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Porcine epidemic diarrhoea virus (PEDV) infects pigs of all ages causing vomiting and diarrhoea. PEDV is transmitted via the oral-faecal route, and a very low dose is enough to infect susceptible pigs, resulting in significant production losses. This short communication aims to describe the introduction of PEDV into a 10,000-sow farrow-to-wean farm located in northwest Mexico. Following the onset of clinical signs, an outbreak investigation was conducted to determine the most probable route of introduction. Based on data collected from interviews, construction of a timeline of events, and the detection of PEDV RNA in feed samples and samples collected from various surfaces of feed transport vehicles, it was concluded that the most probable route for PEDV incursion into this breeding herd was contaminated feed or a contaminated feed transport vehicle. This paper describes how feed or feed transport could serve as potential routes of PEDV infection to a farm and highlights the importance of establishing biosecurity programs to mitigate these risks.

Background Influenza A virus (IAV) is an important pathogen in pigs that affects productivity and has important public health implications because of its zoonotic nature. Surveillance is central to the control of influenza, however, detection of IAV infections can be challenging in endemically infected herds with low prevalence of infection. Methods In groups of suckling (18–21 days of age) and growing (35–45 days of age) pigs, we compared various sampling approaches to detect, isolate and sequence IAV using individual (nasal swabs, nasal wipes and oropharyngeal swabs), group (oral fluids, surface wipes and sow udder skin wipes) and environmental (airborne particles deposited on surfaces and air samples) sampling approaches. All samples were tested by IAV rRT-PCR and a subset was used for virus isolation and direct sequencing. Results In general, environmental and group samples resulted in higher odd ratios (range = 3.87–16.5, p-value

Porcine epidemic diarrhea virus (PEDV) produces infection in pigs characterized by vomiting and diarrhea. PEDV is transmitted via oral-fecal and a very low oral dose is enough to infect susceptible pigs, causing devastating consequences in production. A 10,000-sow farrow-to-wean farm located in northwest Mexico was infected with PEDV. After the observation of the first clinical signs, an outbreak investigation take into place to determine the most probably source of infection. A systematic collection of samples including rectal swabs, gestation and lactation feed, surface swabs from the interior or feed bins and many points of the feed truck delivering the implicated feed was performed. Samples were tested for PEDV polymerase chain reaction (PCR). Positive PCR results showed the evidence of PEDV RNA in lactating feed, the interior walls of the feed bins and in the interior of the auger boom of the feed truck. This, connected with the location of first clinical signs point that the most probably incursion of PEDV in to this breeding herd was contaminated feed. This paper shows how feed or feed transport can be a potential source of PEDV infection in farms and highlight the importance of stablishing biosecurity programs to mitigate the risk of PEDV infections.

Antemortem detection of Mycoplasma hyopneumoniae infection in swine production systems has relied on antibody testing, but the availability of tests based on DNA detection and novel diagnostic specimens, e.g., tracheal swabs and oral fluids, has the potential to improve M. hyopneumoniae surveillance. A field study was performed over a 14-week period during which 10 pigs in one pen at the center of a room with 1,250 6-week-old pigs housed in 46 pens were intratracheally inoculated with M. hyopneumoniae., Antemortem detection of Mycoplasma hyopneumoniae infection in swine production systems has relied on antibody testing, but the availability of tests based on DNA detection and novel diagnostic specimens, e.g., tracheal swabs and oral fluids, has the potential to improve M. hyopneumoniae surveillance. A field study was performed over a 14-week period during which 10 pigs in one pen at the center of a room with 1,250 6-week-old pigs housed in 46 pens were intratracheally inoculated with M. hyopneumoniae. Thereafter, one tracheal sample, four serum samples, and one oral fluid sample were collected from every pen at 2-week intervals. Tracheal and oral fluid samples were tested for M. hyopneumoniae DNA and serum samples for M. hyopneumoniae antibody. Test results were modeled using a hierarchical Bayesian model, based on a latent spatial piecewise exponential survival model, to estimate the probability of detection by within-pen prevalence, number of positive pens in the barn, sample allocation, sample size, and sample type over time. Analysis showed that tracheal samples provided the earliest detection, especially at large sample sizes. While serum samples are more commonly collected and are less expensive to test, high probability of detection estimates were only obtained 30 days postexposure at large sample sizes. In all scenarios, probability of detection estimates for oral fluids within 30 days were significantly lower than those for tracheal and serum samples. Ultimately, the choice of specimen type, sample number, and assay will depend on testing objectives and economics, but the estimates provided here will assist in the design of M. hyopneumoniae surveillance and monitoring programs for different situations.

