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OBJECTIVES: To evaluate the effectiveness of a prevention programme against the vertical transmission of HIV in a resource-limited setting and to investigate variables associated with compliance. PATIENTS AND METHODS: The Kento-Mwana project (2005-2008) provided counselling, serological and biomolecular testing and prophylaxis/therapy to HIV-positive pregnant women and their children attending four antenatal clinics in Pointe Noire, Republic of Congo. Expected and actual rates of vertical transmission of HIV were compared. Univariate and multivariate analyses were performed in order to identify variables associated with non-compliance. RESULTS: The observed transmission rate in the group who completed follow-up was 5/290 (1.7%, 95% CI 0.6%-4.1%). The overall estimated transmission rate in the target population, computed taking into account the expected vertical transmission of HIV among drop-outs, was 67-115/638 (10.5%-18.0%). A comparison between this rate and the expected transmission rate in the absence of intervention (25%-40%) showed that the programme was able to prevent approximately 50% of vertical transmissions. Older age (OR 0.33, 95% CI 0.16-0.66, P = 0.002), telephone availability (OR 0.42, 95% CI 0.24-0.72, P = 0.002) and occupation (OR 0.57, 95% CI 0.29-1.10, P = 0.092) were associated with better compliance. CONCLUSIONS: Despite the vast majority of women accepting counselling and testing, many of them refused prophylaxis or dropped out, thus reducing the effectiveness of the intervention from an ideal 2% to a still important but less impressive median transmission rate of 15% (range 10.5%-18%). Promoting participation and compliance, rather than increasing the potency of antiretroviral regimens, is crucial for preventing the vertical transmission of HIV in Africa

Migrants account for approximately 8.7 % of the resident population in Italy. The immigration status deeply influences access to prevention and care, thus contributing to increase the burden of HIV/AIDS among such a fragile category. The aim of this study was to investigate socio-demographic and baseline clinical and immunological features of HIV-infected migrants, as compared to Italians. We retrospectively analysed data for all the 1,611 HIV-infected migrant patients and a random sample of 4,230 HIV-infected Italian patients aged 18 or older who first accessed nine Italian clinical centres in 2000–2010 and were followed up at least 1 year. Differences in baseline characteristics between migrants and Italians were evaluated in univariate analysis, while factors associated with late presentation were evaluated in multivariate analysis using logistic regression models. The baseline profile differs between the HIV-infected migrant and Italian patients, substantially reflecting what reported by current statistics in terms of gender, age, risk category as well as clinical features. Late presenters were more frequent among migrants as compared to Italians (53.0 vs 45.8 %; adjusted odds ratio [(AOR) = 1.55, 95 % confidence interval (CI) 1.34–1.78]. Other factors associated with late presentation included increasing age, as well as undocumented legal status among foreign-born subjects (AOR = 1.41, 95 % CI 0.97–2.04), though of borderline significance. Late presentation still represents a relevant problem despite the advances in the management of HIV infection. More efforts are needed to allow early diagnosis and access to care among the most vulnerable, such as undocumented foreign-born subjects in a country where migration flows are on the rise.

This multicentre, double-blind study compared (100 mg) sumatriptan administered orally with placebo in treating an acute attack of migraine; 238 patients were studied over a 48-h period. Four hours after treatment, 92 of the 142 evaluable sumatriptan patients (65%) showed significant reductions ( P < 0.001) in headache severity, clinical disability and accompanying symptoms compared with 32 of the 80 evaluable placebo-treated patients (40%). The duration of attack prior to taking medication and the history of persistent migraine do not influence the observed difference between the two treatment regimens (sumatriptan and placebo), which remained statistically significant ( P < 0.001) in both cases. The incidence of headache recurrence in patients who experienced relief 4 h after initial treatment was low, occurring in 16 (17%) and 4 (13%) of the sumatriptan- and placebo-treated patients, respectively. Only patients with a history of migraine attacks lasting longer than 24 h suffered headache recurrences, and these recurrences were not consistent with the International Headache Society definition of migraine. Treatment with sumatriptan was well tolerated.

Sperm mediated gene transfer (SMGT) has been reported to be a powerful tool for producing transgenic livestock with applications in biomedicine and agriculture To date, two studies have reported the production of transgenic equine embryo with, however, low efficiency in blastocyst production and transgene expression The aim of the present study was to develop a method which allowed the efficient production of transgene-expressing embryos of the equine species through SMGT To overcome problems due to in vitro fertilization (IVF) in horses, the ICSI procedure was associated with SMGT The uptake of exogenous DNA in equine spermatozoa was assessed using a spectrophotometric approach and its internalisation using real time PCR and confocal laser scanning microscopy (CLSM) Embryos obtained from the ICSI-MGT procedure were analysed for the expression of eGFP and then for the transmission of the transgene.Our results suggested that the maximal uptake of exogenous DNA in equine spermatozoa occurs from 30 to 60 min of co-incubation Furthermore, real time PCR analysis suggested that the internalisation of exogenous DNA in the highest quality spermatozoa was slightly greater than in those having the poorest quality parameters Confocal laser scanning microscopy analysis confirmed that exogenous DNA is internalised by membrane intact spermatozoa In this study, 22 embryos were produced, 8 of which reached the 8-cell stage or greater Our data confirmed the transmission of the transgene in 86.3% of the cleaved embryos and the expression of the transgene in 25% of the embryos These data allowed us to affirm that this method is highly efficient in producing equine embryos which are able to express high levels of the exogenous protein © 2012 Elsevier B.V.

A simple and efficient method for producing multitransgenic animals is required for medical and veterinary applications. Sperm-mediated gene transfer (SMGT) is an effective method for introducing multiple genes into pigs (Sus, Sus scrofa). The major benefits of this technique are the high efficiency, low cost, and ease of use compared with that of other methods: Sperm-mediated gene transfer does not require embryo handling or expensive equipment. The aim of this study was to investigate the influence of SMGT treatment and exogenous DNA uptake on sperm quality. Even after a coincubation with a 20-fold larger amount (100 microg/mL) of DNA than usual (5 microg/mL), sperm quality parameters were not significantly affected, confirming the hypothesis that the SMGT protocol itself or the amount of bound DNA do not compromise the possibility of an extended employment of SMGT. More importantly, we found that semen used for in vitro fertilization 24h after DNA uptake gave good cleavage (60% vs. 58%, treated vs. control) and developmental rates definitely positive (41% vs. 48%, treated vs. control). These good results are connected to a competitive efficiency of transformation (62%) due to the numerous improvements in SMGT technique. We demonstrate that SMGT-treated spermatozoa retain good quality and fertilization potential for at least 24h, expanding the possibility to apply transgenesis in field conditions in swine, where the greatest hurdles are fertilization timing and plain procedure.

