Fabrication and in-vitro characterization of a polymeric aortic valve for minimally invasive valve replacement.

The valve replacement therapy is the standard treatment for severe heart valve diseases. Nowadays, two types of commercial prosthesis are available: mechanical and biological, but both of them have severe limitations. Moreover, alternative therapeutic approach for valve replacement, based on minimally invasive techniques (MIAVR), motivates the search for new valve materials. In this study a polyurethane-based self-expandable tri-leaflets heart valve compatible with MIAVR procedure is proposed. The device is based on the development, fabrication and characterization of three different elements: the leaflets, the polymeric stent for supporting the leaflets, and the external metallic stent for anchoring the valve to the native aortic root. The polymeric stent and the valve leaflets were fabricated using a thermoplastic silicone-polycarbonate-urethane using 3D printing and spray technology while the external metallic stent was made in nickel titanium (Nitinol) to obtain a self-expandable valve after the crimping process. The three elements were assembled in the completed device and tested by crimping, fatigue and fluid-dynamic test. The novel polymeric valve proposed showed promising results about valve crimping capabilities, durability and fluid dynamic performances. This approach could offer advantages such as low cost and to produce a tailor-made device basing on patient's imaging data. Moreover, the selected biomaterial offers the potential to have a device that could need of permanent anticoagulation and lack of calcification.

Fibrin acts as biomimetic niche inducing both differentiation and stem cell marker expression of early human endothelial progenitor cells.

OBJECTIVES: Transplantation of endothelial progenitor cells (EPCs) is a promising approach for revascularization of tissue. We have used a natural and biocompatible biopolymer, fibrin, to induce cell population growth, differentiation and functional activity of EPCs. MATERIALS AND METHODS: Peripheral blood mononuclear cells were cultured for 1 week to obtain early EPCs. Fibrin was characterized for stiffness and capability to sustain cell population expansion at different fibrinogen-thrombin ratios. Viability, differentiation and angiogenic properties of EPCs were evaluated and compared to those of EPCs grown on fibronectin. RESULTS: Fibrin had a nanometric fibrous structure forming a porous network. Fibrinogen concentration significantly influenced fibrin stiffness and cell growth: 9 mg/ml fibrinogen and 25 U/ml thrombin was the best ratio for enhanced cell viability. Moreover, cell viability was significantly higher on fibrin compared to being on fibronectin. Even though no significant difference was observed in expression of endothelial markers, culture on fibrin elicited marked induction of stem cell markers OCT 3/4 and NANOG. In vitro angiogenesis assay on Matrigel showed that EPCs grown on fibrin retain angiogenetic capability as EPCs grown on fibronectin, but significantly better release of cytokines involved in cell recruitment was produced by EPC grown on fibrin. CONCLUSION: Fibrin is a suitable matrix for EPC growth, differentiation and angiogenesis capability, suggesting that fibrin gel may be very useful for regenerative medicine.

Pharmacokinetics of methylphenidate after oral administration of immediate and sustained-release preparations in Beagle dogs.

Methylphenidate (MPH) is a drug administered either as an immediate- or sustained-release preparation for the treatment of attention deficit hyperactivity disorder in humans. The aim of this study was to determine the pharmacokinetics of two different MPH formulations in the dog. Eight dogs were randomly assigned to two treatment groups using a two-part randomised, cross-over experimental design. Each subject received a single dose of 20 mg d,l-MPH as an immediate- (IR) or sustained-release (SR) tablet. Blood was collected at specific times, and the plasma concentrations of d,l-MPH were evaluated using high performance liquid chromatography. There were no adverse effects following the oral administration of d,l-MPH in either the IR or SR groups, apart from mild hyperkinesia which was observed in some of the IR group. The plasma concentration data of d,l-MPH were best described by a one-compartment model. There were significant differences in the maximum concentration (C(max)), time to C(max) (T(max)), area under the curve (AUC) and clearance (Cl) between the two formulations. The relative bioavailability of the SR formulation was 30.58±13.73% and, despite low drug plasma concentrations, the SR formulation resulted in uniform plasma concentrations of d,l-MPH. However, the dose rate of the SR formulation used in this study resulted in plasma concentrations that were below effective levels for clinical efficacy, so further studies are required to confirm the suitability of higher dose rates for clinical use.

Pharmacokinetics of methylphenidate following two oral formulations (immediate and sustained release) in the dog.

