Effects of the supplementation with an high-polyphenols extra-virgin olive oil on kinetic sperm features and seminal plasma oxidative status in healthy dogs.

The aim of this work was to evaluate the effects of the supplementation of two extra-virgin olive oils (EVOO) having different polyphenols content, on canine spermatozoa kinetic parameters and seminal plasma oxidative status. The study was conducted on 12 clinically healthy dogs of different breeds (2-7 years, 5-48 kg of body weight) divided into two groups: an experimental group supplemented with EVOO (Coratina cultivar) high in polyphenols (H-P) and a control group fed EVOO (Cima di Bitonto cultivar) low in polyphenols (L-P). The oil was daily administered per os (1 ml/3 kg BW) before meal. Semen collection was made twice at 15 days distance (D01 and D02 ) and then at 30 (D30), 60 (D60) and 90 (D90) days. Semen concentration and kinetic parameters were measured using computer-assisted sperm analysis (CASA) system to evaluate: sperm total count, sperm motile (MOT%), progressive motility (PROGR%) and its fractions, straight-line velocity (VSL, µm/s), curvilinear velocity (VCL, µm/s), average path velocity (VAP, µm/s), amplitude of lateral head displacement (ALH, µm), beat cross frequency (BCF, Hz), straightness (STR%) and linearity (LIN%). On seminal plasma, reactive oxygen species (ROS) and biological antioxidant potential (BAP) were tested. From findings, no differences were found for sperm MOT, VSL, VCL, VAP, ALH, BCF, STR, LIN and BAP. A gradual enhancement of PROGR% was observed in H-P group (p < .01). The ROS levels were higher in dogs H-P compared to the other group (p < .05). In conclusion, our results highlight the positive effects of EVOO polyphenols on sperm PROGR% in healthy dogs.

Rationale, design and methods of CTCA-PRORECAD (Computed Tomography Coronary Angiography Prognostic Registry for Coronary Artery Disease): a multicentre and multivendor registry.

PURPOSE: This study was done to assess the prognostic value of computed tomography coronary angiography (CTCA) in a large multicentre population of patients with suspected coronary artery disease (CAD) and, in particular, its incremental value compared with traditional methods for risk stratification. MATERIALS AND METHODS: This is a retrospective observational study that began in January 2003 conducted on patients with suspected CAD assessed with CTCA on the basis of symptoms (chest pain, dyspnoea) and/or abnormal or equivocal stress test and/or a high cardiovascular risk profile. The participating centres will provide data obtained with CTCA performed with 16-slice or higher equipment. Exclusion criteria are renal insufficiency, allergy to iodinated contrast material, pregnancy and previous myocardial infarction or revascularisation (percutaneous coronary intervention and/or coronary artery bypass graft). All patients are stratified by means of clinical assessment and/or data retrieved from a clinical database. Risk factors considered are hypertension, dyslipidaemia, diabetes mellitus, smoking, family history and obesity. Symptoms are classified as absent, typical chest pain, atypical chest pain and dyspnoea. Primary endpoints are death, major adverse cardiovascular events (cardiac death, unstable angina requiring hospitalisation, acute myocardial infarction) and shifting of cardiovascular risk category on the basis of coronary plaque burden. The secondary endpoint is coronary revascularisation. Telephone interviews and/or clinical databases are used for the follow-up. The study will be conducted on a population >1,000 patients. CONCLUSIONS: The information collected from the Prognostic Registry for Coronary Artery Disease (PRORECAD) will provide insight into the prognostic value of CTCA in addition to demographic and clinical features. The results will allow for better use and interpretation of CTCA for prognostic purposes.

Computed tomography coronary angiography in the selection of outlier patients: a feasibility report.