New biotechnologies, such as sperm-mediated gene transfer (SMGT), spermatozoa freezing and spermatozoa sorting have improved the possibilities to produce animals with desirable features. The main problem associated with these technologies is the scarce availability of spermatozoa for insemination. The objective of this study was to develop a laparoscopic insemination (LI) technique in gilt that allows the use of low semen doses resulting in high fertilization rates (FR) and minimal distress to the animal; the efficiency of this technique was compared to conventional artificial insemination (AI). Ten gilts were inseminated 36 h post hCG treatment near both utero-tubal junctions (UTJ) with 1.5 x 10(9)spermatozoa/5 mL per horn and 10 gilts (C) underwent conventional AI. Embryos were collected either at two to four cell stage (LI, n = 5; C, n = 5) for determination of fertilization rate or at day 6 for evaluation of developmental competence (LI, n = 5; C, n = 5). LI gilts showed a slightly higher FR than control animals. In a second trial, 24 gilts underwent LI with varying doses (1.5 x 10(8), 1.5 x 10(7), 1 x 10(7), 5 x 10(6) or 1 x 10(6)) of semen. Two to four stage embryos were collected and FR was evaluated in each tube. FR obtained with the lowest dose was significantly different from that with other dosages (P0.05). Embryos were cultured in vitro to blastocyst stages (percentage of blastocysts: 79.2 +/- 3.6%). In a third trial, five gilts were inseminated with semen processed by SMGT technique; both FR (86.1 +/- 9.9%) and transgene protein expression were satisfactory. In conclusion, this study shows that LI can be a useful tool for reducing doses of insemination, without affecting the efficiency of fertilization; this technique could have a wide range of biotechnological applications.

Introduction Delirium and sarcopenia are common, although underdiagnosed, geriatric syndromes. Several pathological mechanisms can link delirium and low skeletal muscle mass, but few studies have investigated their association. We aimed to investigate (1) the association between delirium and low skeletal muscle mass and (2) the possible role of calf circumference mass in finding cases with delirium. Methods The analyses were conducted employing the cross-sectional “Delirium Day” initiative, on patient 65 years and older admitted to acute hospital medical wards, emergency departments, rehabilitation wards, nursing homes and hospices in Italy in 2017. Delirium was diagnosed as a 4 + score at the 4-AT scale. Low skeletal muscle mass was operationally defined as calf circumference ≤ 34 cm in males and ≤ 33 cm in females. Logistic regression models were used to investigate the association between low skeletal muscle mass and delirium. The discriminative ability of calf circumference was evaluated using non-parametric ROC analyses. Results A sample of 1675 patients was analyzed. In total, 73.6% of participants had low skeletal muscle mass and 24.1% exhibited delirium. Low skeletal muscle mass and delirium showed an independent association (OR: 1.50; 95% CI 1.09–2.08). In the subsample of patients without a diagnosis of dementia, the inclusion of calf circumference in a model based on age and sex significantly improved its discriminative accuracy [area under the curve (AUC) 0.69 vs 0.57, p Discussion and conclusion Low muscle mass is independently associated with delirium. In patients without a previous diagnosis of dementia, calf circumference may help to better identify those who develop delirium.

Roe deer are seasonal breeders with a complete yearly testicular cycle. The peak in reproductive activity is recorded during summer, the rutting period, with the highest levels of androgens and testicular weight. Melatonin plays a pivotal role in seasonal breeders by stimulating the hypothalamus–pituitary–gonads axis and acting locally, in different species, its synthesis within testes has been reported. The aim of this study was to evaluate the physiological melatonin pattern within roe deer testes by comparing data obtained from animals sampled during pre- and post-rut periods. Melatonin was quantified in testicular parenchyma, along with the genetic expression of enzymes involved in its local synthesis (AANAT and ASMT) and function (UCP1). Melatonin receptors, MT1-2, were quantified both at protein and gene expression levels. Finally, to assess changes in reproductive hormonal profiles, testicular dehydroepiandrosterone (DHEA) was quantified and used for a correlation analysis. Melatonin and AANAT were detected in all samples, without significant differences between pre- and post-rut periods. Despite DHEA levels confirming testicular involution during the post-rut period, no correlations appeared between such involution and melatonin pathways. This study represents the first report regarding melatonin synthesis in roe deer testes, opening the way for future prospective studies in the physiology of this species.

Background Mesenchymal stromal cell (MSC)-based therapy is currently under study to treat inflammatory bowel diseases. MSC bioactive products could represent a valid alternative to overcome issues associated with systemic whole-cell therapies. However, MSC anti-inflammatory mechanisms differ between rodents and humans, impairing the reliability of preclinical models. Aim To evaluate the effect of conditioned medium (CM) derived from porcine vascular wall MSCs (pVW-MSCs) on survival and differentiation of porcine and guinea pig enteric ganglia exposed to lipopolysaccharide (LPS). Methods Primary cultures of enteric ganglia were obtained by mechanic and enzymatic digestion of ileum resections from guinea pigs (Cavia porcellus) (GPEG) and pigs (Suus scrofa) (PEG). pVW-MSCs were derived by enzymatic digestion from vascular wall resections of porcine aorta and tested by immunoflowcytometry for MSC immune profile. Enteric ganglia were treated with increasing concentrations of LPS, CM derived by pVW-MSCs or a combination of CM and LPS 1 µg/mL. Cell count and morphometric analysis of HuD positive neurons and glial fibrillary acidic protein positive glial cells were performed by immunofluorecent staining of cultured ganglia. Results PEG showed a higher number of neurons compared to GPEG. Overall, CM exerted a protective role on LPS-treated enteric ganglia. CM in combination with LPS increased the number of glial cells per ganglion in both cultures evoking glial cells differentiation in porcine cultures. Conclusion These findings suggest an immunomodulating activity of pVW-MSCs mediators on the enteric nervous system in inflammatory conditions.

Objective: Sensory deficits are important risk factors for delirium but have been investigated in single-center studies and single clinical settings. This multicenter study aims to evaluate the association between hearing and visual impairment or bi-sensory impairment (visual and hearing impairment) and delirium. Design: Cross-sectional study nested in the 2017 "Delirium Day" project. Setting and participants: Patients 65 years and older admitted to acute hospital medical wards, emergency departments, rehabilitation wards, nursing homes, and hospices in Italy. Methods: Delirium was assessed with the 4AT (a short tool for delirium assessment) and sensory deficits with a clinical evaluation. We assessed the association between delirium, hearing and visual impairment in multivariable logistic regression models, adjusting for: Model 1, we included predisposing factors for delirium (ie, dementia, weight loss and autonomy in the activities of daily living); Model 2, we added to Model 1 variables, which could be considered precipitating factors for delirium (ie, psychoactive drugs and urinary catheters). Results: A total of 3038 patients were included; delirium prevalence was 25%. Patients with delirium had a higher prevalence of hearing impairment (30.5% vs 18%; P < .001), visual impairment (24.2% vs 15.7%; P < .01) and bi-sensory impairment (16.2% vs 7.5%) compared with those without delirium. In the multivariable logistic regression analysis, the presence of bi-sensory impairment was associated with delirium in Model 1 [odds ratio (OR) 1.5, confidence interval (CI) 1.2-2.1; P = .00] and in Model 2 (OR 1.4; CI 1.1-1.9; P = .02), whereas the presence of visual and hearing impairment alone was not associated with delirium either in Model 1 (OR 0.8; CI 0.6-1.2, P = .36; OR 1.1; CI 0.8-1.4; P = .42) or in Model 2 (OR 0.8, CI 0.6-1.2, P = .27; OR 1.1, CI 0.8-1.4, P = .63). Conclusions and implications: Our findings support the importance of routine screening and specific interventions by a multidisciplinary team to implement optimal management of sensory impairments and hence prevention and the management of the patients with delirium.