Methylphenidate (MPH) is an immediate-release (IR) or sustained-release (SR) drug used to treat attention-deficit hyperactivity disorder. Eight dogs were randomly assigned to two treatment groups, using an open, single-dose, two-treatment, two-period, randomized, crossover design. Each subject received a single dose of 20 mg d,l-MPH IR or SR tablet. After blood collections at specific times, the concentrations of d,l-MPH in plasma were evaluated by high-performance liquid chromatography. Following both IR and SR oral administration of d,l-MPH, the animals did not show any side effects, except that mild hyperkinesia was observed in a few subjects belonging to the IR treatment group. After both administrations, the concentration data for d,l-MPH in plasma displayed a characteristic, one-compartment drug model. The relative bioavailability of the SR formulation was 30.58 +/- 13.73%. Significant differences between the two administrations were found in T(max), C(max), AUC, and Cl. Despite low drug concentrations in the blood, the SR formulation ensured uniformity of d,l-MPH plasma concentrations and, thus, a simpler and easier titration. In conclusion, the tested dosage appears to be too low for clinical application in canines, and an increase in dosing is suggested. Further pharmacodynamics studies are necessary to support this speculation.

Evaluation of methicillin resistance in Staphylococcus intermedius isolated from dogs.

Methicillin and multi-drug resistance were investigated in 136 Staphylococcus intermedius strains of canine origin. The large majority of isolates were found to be mecA-negative by polymerase chain reaction, whereas only four strains were positive for the mecA gene. All mecA-positive strains were confirmed as methicillin-resistant by complementary tests, except for oxacillin disk diffusion, which yielded one false-negative result. A significantly higher resistance to fusidic acid, lincosamides, and cotrimoxazole was observed in methicillin-resistant S. intermedius (MRSI) compared with methicillin-susceptible strains. Although the prevalence of MRSI in dogs appeared to be low, methicillin resistance was confirmed to be associated with multi-drug resistance, suggesting the importance of antimicrobial susceptibility testing of canine S. intermedius strains.

Mycotoxin detection in infant formula milks in Italy.

After birth, infant formulas constitute an important or often sole food source for infants during the first months of life. In this study, a survey on the presence of aflatoxin M1 (AFM1) and ochratoxin A (OTA) in the 14 leading brands of infant formulas marketed in Italy was conducted. Mycotoxins were determined by immunoaffinity column clean-up and high-performance liquid chromatography (HPLC) with fluorescence detection. AFM1 was found in two of 185 samples, but at levels below the European legislation limit of 25 ng l(-1). OTA was detected in 133 (72%) samples (range = 35.1-689.5 ng l(-1)). It has been observed that OTA contamination was 80% in the ready-to-use preparations and 63% in the powdered samples. The Scientific Committee for Food (SCF) reviewed the toxicology on OTA and concluded that it would be prudent to reduce exposure to OTA ensuring that exposure is towards the lower end of the range of tolerable daily intakes of 1.2-14 ng kg(-1) body weight day(-1). OTA was also evaluated by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) and a provisional tolerable weekly intake (PTWI) of 100 ng kg(-1) body weight was established. The OTA levels in pre-term ready-to-use infant formulas were sufficient to cause a higher OTA intake than the suggested TDI. The results point out the need to perform controls for prevention programmes especially when attempting to identify risk markers of the infant feed quality.

In vitro evaluation of the PEtU-PDMS material immunocompatibility: the influence of surface topography and PDMS content.

The aim of the present work is to evaluate the in vitro immunocompatibility of an elastomeric material with feasible applications in the cardiovascular field. In particular, since it is well known that surface chemistry and topography play a key role in the foreign body response, their influence on human monocytes was evaluated. The material, constituted by a poly(ether)urethane (PEtU) and a polydimethylsiloxane (PDMS), was synthesized to manufacture films and small-diameter vascular grafts with three different surface topographical features, smooth, rough and porous, and siloxane rates, 10, 30 and 40. Human THP-1 monocytes have been cultured for 72 h on the films and human blood has been circulating for 2 h into the grafts to assess leukocyte adhesion and cytokine releases. Materials extracts were utilized to evaluate monocyte apoptosis. Smooth films showed lower cell adhesion degrees than rough and porous ones. All the PEtU-PDMS (poly(ether)urethane-polydimethylsiloxane) films and vascular grafts induced a narrow inflammatory response, as demonstrated by slight cytokine secretion levels, in particular samples with the highest PDMS contents (30 and 40%) induced the lowest IL-1b secretion. Moreover, an absence of monocyte apoptosis advises that the negligible release values have not to be ascribed to material toxicity. In the end, surface topography showed to affect only monocyte adhesion while siloxane content the cytokine release. Therefore, the possibility to modify the above tested parameters during material synthesis and manufacture could allow to bound the inflammatory potency of the PEtU-PDMS devices and render them excellent candidates for cardiovascular reconstruction.