PURPOSE: This study evaluated criteria, presence and distribution of outlier patients by means of computed tomography coronary angiography (CTCA) in a large institutional database. MATERIAL AND METHODS: From a population of 2,881 consecutive patients (1,842 men, mean age 62 ± 13 years) in sinus rhythm who underwent CTCA, we extracted data on patients with suspected coronary artery disease (CAD). We selected patient outliers in the fifth and sixth decades of life with the following criteria: ≥ 3 risk factors and absence of CAD, zero to one risk factors and ≥ 5 diseased coronary segments. Diabetes was excluded from risk factors because of the different impact on CAD. RESULTS: The patient population consisted of 2,432 individuals with suspected CAD (1,495 men, age 62 ± 13 years). The prevalence of obstructive CAD (≥ 50% lumen reduction at CTCA) was 36% (863/2,432). Patients with normal coronary arteries accounted for 34% of the total (837/2,432; 431 men, age 55 ± 14 years). Of these, 210 were in the fifth and 231 in the 6th decade (men 196, women 245); those with ≥ 3 risk factors accounted for 4.2% of the total (102/2,432; men 42, women 60). Patients with ≥ 5 diseased coronary segments accounted for 28% of the total (686/2,432; 510 men, age 68 ± 10 years). Of these, 115 were in the fifth and 270 in the sixth decade (men 309, women 76); those with zero to one risk factors accounted for 3.0% (73/2,432; men 66, women 7). CONCLUSIONS: CTCA is a reliable noninvasive diagnostic modality that can be used to identify outlier patients. This will enable dedicated trials aimed at characterising biomarkers and genomics of protective and nonprotective factors against CAD and its complications.

Diagnostic accuracy of 64-slice computed tomography coronary angiography in a large population of patients without revascularisation: registry data on the comparison between male and female population.

PURPOSE: This study was undertaken to evaluate the diagnostic accuracy of computed tomography coronary angiography (CTCA) for detecting significant coronary artery stenosis (≥50% lumen reduction) compared with conventional coronary angiography (CAG) in a male and female population. MATERIAL AND METHODS: A total of 1,372 patients (882 men, 490 women; mean age 59.3 ± 11.9 years) in sinus rhythm imaged with CTCA (64-slice technology) and CAG were enrolled. Diagnostic accuracy and likelihood ratios (LR+ and LR-) of CTCA were assessed against CAG for the male and female populations. RESULTS: The prevalence of obstructive disease was 53% (men 58%; women 43%). CAG demonstrated the absence of significant coronary artery disease (CAD) in 47% (men 42%; women 56%), single-vessel disease in 25% (men 36%; women 22%) and multivessel disease in 29% (men 32%; women 23%) of patients. In the per-patient analysis, sensitivity, specificity and positive (PPV) and negative (NPV) predictive values of CTCA were 99% (men 98%; women 100%), 92% (men 92%; women 92%), 94% (men 95%; women 90%) and 99% (men 98%; women 100%), respectively. The per-patient likelihood ratios (LR) in the total population (LR+=12.4 and LR-=0.011), the male (LR+=12.9 and LR-=0.016) and female (LR =11.9 and LR-=0) populations were very good. We observed no significant differences in diagnostic accuracy between male and female populations. CONCLUSIONS: CTCA is a reliable diagnostic modality with high sensitivity and NPV in the female population.

Diagnostic accuracy of 64-slice computed tomography coronary angiography in a large population of patients without revascularisation: registry data in NSTEMI acute coronary syndrome and influence of gender and risk factors.

PURPOSE: This study sought to evaluate the diagnostic accuracy of computed tomography coronary angiography (CTCA) for detecting significant coronary artery stenosis (≥50% lumen reduction) compared with conventional coronary angiography (CAG) in non-ST-elevation myocardial infarction-acute coronary syndrome (NSTEMI-ACS) and in subgroups selected by gender and number of risk factors (RF). MATERIALS AND METHODS: We selected from a population of 1,500 patients in a multicentre registry with NSTEMI-ACS who had undergone CTCA and CAG, (n=237; 187 men, mean age 63±10 years). Diagnostic accuracy and likelihood ratios (LR) of CTCA were assessed against CAG in the total population and subgroups (men, women: 0 RF = absence of RF, 1-2 RF = presence of one or two RF, >2 RF = presence of more than two RF). RESULTS: The prevalence of obstructive disease was 53%. In the per-patient analysis, sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of CTCA were 100% (men 100%; women 100%; 0 RF 100%; 1-2 RF 100%; >2 RF 100%), 95% (men 98%; women 50%; 0 RF NA% (NA, not assessable); 1-2 RF 96%; >2 RF 96%), 95% (men 98%; women 91%; 0 RF 91%; 1-2 RF 96%; >2 RF 96%), 100% (men 100%; women 100%; 0 RF NV%; 1-2 RF 100%; >2 RF 100%), respectively. The per-segment analysis showed a reduction in PPV (ranging between 56% and 67%). The per-patient LR+ ranged between 18 and 27, whereas LR-were always 0. We observed no significant differences in diagnostic accuracy between subgroups. CONCLUSIONS: CTCA is a reliable diagnostic modality with high sensitivity and NPV in NSTEMI-ACS patients who are not candidates for early revascularisation, regardless of gender and number of risk factors.