Large colon volvulus in horses is associated with a poor prognosis, especially when ischemic-reperfusion injury of the affected intestinal tract develops. Proteinase-activated receptor 2 (PAR2) plays an important role in the pathogenesis of inflammation in the gastrointestinal tract. The aim of this study was to evaluate the distribution and expression of PAR2in colonic pelvic flexure of horses spontaneously affected by large colon volvulus (CVH group). Eight horses admitted for severe abdominal colon volvolus and which underwent surgery were included. Colon samples were collected after enterotomy. Data previously obtained from healthy horses were used as a control group. Histologic evaluation was carried out to grade the severity of the colon lesions. Immunofluorescence, western blot and quantitative polymerase chain reaction (RT-qPCR) were carried out on colon samples to evaluate PAR2expression. In addition, the transcriptional profile of cytokines and chemokines was evaluated using RT2Profiler™ PCR Array Horse Cytokines & Chemokines. Three out of the eight patients were euthanised due to clinical deterioration. Immunostaining for PAR2was observed in the enterocytes, intestinal glands and neurons of the submucosal and myenteric plexi. In the CVH horses, the expression of PAR2mesenger RNA (mRNA) did not differ significantly from that of the healthy animals; western blots of the mucosa of the colon tracts showed a clear band of the expected molecular weight for PAR2(~44 kDa) and a band smaller than the expected molecular weight for PAR2(25kDa), suggesting its activation. The gene expressions for C-X-C motif ligand 1 (CXCL1); interleukin 8 (IL8), macrophage inflammatory protein 2 beta (MIP-2BETA) were upregulated in the colic horses as compared with the colons of the healthy horses. Therefore, in the present study, the expression and activation of PAR2in the colons of horses in the presence of an inflammatory reaction like that occurring in those with spontaneous colon volvulus was confirmed.

Proteinase activated receptor 4 (PAR4) in the gastrointestinal tract is involved in the regulation of inflammation and pain pathways. The aim of the present study was to evaluate the distribution and expression of PAR4 in the jejunum of healthy horses and in the pathologic tracts from horses undergoing surgery for herniation of the small intestine through the epiploic foramen. Eight healthy horses (Group H) and eight horses with epiploic hernia (Group EH) were included; the jejunum samples were collected at the slaughter or intraoperatively after enterectomy, respectively. To evaluate PAR4 expression in sections of the jejunum, immunofluorescence, western blot and quantitative polymerase chain reaction (qRT-PCR) were performed. Immunohistochemistry of PAR4 in the jejunum of the healthy horses showed that receptors are predominantly expressed in the immune cell population scattered throughout the lamina propria of the mucosa and in the submucosa. Quantitative PCR data demonstrated that PAR4 mRNA was detectable in all of the samples analyzed without any difference between the H and the EH groups, however the PAR4 protein level was significantly lower in the jejunums of the EH horses. In the Group EH horses, PAR4 immunoreactivity was mainly expressed in the mast cells and was extensively distributed in the sierosa. In the lamina propria of mucosa of Group EH, leukocytes were less abundant than in Group H. In this study, the distribution and expression of PAR4 in the jejunums of the healthy horses and in those with spontaneous occurring epiploic hernia was demonstrated.

It is widely accepted that mature sperm contains RNA. The first hypothesis was that sperm RNAs have no functions of their own but are simply residues of spermatogenesis reflecting the events that occurred during their formation in the testes. More recently new discoveries have essentially expanded these views, showing that sperm mRNAs constitute a population of stable full-length transcripts, many of which are selectively retained during spermatogenesis and delivered to oocytes contributing to early embryo development. It is well known that semen quality can be influenced by occasional physical stress, infection, and variation in temperature and the definition of new markers for evaluation of semen could offer knowledge about the fertility potential of a semen sample. The aim of the present study was to evaluate the presence and the relative quantity of transcripts and protein of heat shock protein 70 (HSP70), 90 (HSP90) and clusterin (CLU) in Percoll-selected spermatozoa collected from seven adult boars of proven fertility routinely employed for artificial insemination. Our results showed the presence of HSP70, HSP90 and CLU transcripts with different level of expression: high for HSPs and low for CLU transcripts. The transcript level of both HSPs are similar among selected spermatozoa derived from high quality sperm with the exception of one boar that showed a reduced content of HSP70 and HSP90 mRNA together with a lower semen quality. At protein level, both HSPs were detected with similar amount among all seven boars whilst no band was evidenced for CLU protein.

Growing evidence indicates cannabinoid receptors as potential therapeutic targets for chronic pain. Consequently, there is an increasing interest in developing cannabinoid receptor agonists for treating human and veterinary pain. To better understand the actions of a drug, it is of paramount importance to know the cellular distribution of its specific receptor(s). The distribution of canonical and putative cannabinoid receptors in the peripheral and central nervous system of dogs is still in its infancy. In order to help fill this anatomical gap, the present ex vivo study has been designed to identify the cellular sites of cannabinoid and cannabinoid-related receptors in canine spinal ganglia. In particular, the cellular distribution of the cannabinoid receptors type 1 and 2 (CB1 and CB2) and putative cannabinoid receptors G protein-coupled receptor 55 (GPR55), nuclear peroxisome proliferator-activated receptor alpha (PPARα), and transient receptor potential vanilloid type 1 (TRPV1) have been immunohistochemically investigated in the C6-C8 cervical ganglia of dogs. About 50% of the neuronal population displayed weak to moderate CB1 receptor and TRPV1 immunoreactivity, while all of them were CB2-positive and nearly 40% also expressed GPR55 immunolabeling. Schwann cells, blood vessel smooth muscle cells, and pericyte-like cells all expressed CB2 receptor immunoreactivity, endothelial cell being also PPARα-positive. All the satellite glial cells (SGCs) displayed bright GPR55 receptor immunoreactivity. In half of the study dogs, SGCs were also PPARα-positive, and limited to older dogs displayed TRPV1 immunoreactivity. The present study may represent a morphological substrate to consider in order to develop therapeutic strategies against chronic pain.

The link between metabolic remodeling and stem cell fate is still unclear. To explore this topic, the metabolic profile of porcine vascular wall mesenchymal stem cells (pVW-MSCs) was investigated. At the first and second cell passages, pVW-MSCs exploit both glycolysis and cellular respiration to synthesize adenosine triphosphate (ATP), but in the subsequent (third to eighth) passages they do not show any mitochondrial ATP turnover. Interestingly, when the first passage pVW-MSCs are exposed to 0.1 or 10 μg/ml lipopolysaccharides (LPSs) for 4 hr, even if ATP synthesis is prevented, the spare respiratory capacity is retained and the glycolytic capacity is unaffected. In contrast, the exposure of pVW-MSCs at the fifth passage to 10 μg/ml LPS stimulates mitochondrial ATP synthesis. Flow cytometry rules out any reactive oxygen species (ROS) involvement in the LPS effects, thus suggesting that the pVW-MSC metabolic pattern is modulated by culture conditions via ROS-independent mechanisms.