Processing fluid samples are easily collected under field conditions and provide the means to test more piglets more frequently in a practical way, thereby improving PRRSV surveillance. However, a deeper understanding of the diagnostic characteristics of this newly described sample type is still required. Therefore, the objective of this field-based study was to determine the relationship between viremic piglets and the detection of PRRSV RNA in processing fluid samples. In two PRRSV-positive breeding herds, processing fluids (n = 77) and individual piglet serum samples (n = 834) were collected from 77 litters in three sampling events and tested for PRRSV RNA. Among the 77 litters in the study, 55 litters (71.4%) contained no viremic piglets and processing fluids tested negative for PRRSV RNA. Among the 22 (28.6%) litters with ≥1 viremic piglets, 10 litters contained a single viremic piglet and 5 of the 10 processing fluids from this group tested positive for PRRSV RNA. Based on a fitted mixed effects logistic regression model, the probability of detecting PRRSV RNA in processing fluids was highly dependent on the number of viremic piglets contributing to the sample. When the within-litter prevalence was ≥39%, the probability of detecting PRRSV RNA in processing fluids was ≥95%. By extension, the results suggest that pooling processing fluids from several litters increases the probability of PRRSV RNA detection because of the greater likelihood of including multiple litters each with ≥1 viremic piglets. In contemporary breeding herds that use processing fluid samples for PRRSV surveillance, the diagnostic costs associated with testing 100% of the processing-age piglet population can be estimated at €0.077 ($0.086 USD) per pig weaned. In contrast, to achieve an equivalent testing coverage with the use of individual piglet serum samples, the diagnostic costs associated would be €4.48 ($5.00 USD) per pig weaned. Processing fluid represents a practical, reliable and efficient method to surveil breeding herds for PRRSV because it allows for continuous surveillance at a low cost.

There has been a tremendous increase in recent years of population-based diagnostic monitoring and surveillance strategies in swine populations. One example is the use of processing fluids (PF) to screen breeding herds for porcine reproductive and respiratory syndrome virus (PRRSV) activity. An important question from practitioners using such methods is on how intensively can the sample be pooled. More specifically, processing fluids of how many litters can be pooled into a single sample for diagnostic testing to preserve a high probability of PRRSV RNA detection at low prevalence situations? The objective of this study was to model the effect of pooling PF samples on the probability of PRRSV RNA detection. For this study, a PRRSV-positive PF field sample with a RT-rtPCR quantification cycle (Cq) value of 28 was selected to represent a litter of 11 pigs with a single viremic piglet. PF samples from a PRRSV-naïve herd were used to perform 6 replications of 8 two-fold serial dilutions of the PRRSV-positive sample, thus modeling the pooling effect (dilution). Each two-fold dilution represented an increase in the number of PRRS-negative pigs in the sample by a factor of 2. Samples were tested for PRRSV RNA by RT-rtPCR and the data was analyzed using linear and probit regression models. There was an average increment of 1.37 points in Ct for each two-fold dilution. The estimated probability of testing positive on RT-rtPCR was 43 %, 80 %, and 95 % when there was a single PRRSv-positive piglet among 784, 492, and 323 PRRSv-negative piglets contributing to the sample respectively. Results from this study support the practice of collecting and aggregating PF samples from multiple litters for PRRSV RNA testing.

