0-60 days). Two statistical models were built: the first evaluated the effects of AgNPs and LPS (vs. Control), whereas the second evaluated the protective effect of an NSAID and vitamin E against AgNP-induced damage. Three principal component analyses were performed: sperm traits (motility, volume), oxidative status (antioxidants, oxidative metabolites, and redox reactions), and cytokines (TNF-α, IL-8, and IL-6). A negative effect on reproductive traits resulted after NP administration. In particular, an inflammatory/oxidative response took place in the reproductive tract during the first 2-3 wks of AgNP administration (cytokine and oxidative metabolite generation); the inflammatory/oxidative thrust impaired the status of rabbit tissues (seminal plasma, sperm, and blood), inducing a response (increased antioxidant enzymes and redox reactions) at 4-7 wks; oxidative stress, if not totally counteracted, likely induced toxicity in the late phases of AgNP administration (8-9 wks). In conclusion, exposure to silver nanoparticles produced a similar but more persistent effect than that of LPS on rabbit reproductive tissues: AgNP administration triggered a proinflammatory response linked to oxidative thrust, worsening many sperm parameters. However, only anti-inflammatory treatment counteracted the negative effects of AgNPs, whereas vitamin E seemed to act as an adjuvant, attenuating the oxidative cascade.

Despite promising immunotherapy strategies in human melanoma, there are few studies on the immune environment of canine melanocytic tumors. In humans, the activation of immunosuppressive cell subpopulations, such as regulatory T cells (Tregs) that express forkhead box protein P3 (FoxP3), the engagement of immunosuppressive surface receptors like cytotoxic T lymphocyte antigen (CTLA-4), and the secretion of molecules inhibiting lymphocyte activation, such as indoleamine-pyrrole 2,3-dioxygenase (IDO), are recognized as immunoescape mechanisms that allow tumor growth and progression. The aim of our study was to investigate the expression of these immunosuppression markers in canine melanocytic tumors and to postulate their possible role in melanoma biology and progression. Fifty-five formalin-fixed, paraffin-embedded canine melanocytic tumors (25 oral melanomas; 20 cutaneous melanomas; 10 cutaneous melanocytomas) were selected to investigate the expression of FoxP3, CTLA-4, and IDO by immunohistochemistry and RT-qPCR (real-time quantitative polymerase chain reaction). All of the tested markers showed high gene and protein expression in oral melanomas and were differently expressed in cutaneous melanomas when compared to their benign counterpart. IDO expression was associated with an increased hazard of death both in univariable and multivariable analyses ( P < .05). FoxP3 protein expression >6.9 cells/HPF (high-power field) was an independent predictor of death ( P < .05). CTLA-4 gene and protein expressions were associated with a worse prognosis, but only in the univariable analysis ( P < .05). FoxP3, CTLA-4, and IDO likely play a role in canine melanoma immunoescape. Their expression, if supported by future studies, could represent a prognostic tool in canine melanoma and pave the way to future immunotherapeutic approaches in dogs.

Throughout gestation, changes in foetal umbilical cord Doppler parameters in ewes were detected. Doppler ultrasonography of the umbilical artery was performed weekly starting at 18 weeks before parturition until birth. In the foetal umbilical artery (UA), systolic peak velocity (SPV), end diastolic velocity (EDV), increased (p for within‐subjects effect

Inflammation of the uterus and oviduct is associated with reduced reproductive performance in humans and domestic animals. Toll-like receptors are expressed in various immune and non-immune cells and play a crucial role in innate immunity. Toll-like receptor - 4 (TLR4) can detect lipopolysaccharide (LPS) from Gram-negative bacteria leading to the secretion of pro-inflammatory cytokines, chemokines, antimicrobial peptides and other inflammatory mediators. To investigate the effects of a local inflammation on the expression levels of TLR4 and pro-inflammatory cytokines, 12 female rabbits received an intracervical infusion with either saline solution endotoxin-free (carrier, 2 mL; n = 6) or LPS (500 μg diluted in 2 mL of saline solution; n = 6). Blood samples were performed at 0, 30, 60 and 90 min and 2,4,6 and 24 h after treatment to evaluate interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) plasma concentrations. Animals were sacrificed 24 h post-treatment. The uterus and oviducts were immediately collected. The gene expression and protein levels of TLR4 and pro-inflammatory cytokines were detected by quantitative real-time PCR (qRT-PCR) and immuno-histochemical assay, respectively. Our study showed that the intracervical administration of LPS induced local inflammation given that the animals showed no clinical signs, the histological samples revealed signs of inflammation and plasma levels of pro-inflammatory cytokines were unchanged compared to the control. LPS produced an increase in the TLR4 mRNA expression levels in the uterus with respect to the control (P