Canine liposarcoma is an uncommon soft tissue sarcoma usually arising in the subcutis. While liposarcoma classification in dogs is based solely on histology, in humans it depends on the detection of genetic abnormalities that can lead to specific protein overexpression. This study is an immunohistochemical evaluation of MDM2 and CDK4 expression in canine liposarcoma designed to assess the correlation of these proteins with histologic type, grade, mitotic index and Ki67 labeling index and evaluate their utility in improving tumor classification. Fifty-three liposarcomas were retrospectively collected: 24 were well differentiated liposarcomas (WDL), 16 of which expressed MDM2 and 21 CDK4; 7 were myxoid liposarcomas (ML), 1 of which expressed MDM2 and 5 expressed CDK4; 18 were pleomorphic liposarcomas (PL), all were MDM2 negative and 12 expressed CDK4. Four tumors were morphologically consistent with dedifferentiated liposarcoma (DDL) a subtype described only in humans: 3 expressed MDM2 and 4 expressed CDK4. MDM2 expression correlated with histotype (highly expressed in WDL and DDL) and grade (highly expressed in grade 1 tumors). Histotype correlated with the Ki67 labeling index (lowest in WDL and highest in DDL). A revised classification, considering MDM2 expression, allowed 8 WDL to be reclassified as PL and correlated significantly with mitotic and Ki67 labeling index (both significantly lower in WDL and progressively higher in ML and DDL). These results partially parallel data reported for human liposarcomas, suggesting that WDL and DDL are distinct neoplastic entities characterized by MDM2 expression, which may represent a useful diagnostic and potentially prognostic marker for canine liposarcoma.

Background It is widely accepted the key role of endothelium in the onset of many chronic and acute vascular and cardiovascular diseases. In the last decade, traditional compounds utilized in “folk medicine” were considered with increasing interest to discover new bioactive molecules potentially effective in a wide range of diseases including cardiovascular ones. Since ancient times different parts of the Cucumis sativus L. plant were utilized in Ayurvedic medicine, among these, fruits were traditionally used to alleviate skin problem such as sunburn irritation and inflammation. The main purpose of the present research was, in a well-defined in vitro model of endothelial cells, to investigate whether a water/ethanol extract of Cucumis sativus L. (CSE) fruit can attenuate the damaging effect of pro-inflammatory lipopolysaccharide (LPS). Methods Cell viability, gene expression of endothelial cell markers, cytokines secretion and in vitro angiogenesis assay were performed on porcine Aortic Endothelial Cells exposed to increasing doses (0.02; 02; 2 mg/ml) of CSE in the presence of pro-inflammatory lipopolysaccharide (LPS 10 μg/ml). Results CSE reduced LPS-induced cytotoxicity and decreased the cellular detachment, restoring the expression of tight junction ZO-1. The increase of TLR4 expression induced by LPS was counterbalanced by the presence of CSE, while the protective gene Hemeoxygenase (HO)-1 was increased. Cucumis sativus L. inhibited the early robust secretion of inflammatory IL-8 and GM-CSFs, furthermore inhibition of inflammatory IL-6 and IL-1α occurred late at 7 and 24 h respectively. On the contrary, the secretion of anti-inflammatory IL-10, together with IL-18 and IFN-γ was increased. Moreover, the in vitro angiogenesis induced by inflammatory LPS was prevented by the presence of Cucunis sativus L. extract, at any doses tested. Conclusions Our results have clearly demonstrated that Cucumis sativus L. extract has attenuated lipopolysaccharide-induced inflammatory response in endothelial cells.

Tributyltin (TBT), is a man-made pollutants, known to accumulate along the food chain, acting as an endocrine disruptor in marine organisms, with toxic and adverse effects in many tissues including vascular system. Based on the absence of specific studies of TBT effects on endothelial cells, we aimed to evaluate the toxicity of TBT on primary culture of porcine aortic endothelial cells (pAECs), pig being an excellent model to study human cardiovascular disease. pAECs were exposed for 24h to TBT (100, 250, 500, 750 and 1000nM) showing a dose dependent decrease in cell viability through both apoptosis and necrosis. Moreover the ability of TBT (100 and 500nM) to influence endothelial gene expression was investigated at 1, 7 and 15h of treatment. Gene expression of tight junction molecules, occludin (OCLN) and tight junction protein-1 (ZO-1) was reduced while monocyte adhesion and adhesion molecules ICAM-1 and VCAM-1 (intercellular adhesion molecule-1 and vascular cell adhesion molecule-1) levels increased significantly at 1h. IL-6 and estrogen receptors 1 and 2 (ESR-1 and ESR-2) mRNAs, after a transient decrease, reached the maximum levels after 15h of exposure. Finally, we demonstrated that TBT altered endothelial functionality greatly increasing monocyte adhesion. These findings indicate that TBT deeply alters endothelial profile, disrupting their structure and interfering with their ability to interact with molecules and other cells.

Recent findings suggest that progenitor and multipotent mesenchymal stromal cells (MSCs) are associated with vascular niches. Cells displaying mesenchymal properties and differentiating to whole components of a functional blood vessel, including endothelial and smooth muscle cells, can be defined as vascular stem cells (VSCs). Recently, we isolated a population of porcine aortic vascular precursor cells (pAVPCs), which have MSC- and pericyte-like properties. The aim of the present work was to investigate whether pAVPCs possess VSC-like properties and assess their differentiation potential toward endothelial and smooth muscle lineages. pAVPCs, maintained in a specific pericyte growth medium, were cultured in high-glucose DMEM + 10% FBS (long-term medium, LTM) or in human endothelial serum-free medium + 5% FBS and 50 ng/ml of hVEGF (endothelial differentiation medium, EDM). After 21 days of culture in LTM, pAVPCs showed an elongated fibroblast-like morphology, and they seem to organize in cord-like structures. qPCR analysis of smooth muscle markers [α-smooth muscle actin (α-SMA), calponin, and smooth muscle myosin (SMM) heavy chain] showed a significant increment of the transcripts, and immunofluorescence analysis confirmed the presence of α-SMA and SMM proteins. After 21 days of culture in EDM, pAVPCs displayed an endothelial cell-like morphology and revealed the upregulation of the expression of endothelial markers (CD31, vascular endothelial-cadherin, von Willebrand factor, and endothelial nitric oxide synthase) showing the CD31-typical pattern. In conclusion, pAVPCs could be defined as a VSC-like population considering that, if they are maintained in a specific pericyte medium, they express MSC markers, and they have, in addition to the classical mesenchymal trilineage differentiation potential, the capacity to differentiate in vitro toward the smooth muscle and the endothelial cell phenotypes.