Isolation, proliferation, cytogenetic, and molecular characterization and in vitro differentiation potency of canine stem cells from foetal adnexa: a comparative study of amniotic fluid, amnion, and umbilical cord matrix.

The possibility to isolate canine mesenchymal stem cells (MSCs) from foetal adnexa is interesting since several canine genetic disorders are reported to resemble similar dysfunctions in humans. In this study, we successfully isolated, cytogenetically and molecularly characterized, and followed the differentiation potency of canine MSCs from foetal adnexa, such as amniotic fluid (AF), amniotic membrane (AM), and umbilical cord matrix (UCM). In the three types of cell lines, the morphology of proliferating cells typically appeared fibroblast-like, and the population doubling time (DT) significantly increased with passage number. For AF- and AM-MSCs, cell viability did not change with passages. In UCM-MSCs, cell viability remained at approximately constant levels up to P6 and significantly decreased from P7 (P < 0.05). Amnion and UCM-MSCs expressed embryonic and MSC markers, such as Oct-4 CD44, CD184, and CD29, whereas AF-MSCs expressed Oct-4, CD44. Expression of the hematopoietic markers CD34 and CD45 was not found. Dog leucocyte antigens (DLA-DRA1 and DLA-79) were expressed only in AF-MSCs at P1. Isolated cells of the three cell lines at P3 showed multipotent capacity, and differentiated in vitro into neurocyte, adipocyte, osteocyte, and chondrocyte, as demonstrated by specific stains and expression of molecular markers. Cells at P4 showed normal chromosomal number, structure, and telomerase activity. These results demonstrate that, in dog, MSCs can be successfully isolated from foetal adnexa and grown in vitro. Their proven stemness and chromosomal stability indicated that MSCs could be used as a model to study stem cell biology and have an application in therapeutic programs.

Diagnostic accuracy of 64-slice computed tomography coronary angiography in a large population of patients without revascularisation: registry data on the impact of calcium score.

PURPOSE: This study evaluated the diagnostic accuracy of computed tomography coronary angiography (CTCA) for detecting significant coronary artery stenosis (≥50% lumen reduction) at different coronary calcium score (CACS) values with conventional coronary angiography (CAG) as the reference standard. MATERIAL AND METHODS: A total of 1,500 patients (928 men, mean age 58.2±12.5 years) in sinus rhythm who underwent CTCA (64-slice technology) and CAG were enrolled. Diagnostic accuracy and likelihood ratios (LR) of CTCA were evaluated against CAG for the total population and in different CACS classes (0; 1-10; 11-100; 101-400; 401-1,000; >1,000). RESULTS: The prevalence of obstructive disease was 51% (23.5% single vessel; 27.5% multivessel; progressive increase from 17.9% to 94% through the CACS classes). In the per-patient analysis, sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of CTCA were 99%, 92%, 94% and 99%, respectively. Per-patient analysis showed a worse PPV of CTCA (76-77%) in classes with low CACS (1-10/11-100). Per-patient LR were higher in classes with extreme CACS values (0 = LR+ 18.3 and LR- = 0.0; c1,000 = LR+ 17.0 and LR- = 0.0) with values always >7 for LR+ and <0.033 for LR- for all CACS classes. CONCLUSIONS: CTCA is a reliable diagnostic modality, with high sensitivity and NPV regardless of CACS.