Human melanoma is one of the deadliest forms of cancer, with poor prognosis and high resistance to chemotherapy and radiotherapy. The discovery of immunosuppressive mechanisms in the human melanoma microenvironment led to the use of new prognostic markers and to the development of immunotherapies targeting immune checkpoint molecules. Immunoescape mechanisms in canine melanoma have not yet been investigated, and no such immunotherapy has been tested. The aim of this study was to provide preliminary data on the expression of transcription factor forkhead box protein P3 (FoxP3) and indoleamine 2,3-dioxygenase (IDO) in primary canine melanocytic tumors and to investigate their prognostic role. Formalin-fixed, paraffin-embedded samples from 74 canine melanocytic tumors (26 oral melanomas, 23 cutaneous melanomas, and 25 cutaneous melanocytomas) were retrospectively evaluated by immunohistochemistry to explore the expression of FoxP3 and IDO. An increased risk of death due to melanoma was associated with a higher number of FoxP3+ cells per high-power field (FoxP3+/HPF), a higher percentage of CD3+ cells that were also FoxP3+ infiltrating and surrounding the tumor (%FoxP3), and a higher number of IDO+ cells/HPF (IDO+/HPF). A prognostic value for FoxP3 and IDO is suggested by our study, with optimal cutoffs of 14.7 FoxP3+ cells/HPF, 6.1 IDO+ cells/HPF, and 12.5% FoxP3+ cells. Both markers were also associated with tumor type. Multivariable analysis identified IDO+/HPF ( P < .001) as an independent prognostic marker. Even though stratification by diagnosis caused a loss of significance, results from the present study suggest a prognostic role for IDO and FoxP3, possibly related to the establishment of an immunosuppressive microenvironment.

Resveratrol, because of its low solubility in water and its high membrane permeability, is collocated in the second class of the biopharmaceutical classification system, with limited bioavailability due to its dissolution rate. Solid dispersion of resveratrol supported on Magnesium DiHydroxide (Resv@MDH) was evaluated to improve solubility and increase bioavailability of resveratrol. Fluorimetric microscopy analysis displays three types of microparticles with similar size: Type 1 that emitted preferably fluorescence at 445 nm with bandwidth of 50 nm, type 2 that emitted preferably fluorescence at 605 nm with bandwidth of 70 nm and type 3 that is non-fluorescent. Micronized pure resveratrol displays only microparticles type 1 whereas type 3 are associated to pure magnesium dihydroxide. Dissolution test in simulated gastric environment resveratrol derived from Resv@MDH in comparison to resveratrol alone displayed better solubility. A 3-fold increase of resveratrol bioavailability was observed after oral administration of 50 mg/kg of resveratrol from Resv@MDH in rabbits. We hypothesize that type 2 microparticles represent magnesium dihydroxide microparticles with a resveratrol shell and that they are responsible for the improved resveratrol solubility and bioavailability of Resv@MDH. © 2018 by the authors. Licensee MDPI, Basel, Switzerland.

Aims: Genetic diversity and antimicrobial resistance of Campylobacter coli and Campylobacter jejuni were investigated along the broiler chicken production chain in central Italy. Methods and Results: Campylobacter sp. isolated from cloacal swabs in farms (n=116) and from the neck skin of chilled and eviscerated carcasses at slaughter (n=24) were identified as C. coli (n=99) and C. jejuni (n=41) by multiplex PCR. Characterization by single amplified fragment length polymorphism (s-AFLP) revealed a specific genotype of Campylobacter for each farm. Minimal inhibitory concentration showed high prevalence of fluoroquinolones (70%), tetracycline (70%) and erythromycin (30%) resistance among C. coli isolates. Campylobacter jejuni isolates showed lower prevalence of fluoroquinolone (39%) and tetracycline (10%) resistance, and all isolates were susceptible to erythromycin. The S-AFLP types of the C. coli and C. jejuni isolates were associated with their antimicrobial resistance profiles (P&nbsp