To control and eliminate porcine reproductive and respiratory syndrome virus (PRRSv) from breeding herds, some veterinarians adopt a strategy called load-close-expose which consists of interrupting replacement pig introduction for several months and exposing the pigs to a replicating PRRSv. This was a prospective quasi-experiment that followed 61 breeding herds acutely infected with PRRSv that adopted one of two exposure programs: modified-live virus (MLV) vaccine or live-resident virus inoculation (LVI). Treatment groups (load-close-expose with MLV or LVI) were compared for: (a) time-to-PRRSv stability (TTS), defined as time in weeks it took to produce PRRSv negative pigs at weaning; (b) the time-to-baseline production (TTBP), defined using statistical process control methods to represent time to recover to the number of pigs weaned per week that herds had prior to PRRSv-detection; and (c) the total production loss in terms of number of pigs weaned per week. TTS and TTBP were compared between treatments using survival analysis. Day 1 of the program was considered to be the day that treatment was administered. Sampling at herds consisted of bleeding 30 due-to-wean piglets on a monthly basis. Serum was tested for PRRSv RNA by RT-PCR. Herds in which PRRSv was not detected over a 90-day period were classified as reaching stability. Multivariate analysis using proportional hazards regression was performed adjusting the effect of treatment on TTBP and TTS to 'severity of PRRSv infection', 'number of whole-herd exposures', 'days from PRRSv-detection to intervention', 'prior PRRSv-infection status' and 'veterinary clinic associated with the herd'. Total loss was compared between groups using multivariate regression analysis adjusted by selected covariates. The median TTS among participating herds was 26.6 weeks (25th to 75th percentile, 21.6-33.0 weeks). The overall TTBP was 16.5 weeks (range 0-29 weeks). The magnitude of production losses following whole-herd exposure averaged 2217 pigs not weaned/1000 sows and was correlated with TTBP. Herds in the MLV group recovered production sooner and had less total loss than herds in the LVI group. TTBP and TTS were significantly shorter and the total loss was significantly less in herds assisted by a specific veterinary clinic and herds that were infected with PRRSv in the 3 years prior to the study. This study provided new metrics to assist veterinarians to decide between methods of exposure to control and eliminate PRRSv from breeding herds.

This paper describes a polymer manufacturing technique for the realization of sealed liquid tanks with a high filling ratio. It is based on a lithographic process combined with lamination and can be performed on any type of substrate: Si, glass or polymer films. We demonstrated the encapsulation of solvents or inks in multilevel tanks on large surface area (>118 cm2) without any damage of the stored liquid. This micro-fabrication technology proposes a significant breakthrough for micro-fluidic applications. We propose a one step process for the filling and sealing of tanks which are performed simultaneously. Tanks can be integrated at any level in the micro-fluidic system with a perfect filling rate and an excellent tightness.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes an acute, viremic infection of 4 to 6 weeks, followed by a persistent infection lasting for several months. We characterized antibody and B-cell responses to viral proteins in acute and persistent infection to better understand the immunological basis of the prolonged infection. The humoral immune response to PRRSV was robust overall and varied among individual viral proteins, with the important exception of a delayed and relatively weak response to envelope glycoprotein 5 (GP5). Memory B cells were in secondary lymphoid organs, not in bone marrow or Peyer's patches, in contrast to the case for many mammalian species. Potent anti-PRRSV memory responses were elicited to recall antigen in vitro, even though a second infection did not increase the B-cell response in vivo, suggesting that productive reinfection does not occur in vivo. Antibody titers to several viral proteins decline over time, even though abundant antigen is known to be present in lymphoid tissues, possibly indicating ineffective antigen presentation. The appearance of antibodies to GP5 is delayed relative to the resolution of viremia, suggesting that anti-GP5 antibodies are not crucial for resolving viremia. Lastly, viral infection had no immunosuppressive effect on the humoral response to a second, unrelated antigen. Taking these data together, the active effector and memory B-cell responses to PRRSV are robust, and over time the humoral immune response to PRRSV is effective. However, the delayed response against GP5 early in infection may contribute to the prolonged acute infection and the establishment of persistence.

Objective—To determine effects of vaccination protocols with modified-live porcine reproductive and respiratory syndrome virus (PRRSV) vaccine on persistence and transmission of virus in pigs infected with a homologous isolate and determine clinical and virologic responses following heterologous viral challenge. Animals—Four hundred forty 6- to 8-week-old PRRSV-naïve pigs. Procedures—Pigs were allocated into 5 groups. Groups A to D were inoculated with wild-type PRRSV VR2332. Group A (positive control pigs) received PRRSV only. Groups B, C, and D received modified-live PRRSV vaccine (1, 2, or 3 doses). Group E served as a negative control group. To evaluate viral transmission, sentinel pigs were introduced into each group at intervals from 37 to 67, 67 to 97, and 97 to 127 days postinoculation (DPI). To evaluate persistence, pigs were euthanized at 37, 67, 97, or 127 DPI. To assess clinical and virologic response after challenge, selected pigs from each group were inoculated at 98 DPI with a heterologous isolate (PRRSV MN-184). Results—Mass vaccination significantly reduced the number of persistently infected pigs at 127 DPI. Vaccination did not eliminate wild-type PRRSV; administration of 2 or 3 doses of modified-live virus vaccine reduced viral shedding after 97 DPI. Previous exposure to wild-type and vaccine virus reduced clinical signs and enhanced growth following heterologous challenge but did not prevent infection. Conclusions and Clinical Relevance—Findings suggest that therapeutic vaccination may help to reduce economic losses of PRRSV caused by infection; further studies to define the role of modified-live virus vaccines in control-eradication programs are needed.