Antimicrobial susceptibility of Staphylococcus intermedius and Staphylococcus schleiferi isolated from dogs.

The susceptibility to 23 antimicrobial agents was determined in 114 isolates of Staphylococcus intermedius and eight isolates of Staphylococcus schleiferi of canine origin. Overall, 73% of S. intermedius isolates and 37.5% of S. schleiferi isolates were susceptible to all the 23 antimicrobials tested. The large majority of S. intermedius strains retained susceptibility to antimicrobials currently employed in treatment of pyoderma (cephalosporins, cotrimoxazole and association amoxicillin-clavulanic acid) as well as to those effective against staphylococci (fusidic acid, rifampicin and fluoroquinolones). Resistance in S. intermedius was observed mainly against macrolides, chloramphenicol and lincosamides, while S. schleiferi isolates retained susceptibility to all antimicrobials except three of six fluoroquinolones. Although, our results confirm susceptibility to antimicrobials currently employed in pyoderma treatment, the several different resistance patterns observed for S. intermedius emphasize the importance of antimicrobial susceptibility testing of canine staphylococci to choose the most appropriate treatment of infections and to allow the prudent use of antimicrobial drugs in companion animals.

An optimized digestion method coupled to electrochemical sensor for the determination of Cd, Cu, Pb and Hg in fish by square wave anodic stripping voltammetry.

An optimized digestion method coupled to electrochemical detection to monitor lead, copper, cadmium and mercury in fish tissues was developed. Square wave anodic stripping voltammetry (SWASV) coupled to disposable screen-printed electrodes (SPEs) was employed as fast and sensitive electroanalytical method for heavy metals detection. Different approaches in digestion protocols were assessed. The study was focused on Atlantic hake fillets because of their wide diffusion in the human nutrition. Best results were obtained by digesting fish tissue with hydrogen peroxide/hydrochloric acid mixture coupled to solid phase (SP) purification of the digested material. This combined treatment allowed quantitative extraction from fish tissue (muscle) of the target analytes, with fast execution times, high sensitivity and avoiding organic residues eventually affecting electrochemical measurements. Finally, the method has been validated with reference standard materials such as dogfish muscle (DORM-2) and mussel tissues (NIST 2977).

Pharmacokinetic study of clazuril (Appertex) in eggs and plasma from laying hens after single or multiple treatments, using a new HPLC method for detection.

1. Anticoccidials are widely used as food additives to prevent and treat coccidiosis. They are licensed for use in a prescribed concentration and during a specific time interval with broilers and pullets, but not for laying hens. 2. This study was set up to develop a new high pressure liquid chromatography (HPLC) method to detect clazuril (CZ: (+/-)-2-chloro-alpha-(4-chlorophenyl)-4-(4,5-dihydro-3,5-dioxo-1,2,4-triazin-2(3H)-yl)-benzeneacetonitrile) in egg yolk and albumen and in plasma; to investigate both the presence of residues of CZ in eggs and its pharmacokinetic behaviour in laying hens. 3. A single oral dose (3 mg/kg BW) and multiple oral doses (3 mg/kg BW for 5 d) were investigated. The analytical method gave very good recovery (64 to 74%) in the three different matrices (yolk, albumen and plasma); precision and accuracy were within 11%. 4. After a single dose no residue was detected in eggs collected for up to 10 d, while following multiple dose treatment, CZ residues were detected until 10 d after the end of treatment. The concentration of the drug was higher in yolk than in albumen with a maximum ratio of 10 : 1. 5. Pharmacokinetics of CZ in laying hens after a single dose showed a detectable concentration of the drug up to 24 h. It reached a steady state after the third administration in multiple dosing. 6. Although further studies are necessary, these results indicate that a single oral dose of CZ could be used as an anticoccidial for laying hens due to the lack of residues in eggs.

Antimicrobial susceptibility and mechanism of resistance to fluoroquinolones in Staphylococcus intermedius and Staphylococcus schleiferi.