Prostasome-like vesicles (PV) are components of the canine prostatic fluid during ejaculation and despite some enzymatic activities have been identified recently, their functions in the reproductive events in this species are still poorly understood. In this study, we evaluated at neutral or slightly alkaline pH, the effects of different concentrations of purified PV on sperm cell motility characteristics and the impact on the short- and long-term preservation of preserved semen maintained at room temperature. Two different experiments were performed. In the first experiment, purified PV were added at increasing concentration (1.25, 2.5, 5, 10, and 20 μL equivalent to 1, 2, 4, 8, and 16 μg of protein, respectively) to aliquots of 100 μL of preserved semen maintained at 22 °C at the following time points: 0, 30, 60, 120, 240, and 480 min. Computer-assisted sperm motility characteristics and pH were assessed three times at each time points, for each sample and for every concentrations. In the second experiment, the purified PV were added with the same methods as described above but only at time 0. Sperm motility characteristics and pH were assessed over the time. This study showed how the addition of purified PV to preserved semen affects negatively (p < 0.05) at neutral and alkaline pH, both total and progressive motility in a concentration depending manner. Furthermore, prostasome addition was demonstrated to change the quality of sperm movement which may represent a mechanism facilitating sperm cells attachment to the uterine epithelium and facilitating energy preservation before fertilization.

For the first time in literature this study describes the pregnancy-associated glycoprotein (PAG) profile of buffalo cows during gestation and the post-partum period using antiserum raised against PAG-molecules purified from buffalo placenta (AS#860). Ninety-eight buffalo cows, belonging to a buffalo herd subjected to a synchronization and artificial insemination (AI) program, were enrolled in this study. Blood samples were taken on days 0 (AI), 23, 25, 28, 30 and then biweekly until the end of pregnancy. Pregnancy was confirmed by ultrasonography on days 28 and 45, and by rectal palpation from day 60 onwards. Blood samples were suspended for the non-pregnant cows on day 45, while the blood of 20 buffaloes that had calved was tested every five days from the day of calving until day 50 post-calving. A cut-off value of 1.0 ng/mL was used in order to discriminate between pregnant and non-pregnant buffaloes. We used Linear Mixed models after Log(x+1) transformation to analyse the PAG concentrations. Fifty-two buffalo cows had become pregnant out of 98 synchronized (53%) and 46 remained non-pregnant (47%) as shown by ultrasonography and the PAG analysis. Significant differences (P < 0.001) in PAG concentrations were observed between the pregnant and non-pregnant buffaloes from day 23 as the PAG of the non-pregnant cows was always close to zero. Conversely, the PAG of the pregnant cows increased progressively from day AI until day 105 post-insemination and then stabilized until the end of pregnancy. Regarding pregnancy diagnosis, the sensitivity of PAG-RIA 860 system (ability of the test to correctly identify pregnant buffalo) ranged from 23% on day 23–98% on day 28 post AI; the specificity (ability to correctly identify non-pregnant buffaloes) was 100% throughout the sampling period. PAG progressively decreased from parturition to day 25 post-partum; from day 30 post-partum, the concentrations fell below 1 ng/mL and were close to 0 on the last day of observation (50 d post-partum). In conclusion, our results showed that RIA-860 is highly accurate for diagnosing pregnancy in buffaloes starting from day 28 of gestation. Furthermore, the rapid disappearance of PAG concentration after calving means that a cut-off limit in post-partum for detecting a new pregnancy is not required.

This study investigates for the first time mRNA pregnancy-associated glycoprotein 2 (PAG-2) expression in blood cells during early pregnancy in water buffalo. The PAGs constitute a large family of glycoproteins expressed in the outer epithelial layer of the placenta in eutherian species. All PAGs are not concomitantly expressed throughout pregnancy; some of them are expressed in the earlier phases, whereas others appear later and are expressed over a shorter period. Twenty-one lactating buffaloes were analyzed—17 females were synchronized with PRID and artificially inseminated (AI), whereas four females were synchronized but not inseminated (control group). Blood was collected at Days 0, 18, 28, 40, and 75 from AI (AI = Day 0). Expression of PAG-2 mRNA in blood samples was measured with real-time polymerase chain reaction. Pregnancy diagnosis was performed on Day 28 (D28) and Day 40 (D40) after AI by ultrasonography (US) and by PAG-1 RIA method. The females diagnosed pregnant at D28 and confirmed pregnant at D40 were defined as D28(+)D40(+) group; the females diagnosed pregnant at D28 but not confirmed pregnant at D40 were defined as D28(+)D40(−) group; and the females that were diagnosed as nonpregnant on either days were defined as D28(−)D40(−) group. PAG-2 mRNA at Day 0 was not observed in any groups. The D28(+)D40(+) group showed the highest expression, starting on Day 18 and increasing progressively up to Day 75. PAG-2 mRNA was also expressed on Day 18 in both D28(+)D40(−) and D28(−)D40(−) groups, but their levels were lower than those of D28(+)D40(+) group and almost constant over time. PAG-2 mRNA was never detected in the control group. The significant difference in the expression of PAG-2 mRNA between the D28(+)D40(+) group and the D28(−)D40(−) group, starting from Day 18, suggests that these animals might have conceived, but have experienced early embryonic loss; therefore, the PAG-2 mRNA was still present in blood circulation although at lower levels, as found in the D28(+)D40(−) group. In conclusion, this study shows that PAG-2 mRNA can be detected in peripheral maternal blood cells earlier than circulating PAG-1 molecules and could be useful for studies on early pregnancy and embryonic mortality.