Prognostic value of computed tomography coronary angiography in patients with chest pain of suspected cardiac origin.

PURPOSE: The authors sought to determine the prognostic value of computed tomography coronary angiography (CTCA) in patients with acute chest pain (ACP). MATERIALS AND METHODS: A total of 145 consecutive patients (75 men; 64±12 years) with ACP were referred from the Emergency Department for CTCA, which was performed with a standard protocol using a 64-slice scanner. Patients were stratified according to the Morise clinical score (low, intermediate, high) and to the CTCA findings [absence of coronary artery disease (CAD), nonobstructive CAD, obstructive CAD]. Patients were followed up for the occurrence of major events: cardiac death, nonfatal myocardial infarction, unstable angina and revascularisation. RESULTS: One hundred and twenty-seven (87.6%) patients were without a history of CAD, and 18 (12.4%) patients had a history of CAD. Obstructive CAD (>50% luminal narrowing) was detected in 35 (24%) patients; nonobstructive CAD (≤ 50% luminal narrowing) in 62 (43%) and absence of CAD in 48 (33%) patients. During a mean follow-up of 20 ± 3 months, 20 events occurred (four hard events). Sixteen events (three hard events) occurred in patients without a history of CAD, and four events (one hard event) occurred in patients with a history of CAD. In patients with absence of CAD as detected by CTCA, the rate of events was 0%. At multivariate analysis, hypercholesterolaemia and obstructive CAD were significant predictors of events (p<0.05). CONCLUSIONS: An excellent prognosis was observed in patients with ACP and normal CTCA. CTCA shows the potential for optimal stratification of patients with ACP.

Low dose CT of the heart: a quantum leap into a new era of cardiovascular imaging.

In 10 years, computed tomography coronary angiography (CTCA) has shifted from an investigational tool to clinical reality. Even though CT technologies are very advanced and widely available, a large body of evidence supporting the clinical role of CTCA is missing. The reason is that the speed of technological development has outpaced the ability of the scientific community to demonstrate the clinical utility of the technique. In addition, with each new CT generation, there is a further broadening of actual and potential applications. In this review we examine the state of the art on CTCA. In particular, we focus on issues concerning technological development, radiation dose, implementation, training and organisation.

Changes in the expression of the miu-opioid receptor in the mare oviduct during oestrus and anoestrus.

The presence of the mu-opioid receptor (MOR) was investigated in the mare oviduct during oestrus and anoestrus, by means of immunoblotting and immunohistochemistry. Immunoblotting analysis showed that the MOR protein is expressed as 65, 50 and 30 kDa forms in the infundibulum and ampulla both in oestrus and anoestrus, while the 30 kDa form is absent in the isthmus. Moreover, different levels of expression were observed along the ampulla in the two periods examined. Immunohistochemistry revealed MOR in the mucosal epithelium, stromal cells, myocytes and blood vessels. Ciliated cells expressed MOR in the apical cytoplasm and, except for the isthmus of oestrous mares, also in the nucleus. Non-ciliated cells showed MOR only in the isthmus segment during oestrus. Stromal cells showed different immunoreactivity along the oviduct segments and during the oestrous and anoestrous phases. The myosalpinx displayed immunostained myocytes in the intrinsic musculature of the ampulla and in the extrinsic and intrinsic musculature of the isthmus without significant differences between anoestrus and oestrus. Blood vessels expressed MOR in endothelial cells and smooth muscle cells in the isthmus myosalpinx of oestrous mares only. In conclusion, these findings show diverse MOR expression in the three segments constituting the oviduct, as well as changes in MOR expression linked to the mare's physiological condition.

Expression of the micro-opioid receptor on Malassezia pachydermatis and its effect in modulating phospholipase production.