Aim of this study was to characterize the effects of an ochratoxin A (181 ± 34 ng/g) contaminated diet on growth performances, blood parameters, systemic cytokine levels, cell stress markers and reactivity of immune system of weaned pigs. Growth performance was not affected by OTA consumption even if OTA levels increased in plasma, kidney and liver. OTA diminished the protein content in the serum and increased levels of TNF-alpha and IL-10 in plasma. HO-1 mRNA, indicative for cells stress, was decreased in the kidney but increased in the liver. Additionally, whole blood of the animals of the OTA-group showed a decreased capacity to respond with cytokine expression (mRNA and protein) to ex vivo challenge with LPS. In conclusion our findings indicate that chronic ingestion with OTA-contaminated feed, even at low level, is hazardous for the animal and virtually for human health, pig being an excellent model for human.

Cells derived from porcine aorta tunica media show mesenchymal stromal-like cell properties in in vitro culture. Am J Physiol Cell Physiol 306: C322–C333, 2014. First published December 4, 2013; doi:10.1152/ajpcell.00112.2013.—Several studies have already described the presence of specialized niches of precursor cells in vasculature wall, and it has been shown that these populations share several features with mesenchymal stromal cells (MSCs). Considering the relevance of MSCs in the cardiovascular physiopathology and regenerative medicine, and the usefulness of the pig animal model in this field, we reported a new method for MSC-like cell isolation from pig aorta. Filling the vessel with a collagenase solution for 40 min, all endothelial cells were detached and discarded and then collagenase treatment was repeated for 4 h to digest approximately one-third of the tunica media. The ability of our method to select a population of MSC-like cells from tunica media could be ascribed in part to the elimination of contaminant cells from the intimal layer and in part to the overnight culture in the high antibiotic/antimycotic condition and to the starvation step. Aorticderived cells show an elongated, spindle shape, fibroblast-like morphology, as reported for MSCs, stain positively for CD44, CD56, CD90, and CD105; stain negatively for CD34 and CD45; and express CD73 mRNA. Moreover, these cells show the classical mesenchymal trilineage differentiation potential. Under our in vitro culture conditions, aortic-derived cells share some phenotypical features with pericytes and are able to take part in the formation of network-like structures if cocultured with human umbilical vein endothelial cells. In conclusion, our work reports a simple and highly suitable method for obtaining large numbers of precursor MSC-like cells derived from the porcine aortic wal

Inherited bleeding disorders including abnormalities of platelet number and function rarely occur in a variety of dog breeds, but are probably underdiagnosed. Genetically characterized canine forms of platelet disorders provide valuable large animal models for understanding similar platelet disorders in people. Breed-specific disease associated genetic variants in only eight different genes are known to cause intrinsic platelet disorders in dogs. However, the causative genetic variant in many dog breeds has until now remained unknown. Four cases of a mild to severe bleeding disorder in Cocker Spaniel dogs are herein presented. The affected dogs showed a platelet adhesion defect characterized by macrothrombocytopenia with variable platelet counts resembling human Bernard-Soulier syndrome (BSS). Furthermore, the lack of functional GPIb-IX-V was demonstrated by immunocytochemistry. Whole genome sequencing of one affected dog and visual inspection of the candidate genes identified a deletion in the glycoprotein IX platelet (GP9) gene. The GP9 gene encodes a subunit of a platelet surface membrane glycoprotein complex; this functions as a receptor for von Willebrand factor, which initiates the maintenance of hemostasis after injury. Variants in human GP9 are associated with Bernard-Soulier syndrome, type C. The deletion spanned 2460 bp, and included a significant part of the single coding exon of the canine GP9 gene on dog chromosome 20. The variant results in a frameshift and premature stop codon which is predicted to truncate almost two-thirds of the encoded protein. PCR-based genotyping confirmed recessive inheritance. The homozygous variant genotype seen in affected dogs did not occur in 98 control Cocker Spaniels. Thus, it was concluded that the structural variant identified in the GP9 gene was most likely causative for the BSS-phenotype in the dogs examined. These findings provide the first large animal GP9 model for this group of inherited platelet disorders and greatly facilitate the diagnosis and identification of affected and/or normal carriers in Cocker Spaniels.

In pig production, artificial insemination is widely carried out and the use of fresh diluted semen is predominant. For this reason, there are increasing interests in developing new extenders and in establishing the optimal storage conditions for diluted spermatozoa. In the last few decades, we utilised a homemade diluent (swine fertilisation medium (SFM)) for spermatozoa manipulation and biotechnological application as the production of transgenic pigs utilising the sperm-mediated gene transfer technique. The purpose of the present study is therefore to analyse the ability of SFM, in comparison to four commercial extenders, in preserving the quality of diluted boar semen stored at 16.58C till 15 days. We utilised some of the main predictive tests as objectively measured motility, acrosome and sperm membrane integrity, high mitochondrial membrane potential and pH. Based on our in vitro study, SFM could be declared as a good long-term extender, able to preserve spermatozoa quality as well as Androhep Enduraguard for up to 6 to 9 days and more.

Several reliable methods to produce transgenic animals utilize the male genome. After penetration into oocyte, sperm DNA undergoes dramatic conformational changes that could represent a great opportunity for exogenous DNA to be integrated in the zygote genome. Among the enzymes responsible for sperm remodeling, a nuclease could be involved. The presence of a DNase I in oocytes has not been much investigated. To date, an immunolocalization of DNase I has been reported only in rat immature oocytes and the presence of nuclease activities has been shown in avian oocytes.The present study was conducted to verify whether a DNase-I like activity is present in MII mature pig oocytes. To do this, oocyte extracts were assessed for nuclease activity by a plasmid degradation assay and by zymography; these analyses evidenced a 33 kDa, Ca2+/Mg2+dependent DNase I-like activity that was inhibited by Zn2+. A further identification of DNase I was achieved by Western blot, immunofluorescence and RT-PCR experiments. Moreover, the presence of the enzyme activity was confirmed by the rapid degradation of exogenous DNA microinjected into the ooplasm. Finally, the exogenous DNA transferred to oocyte by spermatozoa during sperm mediated gene transferin vitrofertilisation protocol seemed to be protected from DNase I degradation and to persist in the ooplasm till 6 h.These results, together with the high efficiency of sperm based transgenesis methods, suggest that the association with spermatozoa protects exogenous DNA from nuclease activities.

Matrix metalloproteinases (MMP) 2 and 9 are enzymes involved in extracellular matrix (ECM) degradation with specific proteolytic activity for type IV collagen and gelatin.MMPs participate in several physiological processes characterised by ECM remodelling and are implicated in various inflammatory, degenerative and neoplastic pathological conditions. The invasive and metastatic potential of several neoplastic lesions in the central nervous system (CNS) has been correlated with MMP expression; therefore, MMP assessment in cerebrospinal fluid (CSF) has gained an important diagnostic and prognostic role in human medicine (Friedberg et al., 1998).MMP2has been evidenced in the CSF of clinically normal dogs; nevertheless, MMP expression has not been evaluated in the CSF of dogs affected by pathological conditions of the CNS (Bergman et al., 2002). The aim of the present work was to retrospectively evaluate, using gelatin-zymography, the MMP2 and 9 activities in CSF samples obtained from dogs with non-inflammatory diseases of the CNS, with particular attention to space-occupying lesions (SOL). This last activity was evaluated in comparison with two other different pathological conditions: idiopathic epilepsy (IE) and disk herniation (DH).