Choroid plexus tumors (CPTs) are reported with an increasing incidence in dogs, and they call for a reexamination of histologic features and criteria of classification corresponding to their biological behavior. In this study, the human World Health Organization classification was applied to 16 canine CPTs, and the expression of molecules involved in neoplastic cell adhesion (E-cadherin, N-cadherin), invasion (doublecortin), and proliferation (Ki-67) was investigated. Mitotic index was found to be the main criterion for grading CPTs. Cell density and multilayering of papillae were also statistically associated with histologic grade. Intraventricular spread and parenchymal invasion was observed for tumors showing histologic benign features. E-cadherin was expressed in all CPT grades, independent of tumor invasion. N-cadherin immunolabeling was more expressed in grade I than high-grade CPTs, whereas doublecortin expression was not detected in CPTs. An increasing proliferative activity was observed in relation with histologic grade.

The aim of this study was to investigate a new approach for equine maxillary nerve blocks, which can facilitate several orofacial surgeries. Current techniques aim at the maxillary foramen and approach via the zygomatic arch, conferring the risk of injury to several delicate structures in the target area. To investigate the feasibility of a retrograde approach from the infraorbital foramen, an anatomic study of the infraorbital canal and its surrounding structures was performed on 13 cadaveric skulls using computed tomography and anatomical dissection. Measurements included canal length and volume, its conformation and relationship with the enclosed structures, and infraorbital foramen diameters. The technical approach to simulate the distribution of local anaesthetic within the infraorbital canal was further defined, including needle selection among seven different needles, evaluating ease of insertion, trauma to surrounding tissues and spread of contrast medium toward the target area. To validate the technique, two Tuohy needles were randomly inserted at 12 infraorbital foramina and 10 mL of contrast medium was injected. CT verified the spread of the solution and possible complications. Each canal had a serpentine-curved pathway. Anatomical dissections evidenced gaps between the infraorbital nerve, vessels and the infraorbital canal. The integrity of these structures had been preserved from the passage of the selected Tuohy needles. This study suggests a feasible approach to the maxillary nerve block within the infraorbital canal providing an appropriate needle selection and technique to limit complications.

International audience; A retrospective study was used to investigate the incidence of prostatic diseases in a large population of dogs at Alfort Veterinary Hospital and to clarify epidemiologic features, which might be of a great help to veterinarians in managing and discriminating prostatic disorders. During the investigation period, a total of 72,300 male dogs (coming mainly from the Ile-de-France region) were registered in the Alfort Veterinary College database, and 481 of them (0.7%) were found to have prostatic disorder. The diagnosis was carried out on the basis of clinical signs and ultrasound findings. Among dogs experiencing a prostatic disorder, most frequently recorded diseases were benign prostatic hyperplasia (45.9%) and prostatitis (38.5%), followed by abscesses (7.7%), cysts (5.0%), neoplasia (2.6%), and squamous metaplasia (0.2%). Our study revealed an incidence of 0.3% of prostatic disorders observed in intact male dogs, except in the case of prostatic neoplasia. The mean age of the dogs experiencing prostatic disorders was 8.6 +/- 3.2 years. This was significantly different (P < 0.001). Large dogs were significantly more affected by prostatic disorders (P < 0.05), except for prostatic neoplasia. A breed predisposition was suspected in German Shepherd (odds ratio [OR] = 2.1; 95% confidence interval [CI]: 1.5-2.9), Rottweiler (OR = 1.8; 95% CI: 1.2-2.7), American Staffordshire Terrier (OR = 3.8; 95% CI: 2.5-5.8), Berger de Beauce (OR = 3.7; 95% CI: 2.2-6.1), and Bernese Mountain Dog (OR = 2.5; 95% CI: 1.3-4.7).