Malassezia spp. may act as opportunistic skin pathogens in humans and animals. Malassezia pachydermatis proliferation and phospholipase production may play a pathogenic role in the occurrence of skin lesions in dogs. This study investigates the presence of mu-opioid receptor (MOR) in M. pachydermatis strains isolated from healthy dogs and dogs with skin lesions and its effects on phospholipase activity (p.a.). P.a. of 64 M. pachydermatis isolates was evaluated using different concentrations of naloxone (Nx), a MOR antagonist. Isolates were divided into Group A (i.e., 40 isolates from 26 dogs with dermatitis) and Group B (i.e., 24 isolates from 12 healthy dogs). The MOR expression was analyzed by Western blot and immunofluorescence. A statistically higher p.a. than that of the controls was found with isolates in Group A at a Nx concentration of 10(-6) M (P<0.05). No isolate in Group B displayed p.a. in either control samples or in the presence of any Nx concentration. Immunoblotting revealed two positive MOR immunoreactive bands of approximately 65 and 98 kDa. MOR expression and localization was also demonstrated by immunofluorescence in isolates from Groups A and B. This study provides the first evidence of MOR expression on M. pachydermatis cell membranes pointing to its possible role in modulating p.a. production in isolates from dogs with skin lesions.

Expression of the mu opioid receptor and effects of the opioid antagonist naloxone on in vitro maturation of oocytes recovered from anoestrous bitches.

The mu-opioid receptor (MOR) is expressed in bovine, human, equine and canine oocytes, and in seasonal breeders, it is expressed with higher intensity during the anoestrous phase. Supplementation of in vitro maturation (IVM) medium with opioid agents, agonists or antagonists, was shown to affect oocyte maturation in several species such as rat, bovine and equine. This study reports the effects of supplementing IVM medium with naloxone (Nx), an opioid antagonist, on nuclear and cytoplasmic maturation rate of oocytes recovered from anoestrous bitches. Cytoplasmic maturation was examined in terms of mitochondrial (mt) distribution. In order to confirm the receptor-mediated action of Nx, in oocytes of anoestrous bitches, MOR expression was analyzed by Western blot. Cumulus-oocyte complexes, recovered from the ovaries of bitches in anoestrous, were cultured in vitro and Nx was added at the concentrations of 1 x 10(-6), 1 x 10(-8) and 1 x 10(-10) M. The rate of oocytes resuming meiosis after culture in presence of 1 x 10(-6) M Nx (29%) was significantly higher than that of oocytes of control group (12%; p < 0.05). However, treatment with Nx did not affect mt distribution pattern. In denuded oocytes and in corresponding cumulus cells, a doublet of 65 and 50 kDa was observed. We conclude that, in oocytes of anoestrous bitches, MOR is expressed and Nx significantly improves nuclear maturation rate. Further studies should be performed to elucidate the expression of other opioid receptors, such as delta and kappa, and possible interactive effects of their antagonists on canine oocyte maturation.

Clinical applications of multi-detector computed tomography coronary angiography.

Cardiac computed tomography (CT) is a fast developing technique. In 10 years it developed from investigative tool into a clinical reality. The technology drive has been the key to success for this technique which is to date the only non invasive clinical tool for coronary angiographic assessment. Technical background is quite complex and the newer solutions are aiming at reducing the scan time and the radiation dose while improving temporal resolution, contrast resolution and ultimately image quality. The key technical development has been in the late 1990, the introduction of ECG triggering/gating techniques. Spatial resolution has also been improved reaching sub-millimeter performance. The latest innovations provide fast coverage with >64 slice detectors, high spatial resolution with 0.5 mm slice thickness, high temporal resolution with <100 ms in hardware, and higher contrast resolution with the forthcoming dual-energy solutions. Recently, also dose issues have been exploited and current state of the art technology are able to perform cardiac CT with 1-2 mSv. These improvements put cardiac CT as the most promising clinical tool for the non invasive assessment of coronary arteries.

DNA sexing in Humboldt Penguins (Spheniscus humboldti) from feather samples.