The objective of the present study was to determine the antimicrobial susceptibility of 136 canine isolates of Staphylococcus intermedius and 10 canine isolates of S. schleiferi subspecies coagulans to 16 fluoroquinolones (FQs), and to investigate the mechanisms of resistance in the nonsusceptible isolates. Of the 136 of S. intermedius tested 98.5% were susceptible to all 16 FQs whereas only 40% of the 10 isolates of S. schleiferi subspecies coagulans were susceptible. Two isolates of S. intermedius and six isolates of S. schleiferi, were found to be resistant to 13 out of 16 FQs, while they retained their susceptibility to fourth generation FQs such as gatifloxacin, moxifloxacin and trovafloxacin. Sequencing of the quinolone-resistance determining regions of gyrA and grlA genes showed that in S. intermedius, dichotomous resistance to FQs was associated with the occurrence of one alteration in GyrA-84 and one in GrlA-80, while in S. schleiferi the same pattern of resistance was observed in isolates showing these changes only in gyrA. This study is the first to screen FQs of the second, third and fourth generation for antimicrobial resistance in clinical isolates of S. intermedius and S. schleiferi of canine origin, and to describe mutations in gyrA and grlA associated with FQ resistance in these bacterial species.

An optical immunosensor for rapid vitellogenin detection in plasma from carp (Cyprinus carpio).

Vitellogenin (vtg) has proven to be a sensitive and simple biomarker for assessing exposure of fish to environmental estrogens. The aim of this work was to develop a rapid, in the order of minutes, screening method for the detection of fish vtg. The surface plasmon resonance technique (Biacore Xtrade mark) was coupled with immunodetection for the determination of fish vtg in plasma and mucus from carp (Cyprinus carpio). Monoclonal anti-vtg antibodies were linked on the sensor surface through chemical cross-linking via a capturing antibody. A simple regeneration process allowed the reuse of the sensor surface. Sensor optimisation was carried out using carp vtg. The developed immunosensor was tested with vtg spiked samples and with plasma and mucus from fish exposed to 17beta-estradiol (E2). Vitellogenin could be detected in the ppm range in buffer as well as in plasma and mucus. Good discrimination between control and exposed samples was obtained. The results were compared with ELISA and a correlation coefficient of R(2)=0.85 (n=9) between the two methods indicated that the immunochemical biosensor could be used for the analysis of vtg in fish plasma samples. The assay time was 20min hence allowing for rapid sample screening.

Pharmacokinetics of clomipramine and desmethylclomipramine after single-dose intravenous and oral administrations in cats.

A cross-over study was performed in six adult spayed cats to determine the pharmacokinetics of clomipramine and its metabolite, desmethylclomipramine (DCMP) after intravenous (0.25 mg/kg) and oral (0.5 mg/kg) single-dose administrations. Plasma clomipramine and DCMP were measured by high-performance liquid chromatography at regular intervals for up to 30 h. Intravenous clomipramine best fit a two-compartmental model yielding an elimination rate constant of 0.037-0.09 h(-1) from which a mean half-life of 12.3 h was calculated. Mean clomipramine AUC(0--infinity) (ngxh/mL), clearance (L/hxkg), V(ss) (L/kg) and MRT (h) values were 652.5, 0.393, 5.0, and 13.5, respectively. Compartmental modeling for clomipramine, after oral administration, and DCMP after both administrations, produced wide parameter estimates and plots of residuals indicated poor goodness of fit. Noncompartmental analysis yielded mean AUC(0--30 h) (ngxh/mL), C(max) (ng/mL) and T(max) (h) of 948.3, 87.5 and 6.2 for clomipramine, and 613.8, 34.8, and 12.8 for DCMP respectively after oral administration. Clomipramine bioavailability was 90%. The present study showed marked pharmacokinetic variability for clomipramine and DCMP through biphasic absorption and potential genetic variability in clomipramine metabolism. It was concluded that population pharmacokinetics would allow better characterization of clomipramine variability that may explain the variability in clinical response noted in cats.

Silicone based polyurethane materials: a promising biocompatible elastomeric formulation for cardiovascular applications.