The objective of this study was to perform global gene expression profiling of patients affected by narcolepsy with cataplexy (NRLCP). This enabled identifying new potential biomarkers and relevant molecules possibly involved in the disease pathogenesis. In this study 10 NRLCP patients and 10 healthy controls were compared. Total RNA isolated from blood specimens was analyzed using microarray technology followed by statistical data analysis to detect genome-wide differential gene expression between patients and controls. Functional analysis of the gene list was performed in order to interpret the biological significance of the data. One hundred and seventy-three genes showed significant (p < 0.01) differential expression between the two tested conditions. The biological interpretation allowed categorizing differentially expressed genes involved in neurite outgrowth/extension and brain development, which could be possibly regarded as peripheral markers of the disease. Moreover, the NRLCP-related gene expression profiles indicated a dysregulation of metabolic and immune-related mechanisms. In conclusion, the gene expression profile associated to NRLCP suggested that molecular markers of neurological impairment, dysmetabolic and immune-related mechanisms, can be detected in blood of NRLCP patients.

Flow cytometric separation of X and Y chromosome-bearing spermatozoa has been demonstrated to be effective in pigs, allowing the use of boar sexed semen in in vitro trials. Sperm Mediated Gene Transfer (SMGT) is a widely used and efficient technique for the creation of transgenic animals. The present research intended to prove that it is possible to associate sperm sexing with the SMGT technique in order to speed up the assessment of homozygous lines of transgenic pigs. In the first experiment, the sorting protocol was modified in order to obtain the highest DNA uptake by sorted spermatozoa. In the second experiment, spermatozoa that had undergone only sperm sorting, only SMGT, or both procedures (Sorted-SMGT) were used for in in vitro fertilization of in vitro matured oocytes. In the third experiment, transformed blastocysts of the desired gender (male) were obtained with Sorted-SMGT in an in vitro fertilization trial. The method we developed here allowed us to produce transgenic swine blastocysts of pre-determined gender, giving a positive answer at the aim to couple SMGT and sperm sorting in swine, obtaining fertile spermatozoa able to produce transgenic embryos of pre-determined gender.

Lim mineralization protein-3 (LMP3) induces osteoblast differentiation by regulating the expression and activity of certain molecules involved in the osteogenic cascade, including those belonging to the bone morphogenetic protein (BMP) family. The complete network of molecular events involved in LMP3-mediated osteogenesis is still unknown. The aim of this study was to analyze the genome-wide gene expression profiles in human mesenchymal stem cells (hMSC) induced by exogenous LMP3 to mediate osteogenesis. For this purpose hMSC were transduced with a defective adenoviral vector expressing the human LMP3 gene and microarray analysis was performed 1 day post-adenoviral transduction. Cells transduced with the vector backbone and untransduced cells were used as independent controls in the experiments. Microarray data were independently validated by means of real-time PCR on selected transcripts. The statistical analysis of microarray data produced a list of 263 significantly (p < 0.01) differentially expressed transcripts. The biological interpretation of the results indicated, among the most noteworthy effects, the modulation of genes involved in the TGF-beta1 pathway: 88 genes coding for key regulators of the cell cycle regulatory machinery and 28 genes implicated in the regulation of cell proliferation along with the development of connective, muscular, and skeletal tissues. These results suggested that LMP3 could affect the fine balance between cell proliferation/differentiation of mesenchymal cells mostly by modulating the TGF-beta1 signaling pathway.

Treponema denticola is an oral spirochete that is associated with periodontal disease and detected occasionally in extraoral lesions associated with systemic disorders such as cardiovascular diseases. The effect of specific bacterial products from oral treponemes on endothelium is poorly investigated. This study analyzed the ability of components of the outer membrane of T. denticola (OMT) to induce apoptosis and heat shock proteins (HO-1 and Hsp70) in porcine aortic endothelial cells (pAECs), compared with results obtained with classical pro-inflammatory lipopolysaccharide (LPS) treatment. Cellular apoptosis was detected when pAECs were treated with either OMT or LPS, suggesting that OMT can damage endothelium integrity by reducing endothelial cell vitality. Stimulation with OMT, similarly to LPS response, increased HO-1 and Hsp-70 protein expression in a time-dependent manner, correlating with a rise in HO-1 and Hsp-70 mRNA. Collectively, these results support the hypothesis that T. denticola alters endothelial cell function. Moreover, our in vitro experiments represent a preliminary investigation to further in vivo study using a pig model to elucidate how T. denticola leaves the initial endodontic site and participates in the development of several systemic diseases.

Daidzein, an isoflavone abundant in soybeans and other legumes, displays estrogen like properties. This study was aimed at evaluating the effect of daidzein (1 and 10 M ) on nuclear and cytoplasmic maturation of pig oocytes and on steroidogenic activity of cumulus cells. Daidzein supplementation during IVM had no effect on nuclear maturation and on fertilization traits. By contrast, both concentrations significantly (P < 0.05) inhibited progesterone production by cumulus cells after 24 and 48 h of culture while they did not induce any effect on estradiol production. Furthermore, daidzein did not exert any effect on the percentage of embryos that developed to blastocyst stage, on the number of blastomeres per blastocyst, or on the level of Hsp-70 and -90 gene transcript. Overall, our data demonstrate that daidzein added during oocyte maturation does not affect pig embryo development even if it markedly inhibits progesterone production by cumulus cells. Further studies are needed to evaluate the possible effect of daidzein during embryonic development.

Ribeiro, L.A., Turba, M.E., Bernardini, C., Zannoni, A., Bacci, M.L. and Forni, M., 2007. Matrix metalloproteinases -2 and -9 in swine luteal tissue angiogenesis and angioregression. Veterinary Research Communications, 31(Suppl. 1), 193–196

Selected cases of canine liposarcomas were classified according with the WHO classification of tumors of domestic animals. When this was not possible the human classification was applied. Cross reactivity of antibodies anti-mdm2 and CDK4 was assessed by western blot analysis. Immunohistochemistry was performed of formalin fixed tissue applying an heat induced antigen retrieval. Cases were considered positive when more than 10% of neoplastic cells had nuclear staining. Forty-seven LP were collected: 19 WDLP, 18 PLP, and 7 MLP. Three cases were consistent with DDLP. Fifteen cases expressed MDM2 and 37 CDK4. MDM2 and CDK4 were respectively expressed in 12/19 and 17/19 cases WDLP, 1/7 and 5/7 MLP, 0/18 and 12/18 PLP, 2/3 and 3/3 DDLP. Taken together WDLPs and DDLPs expressed mdm2 in 14/22 (63.6%) and CDK4 in 20/22 cases (90.9%). These results suggest that WDLP/DDLP may represent a biological entity characterized by MDM2 and CDK4 overexpression, paralleling LP in man, and suggesting the presence of similar gene amplifications.