Humboldt Penguins (Spheniscus humboldti) show little sexual dimorphism, and although males are usually heavier and larger than females, sexing by direct observation may be difficult, especially in young subjects. In this paper we evaluate the utility of the molecular approach, for sexing impuberal Humboldt Penguins from feathers. Firstly, a PCR test was used employing primers that amplify the homologous region of the CHD-W gene, unique in female, and the CHD-Z gene, occurring in the two sexes. The analysis of the PCR products showed a band of 370 bp in males and two bands of 370 and 380 bp in females. Additionally, to confirm these results, the PCR products were digested with HaeIII and Asp700 for RFLP analysis. Male PCR products showed two bands (310 and 60 bp) after digestion with HaeIII, and a unique band (370 bp) using Asp700, while all fragments obtained from females resolved into three bands using both HaeIII (380, 310 and 60 bp) and Asp700 (370, 270 and 110 bp), confirming the previous PCR sex determination. Results from these two different DNA-based tests were in accordance, in all cases, with sexes checked by preliminary cloacoscopy. Thus, it was found that the PCR method from feather samples alone is sufficient, reliable and without any risks for a rapid sexing in Humboldt Penguin. This non-invasive sexing technique can be useful at any age to verify the sex ratio in field populations and for gender identification in ex situ conservation programs.

The presence of the mu-opioid receptor in the isthmus of mare oviduct.

The presence of the mu-opioid receptor and the type of glycosylation in the third extra-cellular loop of this receptor was investigated in the isthmus of mare oviduct during oestrus by means of immunoblotting and immunohistochemistry combined with enzymatic (N-glycosidase F and O-glycosidase) and chemical (beta-elimination) treatments. Immunoblotting analysis showed that the mu-opioid receptor consists of two peptides with molecular weights of around 65 and 50 kDa. After N-deglycosylation with N-glycosidase F an additional immunoreactive peptide was observed at around 30 KDa. The cleavage of O-glycans by O-glycosidase failed in immunoblotting as well as in immunohistochemistry investigations, revealing that the third extra-cellular loop of the mu-opioid receptor expressed in mare isthmus oviduct contains some modifications of the Galbeta(1-3)GalNAc core binding to serine or threonine. Immunohistochemistry revealed the mu-opioid receptor in the mucosal epithelium, some stromal cells, muscle cells and blood vessels. In ciliated cells the mu-opioid receptor showed N-linked glycans, since the immunoreactivity was abolished after N-glycosidase F treatment, whereas it was preserved in the apical region after beta-elimination. Most non-ciliated cells expressed the mu-opioid receptor with both N- and O-linked oligosaccharides, as revealed by the abolition of immunostaining after N-glycosidase F and beta-elimination. Stromal cells, endothelial and muscle cells of blood vessels expressed the mu-opioid receptor containing both N- and O-linked oligosaccharides. Myosalpinx myocytes expressed the mu-opioid receptor with O-linked oligosaccharides. The immunopositive myocytes formed a circular coat in the intrinsic musculature, whereas they were arranged in some isolated, oblique bundles in the extrinsic musculature. In conclusion, the mu-opioid receptor could have a role in the production and the movement of isthmus lumen content that contributes to ensuring the effective condition of the sperm in the mare oviduct.

Opioid agonist/antagonist effect of naloxone in modulating rabbit jejunum contractility in vitro.

Opioid peptides are the most effective drugs in controlling pain; their action is elicited by binding to specific membrane receptors. The gastrointestinal tract represents, after the nervous system, the site in which the opioid receptors are expressed at high levels. The opioid agonist morphine has a significant inhibitory effect on intestinal motility, this action is blocked by naloxone an opioid antagonist mainly active at mu and kappa receptors. In this study the presence of mu opioid receptor on rabbit jejunum was investigated by western blot. The effects of beta-endorphin, the endogenous opioid peptide with the highest affinity to the mu opioid receptor and those of naloxone on spontaneous rabbit jejunum contractions were evaluated. Beta-endorphin (10(-6) M) showed a relaxant effect on jejunum contractility while naloxone showed a dual effect inducing an increase of spontaneous contractility at low concentrations (10(-6) M, 10(-7) M, 10(-8) M) and a decrease when high concentrations (10(-3) M, 10(-4) M, 10(-5) M) were utilized. The obtained results demonstrate that mu opioid receptor is expressed in rabbit jejunum and suggest that this receptor may be involved in mediating the effects of both opioid agonist and antagonist on jejunum contractions.