The biocompatibility of a new material for cardiovascular applications constituted by a poly(ether)urethane (PEtU) and a silicone [polydimethylsiloxane (PDMS)] was evaluated. The achieved material shows properties similar to both polyurethanes and silicones. The material was transformed into porous membranes by a spray-deposition technique. Since any material preparation and manufacturing procedure may introduce some toxicity, in vitro cytotoxicity screening tests were carried out. Human umbilical vein endothelial cells (HUVECs) and a mouse fibroblasts cell line (L929) were cultivated with extracts obtained from materials containing 10, 40 and 100% (w/w) of PDMS. The commercially available Estane 5714-F1 and Cardiothane 51 were used as controls. Extracts were incubated up to 72 hours with HUVECs and L929 cells. The cytotoxic effect was evaluated by light microscopy, cell viability (MTT reduction and neutral red uptake) and proliferation (5-bromo-2'-deoxyuridine incorporation) tests. In vivo studies were carried out using materials containing the same PDMS percentages as for in vitro experiments. The same commercial controls were used. Results obtained with cell culture studies agreed with those obtained in the in vivo experiments and showed that the material preparation and manufacturing procedure do not introduce any toxicity in the products at each PDMS concentration investigated.

A new HPLC method to determine glomerular filtration rate and effective renal plasma flow in conscious dogs by single intravenous administration of iohexol and p-aminohippuric acid.

A high-performance liquid chromatography method to determine iohexol (IOX) and p-aminohippuric acid (PAH) in the plasma of dogs is evaluated according to recovery, reproducibility, and linearity utilizing a gradient pump. The mobile phase consists of 50mM sodium dihydrogen phosphate with 0.5mM tetrabutylammonium chloride, the pH is adjusted to 4.1, methanol is added to the final ratio of 90:10 (v/v), the flow rate is set at 1 mL/min, and separation is achieved with an ODS2 Luna column. The UV detector is set at 254 nm. IOX and PAH are used for evaluation of the effective renal plasma flow (ERPF) and glomerular filtration rate (GFR). The present method tested in three dogs demonstrates the accuracy in the evaluation of ERPF and GFR. Because of its precision and simplicity and low cost, it can be considered a good tool for ERPF and GFR in small animal practice.

Safety of azamethiphos in eel, seabass and trout.

The safety of azamethiphos (AZA), an organophosphorous insecticide and the active ingredient of Salmosan, was evaluated in the European eel, seabass and rainbow trout. Fish were bathed in 0.1 ppm AZA for a period of 60, 120 or 240 min. After termination of each treatment fish were transferred to clean aquaria and randomly sampled over 21 days. Compared to controls, brain acetylcholinesterase (AChE) was inhibited up to 44, 56 and 62% in eels, seabass and trout, respectively, with the inhibition being significant for up to 4 days in eels and seabass and 7 days in trout. As result of the AChE depression, fish displayed motor hyperactivity and erratic jumping at the onset of treatment. Mortality was observed only in trout following exposure for 240 min. A variable correlation observed among species between the level of exposure, the reduced activity of brain AChE and the signs of toxicity suggest that brain AChE should be considered as an indicator of exposure rather than as an index of toxicity of AZA. The present data indicate that at the therapeutic dosage of 0.1 ppm AZA for 1h can be safely used in eels, seabass and trout. The extended treatment times up to 240 min were equally safe for eels and seabass but not for trout.

Effects of liquid and freeze-dried grapefruit juice on the pharmacokinetics of praziquantel and its metabolite 4'-hydroxy praziquantel in beagle dogs.

Grapefruit juice changes the pharmacokinetic parameters of a variety of drugs metabolized primarily by cytochrome P450 3A. In a three-phase crossover study, six male beagle dogs were administered 100 ml of water (control), 100 ml of commercial liquid grapefruit juice, or 10 g of freeze-dried grapefruit juice (equivalent to 100 ml of liquid grapefruit juice) with 100 ml of water, followed after 2 h by single oral dose of praziquantel (30 mg kg(-1)). After treatment, the dogs were sampled at different times. Determination of praziquantel and its metabolite 4'-hydroxy praziquantel (identified by GC/MS) was performed by HPLC. Liquid and freeze-dried grapefruit juice preadministration increased the C(max) of praziquantel about three-fold and the AUC 2.5- and 2.3-fold, respectively. The T(max) (0.75 h) was unaffected by liquid or freeze-dried grapefruit juice, while T(1/2) was 2.3- and 1.7-fold higher compared with controls. The amount of 4'-hydroxy praziquantel was also affected by both liquid and freeze-dried grapefruit juice administration: the AUC and C(max) increased four- and three-fold, respectively and the T(max) was significantly enhanced. These findings demonstrate that both freeze-dried grapefruit juice and commercial liquid grapefruit juice significantly increase plasma concentrations and T(1/2) of praziquantel in dogs.