The work was carried out on a total of 26 male dogs that on the basis of clinical examination, prostate ultrasound and prostate biopsy, were divided prospectively into four groups: (1) normal dogs (control group; n ¼ 8); (2) dogs with benign prostatic hyperplasia (group BPH; n ¼ 8); (3) dogs suffering from prostatitis (group prostatitis; n ¼ 4); (4) dogs with prostatic tumors (group tumors; n ¼ 6). The examination of the prostate bymeans of contrast medium and dedicated ultrasound system allowed a detailed qualitative and quantitative analysis of prostatic vessels in normal and diseased conditions, enabling the detection and characterization of different disease states, and quantification of parameters such as peak intensity of perfusion (%), arrival time of the contrast medium to its maximum value of video intensity (time to peak [TTP; seconds]), regional blood volume, regional blood flow, and mean transit time (MTT [seconds]). The hemodynamic indices TTP (P < 0.01) and MTT (P < 0.001) of diseased prostate groups were significantly lower than those in the normal prostate group although there were no differences among diseases. Optimal cutoff values were 31 seconds (Sensitivity: 72%; Specificity: 88%) and 47 seconds (Sensitivity: 100%; Specificity: 88%) while area under receiver operating characteristic curveswere 0.86 (P< 0.01) and 0.97 (P

Systemic and local infections and inflammations are known to cause infertility in humans and animals. However, the mechanisms by which infection/inflammation induces infertility are only partially known. The objectives of this study were: (i) to provide models of systemic (acute) and local (sub-acute) inflammation by intra-peritoneal injection or intra-cervical deposition of lipopolysaccharide (LPS) in the rabbit and (ii) to assess their effects on uterine tissues and sperm transport in the genital tract of rabbit does. Intra-peritoneal administration of different doses of LPS induced systemic effects such as fever, anorexia and changes in white blood cells (WBC) count. In our study, LPS inoculation (100μg/kg) produced an inflammation-like status that lasted for about 3 days, with minimal distress for the animals. Intra-peritoneal administration of LPS 60h before artificial insemination induced a rapid increase of IL-1β concentrations. The intra-cervical inoculation of LPS did not show any systemic effects, as confirmed by the lack of changes in body temperature, feed intake and WBC count. Histological examination of uterine tissues showed an endometritis-like inflammation status in LPS-treated does, more severe in those inoculated intra-cervically. The number of spermatozoa recovered from uterine horns and oviducts of intra-cervically treated does was less than that retrieved from intra-peritoneally treated animals and controls. These results suggest (i) that sub-acute or acute inflammation may cause infertility by compromising the uterine environment and/or impairing sperm transport and (ii) that the LPS-induced -infection/inflammation experimental model is useful for studying the mechanisms involved in reproductive dysfunctions in the rabbit.

Inflammatory bowel diseases (IBD) are characterized by inflammatory conditions of the intestine. Probiotic bacteria (PB) can have beneficial effects in several gastrointestinal disorders. The objectives of this study were: (i) to provide an acute experimental IBD model induced by 2,4,6-trinitrobenzenesulfonic acid (TNBS) in CD-1 mice, and (ii) to assess the preventive effects of Citogenex (Lactobacillus casei and Bifidobacterum lactis) supplementation on intestinal tissues and microbiota. Mice were inoculated intrarectally with saline, ethanol or different TNBS solutions. 1%TNBS induced clinical signs of colitis (P less than 0.01) and histological damage (P less than 0.01). Based on these results, mice were pre-treated with Citogenex or saline for 1, 2 or 3 weeks before 1%TNBS treatment. Probiotic pre-treatment determined a reduction of clinical signs (P less than 0.05), histological alterations of colitis (P less than 0.05) and increased beneficial bacteria (P less than 0.05). This study confirms that TNBS-induced colitis in CD-1 mice is useful for studying the mechanisms involved in IBD pathogenesis, and pre-treatment with Citogenex prevents the intestinal damage induced by TNBS.

This paper reviews the composition of colostrum and the potential preventive and therapeutic use of this "first milk" for treating various gastrointestinal disorders in humans. Colostrum is a complex biological liquid that is richer in antimicrobial peptides, immune-regulating compounds and growth factors than the subsequent mature milk. The main functions of colostrum are to provide essential nutritional components, strengthen the natural defense system, modulate immune response, balance intestinal microbiota and enhance the growth and repair of several tissues. Several studies and clinical trials carried out both in vitro and in vivo on humans and animals suggest the clinical benefits of bovine colostrum (BC) supplementation in gastro-intestinal diseases. Despite the encouraging results, further well-designed studies are required in order to confirm these effects, the dose and duration of treatment. Colostrum is safe since there are no contraindications regarding high dose levels and few side effects of clinical relevance have been reported. In conclusion, in the near future, colostrum-based supplements may play a complementary role to synthetic drugs in the prevention and treatment of various gastrointestinal disorders.