The aim of the present study was to investigate the influence of 50 Hz sinusoidal magnetic field on Hsp27, Hsp70, and Hsp90 expression in a model of primary culture of porcine aortic endothelial cells (PAEC). We took into consideration the Hsp profile in terms of mRNA expression, protein expression and protein localization inside the cells. The choice of the cell system was motivated by the involvement of the endothelial cells in the onset of many diseases; moreover, only few reports describe the effects of extremely low frequency magnetic fields (ELF-MFs) on such cells. ELF-MF exposure induced an increase in the mRNA levels of the three proteins, which was statistically significant for Hsp70. On the contrary, we did not observe any influence on Hsp27, Hsp70, and Hsp90 protein levels. Analysis in situ by immunofluorescence revealed that ELF-MF exposure affected the cellular distribution of Hsp27; in particular a partial relocalization in the nucleus was observed.

Lipopolysaccharide (LPS) is a highly proactive molecule that causes in vivo a systemic inflammatory response syndrome and activates in vitro the inflammatory pathway in different cellular types, including endothelial cells (EC). Because the proinflammatory status could lead to EC injury and apoptosis, the expression of proinflammatory genes must be finely regulated through the induction of protective genes. This study aimed at determining whether an LPS exposure is effective in inducing apoptosis in primary cultures of porcine aortic endothelial cells and in stimulating heat shock protein (Hsp)70 and Hsp32 production as well as vascular endothelial growth factor (VEGF) secretion. Cells between third and eighth passage were exposed to 10 microg/mL LPS for 1, 7, 15, and 24 hours (time-course experiments) or to 1, 10, and 100 microg/mL LPS for 7 and 15 hours (dose-response experiments). Apoptosis was not affected by 1 microg/mL LPS but significantly increased in a dose-dependent manner with the highest LPS doses. Furthermore, apoptosis rate increased only till 15 hours of LPS exposure. LPS stimulated VEGF secretion in a dose-dependent manner; its effect became significant after 7 hours and reached a plateau after 15 hours. Both Hsp70 and Hsp32 expressions were induced by LPS in a dose-dependent manner after 7 hours. Subsequent studies were addressed to evaluate the protective role of Hsp32, Hsp70, and VEGF. Hemin, an Hsp32 inducer (5, 20, 50 microM), and recombinant VEGF (100 and 200 ng/mL), were added to the culture 2 hours before LPS (10 microg/mL for 24 hours); to induce Hsp70 expression, cells were heat shocked (42 degrees C for 1 hour) 15 hours before LPS (10 microg/mL for 24 hours). Hemin exposure upregulated Hsp32 expression in a dose-dependent manner and protected cells against LPS-induced apoptosis. Heat shock (HS) stimulated Hsp70 expression but failed to reduce LPS-induced apoptosis; VEGF addition did not protect cells against LPS-induced apoptosis at any dose tested. Nevertheless, when treatments were associated, a reduction of LPS-induced apoptosis was always observed; the reduction was maximal when all the treatments (HS + Hemin + VEGF) were associated. In conclusion, this study demonstrates that LPS is effective in evoking "the heat shock response" with an increase of nonspecific protective molecules (namely Hsp70 and Hsp32) and of VEGF, a specific EC growth factor. The protective role of Hsp32 was also demonstrated. Further investigations are required to clarify the synergic effect of Hsp32, Hsp70, and VEGF, thus elucidating the possible interaction between these molecules.

Semen quality assessment represents a fundamental step for obtaining successful artificial insemination (AI). In commercial settings, the semen employed for AI should be of high quality but traditional semen quality estimates are not sufficiently sensitive to discriminate between differences among samples in terms of fertilising ability. Therefore, more discriminative sperm characteristics need to be identified in order to better predict fertility outcome. In the present study, a series of molecular aspects of semen, represented by heat shock proteins, oxidative stress status, antioxidant potential and tumour necrosis factor (TNF)-alpha were evaluated and analysed. Several relationships between traditional and investigated molecular semen quality estimates were found by using a multivariate analysis approach. Tumour necrosis factor (TNF)-alpha was identified in boar seminal plasma resulting in positive correlations with several sperm quality aspects and particularly with motility. The protective roles of antioxidant molecules and heat shock proteins have been demonstrated confirming the data previously published in the literature.

Endothelin (ET)-1 is a potent vasoconstrictive peptide and it is involved in the pathogenesis of septic shock. Blockade of ET-1 receptors abolishes the LPS-induced pulmonary hypertension and worsens the LPS-dependent systemic hypotension, but the role of ET-1 in sepsis remains uncertain. To determine the role of ET-1 in cardiovascular and respiratory derangement in a porcine model of endotoxemic shock we evaluated ET-1 plasma levels and ET-1 mRNA and protein levels in lung, liver, and heart as well as Endothelin Converting Enzyme-1, ET A and ET B receptors mRNA in the same tissues. Twelve piglets were randomised to sham operated or to LPS-treated (40 μg/kg/h for 4 h) groups. During the experiment, respiratory and circulatory parameters have been recorded and blood samples collected. At the end of the experiment the animals were sacrificed and tissue samples collected for real-time quantitative PCR and ELISA test. LPS infusion evokes a large increase in ET-1 plasma concentration, and in tissues mRNA levels, associated with an increase in pulmonary arterial pressure, as well as in pulmonary and systemic vascular resistances, and a decrease in stroke volume. LPS infusion caused also a derangement of respiratory mechanics, evidenced by an increase in resistance and a decrease in compliance of the respiratory system. ET A and ET B receptor mRNA levels were markedly decreased in liver and lung and slightly increased in heart, evidencing that ET receptor subtypes were differentially regulated in the major organs of endotoxin treated pigs. In conclusion our data show the presence of a continuative and differentially regulated stimulating mechanism of ET-1 expression during pig endotoxaemia as well as a fundamental role of ET-1 system in the cardiovascular and respiratory derangement.

The presence and cellular distribution of Heat shock protein 70 (Hsp70) in ejaculated, capacitated and acrosome-reacted boar spermatozoa was evaluated by immunofluorescence and Western Blot; the role of Hsp70 during fertilization was also studied. In freshly ejaculated spermatozoa, Hsp70 immunoreactivity is present in a well defined triangular-shaped area in the equatorial segment that seems to correspond to the equatorial sub-segment. The distribution of the fluorescent signal changes in capacitated sperm, that exhibit different patterns probably in relation to the stage of capacitation of individual cells; after acrosome reaction Hsp70 immunoreactivity is localized on both a thick sub-equatorial band and a triangle in the equatorial segment. In reacted spermatozoa, Hsp70 seems to be not only relocalized but also translocated from the inner to the outer leaflet of the sperm plasma membrane, as a significant (P

Stress proteins are induced in response to a wide range of biological and physicochemical stresses; HSC70 (constitutive) and HSP70 (inducible) belong to the HSP70 stress protein family. The present study aimed at investigating whether environmental stress, particularly thermal and oxidative stress, is involved in modulating HSP70/HSC70 expression in in vitro porcine embryos from two/four cell stage to blastocyst. For oxidative stress, embryos were cultured at 38.5 degrees C under a 5% O2-5% CO2 atmosphere or 5% CO2 in air (approximately 20% O2); for thermal stress, embryos were cultured at 38.5 degrees C under 5% O2, 5% CO2, and exposed to heat shock (1 hr at 42 degrees C). At the end of culture, embryos were analysed by Western blotting, using specific antibodies discriminating HSP70 from HSC70. Embryos cultured under 20% O2 showed HSC70 levels significantly higher (P < 0.005) than embryos cultured under 5% O2, while heat shocked embryos presented HSP70 levels higher (P < 0.01) than control group. In addition, the developmental rate of embryos was negatively affected by the higher oxygen tension (P < 0.05). Our data indicate that porcine embryos express both HSP70 and HSC70 and could differentially respond to both oxidative and heat stress by up-regulating HSC70 and HSP70, respectively.

Background It is well known that nitric oxide (NO) is generated by a family of constitutively (nNOS and eNOS) or inducibly (iNOS) expressed enzymes and takes part in different aspects of the inflammatory response; nevertheless, its effective role in the pathogenesis of multiple organ dysfunction and septic shock is not fully understood. Methods To investigate the Nitric Oxide Synthases (NOSs) expression in endothelial cells during endotoxin exposure and the involvement of NO in lipopolysaccharide (LPS)-induced apoptosis, primary cultures of porcine Aortic Endothelial Cells (pAECs) were exposed to LPS for different time periods (1-24 h) and to LPS + L-NAME (15 h). Results Lipopolysaccharide induced an increase in mRNA and protein iNOS expression; on the contrary, the expression of eNOS was decreased. Furthermore, NOSs localisation was in part modified by LPS treatment. No alteration in the total level of Nitric Oxide was observed. L-NAME (5 mM) addition determined a slight decrease of LPS-induced apoptosis. Conclusions Endotoxin treatment strongly influenced NOS expression with an upregulation of iNOS and a simultaneous down regulation of eNOS. Moreover, in our model, the involvement of NO on LPS-induced apoptosis is very modest, suggesting that different pathways are involved in the regulation of this process.

In the field of cardiovascular research, the pig is considered to be an excellent animal model of human diseases. It is well-known that primary cultures of endothelial cells (ECs) are a powerful tool for the study of vascular physiology and pathology, and, according to the principles of the Three Rs, their use results in a substantial reduction in the numbers of experimental animals required. However, a limitation of EC culture is that the cells are not in their physiological context. Here, we describe and characterise a method for the culture of porcine vessels that overcomes the limitation of EC cultures, with the advantage of reducing the number of animals used for research purposes. The organ cultures were set-up by using an aortic cylinder obtained from the arteries of control pigs sacrificed for other experimental purposes. In order to characterise the method, vascular endothelial growth factor (VEGF) secretion, matrix metalloproteinase (MMP) activation and the vessel's structural features were evaluated during organ culture. These analyses confirm that the culture of aortic cylinder lumen, in a medium specific for ECs, results in a stable system in terms of VEGF and MMP secretion. The ECs do not undergo cell division during the organ culture, which is also the case in vivo, if no stimulation occurs. Overall, we show that this novel system closely resembles the in vivo context. Importantly, porcine aortas can be collected from either veterinary surgeries or slaughterhouses, without having to sacrifice animals specifically for the purposes of this type of research.

Breastfeeding plays a major role in the health and wellbeing of mother and infant. However, information on the safety of maternal medication during breastfeeding is lacking for most medications. This leads to discontinuation of either breastfeeding or maternal therapy, although many medications are likely to be safe. Since human lactation studies are costly and challenging, validated non-clinical methods would offer an attractive alternative. This review gives an extensive overview of the non-clinical methods (in vitro, in vivo and in silico) to study the transfer of maternal medication into the human breast milk, and subsequent neonatal systemic exposure. Several in vitro models are available, but model characterization, including quantitative medication transport data across the in vitro blood-milk barrier, remains rather limited. Furthermore, animal in vivo models have been used successfully in the past. However, these models don't always mimic human physiology due to species-specific differences. Several efforts have been made to predict medication transfer into the milk based on physicochemical characteristics. However, the role of transporter proteins and several physiological factors (e.g., variable milk lipid content) are not accounted for by these methods. Physiologically-based pharmacokinetic (PBPK) modelling offers a mechanism-oriented strategy with bio-relevance. Recently, lactation PBPK models have been reported for some medications, showing at least the feasibility and value of PBPK modelling to predict transfer of medication into the human milk. However, reliable data as input for PBPK models is often missing. The iterative development of in vitro, animal in vivo and PBPK modelling methods seems to be a promising approach. Human in vitro models will deliver essential data on the transepithelial transport of medication, whereas the combination of animal in vitro and in vivo methods will deliver information to establish accurate in vitro/in vivo extrapolation (IVIVE) algorithms and mechanistic insights. Such a non-clinical platform will be developed and thoroughly evaluated by the Innovative Medicines Initiative ConcePTION. ispartof: BIOMEDICINE & PHARMACOTHERAPY vol:136 ispartof: location:France status: published

Background The corpus luteum (CL) is a transient endocrine gland and prostaglandin F2-alpha is considered to be the principal luteolysin in pigs. In this species, the in vivo administration of prostaglandin F2-alpha induces apoptosis in large vessels as early as 6 hours after administration. The presence of the prostaglandin F2-alpha receptor (FPr) on the microvascular endothelial cells (pCL-MVECs) of the porcine corpus luteum has not yet been defined. The aim of the study was to assess FPr expression in pCL-MVECs in the early and mid-luteal phases (EL-p, ML-p), and during pregnancy (P-p). Moreover, the effectiveness of prostaglandin F2-alpha treatment in inducing pCL-MVEC apoptosis was tested. Methods Porcine CLs were collected in the EL and ML phases and during P-p. All CLs from each animal were minced together and the homogenates underwent enzymatic digestion. The pCL-MVECs were then positively selected by an immunomagnetic separation protocol using Dynabeads coated with anti-CD31 monoclonal antibody and seeded in flasks in the presence of EGM 2-MV (Microvascular Endothelial Cell Medium-2). After 4 days of culture, the cells underwent additional immunomagnetic selection and were seeded in flasks until the confluent stage. PCR Real time, western blot and immunodetection assays were utilized to assess the presence of FPr on pCL-MVEC primary cultures. Furthermore, the influence of culture time (freshly isolated, cultured overnight and at confluence) and hormonal treatment (P4 and E2) on FPr expression in pCL-MVECs was also investigated. Apoptosis was detected by TUNEL assay of pCL-MVECs exposed to prostaglandin F2-alpha. Results We obtained primary cultures of pCL-MVECs from all animals. FPr mRNA and protein levels showed the highest value (ANOVA) in CL-MVECs derived from the early-luteal phase. Moreover, freshly isolated MVECs showed a higher FPr mRNA value than those cultured overnight and confluent cells (ANOVA). prostaglandin F2-alpha treatment failed to induce an apoptotic response in all the pCL-MVEC cultures. Conclusion Our data showing the presence of FPr on MVECs and the inability of prostaglandin F2-alpha to evoke an in vitro apoptotic response suggest that other molecules or mechanisms must be considered in order to explain the in vivo direct pro-apoptotic effect of prostaglandin F2-alpha at the endothelial level.