Objective—To use mathematical modeling to assess the effectiveness of control strategies for porcine reproductive and respiratory syndrome (PRRS) virus on a swine farm. Sample—A hypothetical small, medium, or large farrow-to-weaning swine farm in the Midwestern United States. Procedures—Stochastic models were formulated to simulate an outbreak of PRRS on a farm. Control strategies assessed in those models included none (baseline) and various combinations of mass immunization, herd closure, and gilt acclimatization. Nine different models resulting from the combination of low, moderate, or high PRRS virus virulence and small, medium, or large herd size were simulated. A stabilized status, the outcome of interest, was defined as the absence of positive PCR assay results for PRRS virus in 3-week-old piglets. For each scenario, the percentage of simulations with a stabilized status was used as a proxy for the probability of disease control. Results—Increasing PRRS virus virulence and herd size were negatively associated with the probability of achieving a stabilized status. Repeated mass immunization with herd closure or gilt acclimitization was a better alternative than was single mass immunization for disease control within a farm. Conclusions and Clinical Relevance—Repeated mass immunization with a PRRS modified-live virus vaccine with herd closure or gilt acclimitization was the scenario most likely to achieve a stabilized status. Estimation of the cost of various PRRS control strategies is necessary.

Transferring a gene into cancer cells in order to sensitize them to drugs is an important approach in human cancer gene-therapy research. Thymidine phosphorylase (TP) is the first enzyme in the metabolic activation pathway of 5-fluorouracil (5-FU) to fluorodeoxyribonucleotides, thus, it could be used to increase the sensitivity of cancer cells to this anti-pyrimidine agent. In this study, an expression vector containing the human TP cDNA was transfected into C26 murine colon-carcinoma cells. Stable transfectants were selected; all showed increased TP activity, ranging from 2- to 10-fold when compared with wild-type cells. The in vitro sensitivity of transfectants to 5-FU and 5'-deoxy-5-fluorouridine (5'-DFUR) was enhanced, in agreement with the observed increase in TP activity. Then, tumors were generated by s.c. injection of TP-transfected or wild-type C26 cells in syngeneic BALB/c mice. 5-FU (25 mg/kg, i.p.) induced a growth delay of TP-transfected C26 tumors as compared with C26 wild-type tumors. These data suggest that TP could be transfected in tumor cells to increase the sensitivity to 5-FU for subsequent cancer gene therapy.

La présente invention a pour objet un procédé de fabrication d'un élément optique comprenant à sa surface des cellules remplies d'un aérogel qui ne se fissure ni lors de sa fabrication, ni lors d'une étape postérieure d'imprégnation de l'aérogel par un liquide. La présente invention a également pour objet les éléments optiques susceptibles d'être obtenus par ce procédé.

Airborne transmission of porcine reproductive and respiratory syndrome virus (PRRSV) is a risk factor for the infection of susceptible populations. Therefore, a long‑term sustainability study of air filtration as a means to reduce this risk was conducted. Participating herds (n = 38) were organized into 4 independent cohorts and the effect of air filtration on the occurrence of new PRRSV infections was analyzed at 3 different levels from September 2008 to January 2012 including the likelihood of infection in contemporary filtered and non-filtered herds, the likelihood of infection before and after implementation of filtration and the time to failure in filtered and non-filtered herds. Results indicated that new PRRSV infections in filtered breeding herds were significantly lower than in contemporary non-filtered control herds (P < 0.01), the odds for a new PRRSV infection in breeding herds before filtration was 7.97 times higher than the odds after filtration was initiated (P < 0.01) and the median time to new PRRSV infections in filtered breeding herds of 30 months was significantly longer than the 11 months observed in non-filtered herds (P < 0.01). In conclusion, across all 3 levels of analysis, the long-term effect of air filtration on reducing the occurrence of new PRRSV infections in the study population was demonstrated.

8 pages; International audience; Mineral liquid crystals are materials in which mineral's intrinsic properties are combined with the self-organization behavior of colloids. However, the use of such a system for practical application, such as optical switching, has rarely been demonstrated due to the fundamental drawbacks of colloidal systems such as limited dispersion stability. Studying colloidal suspensions of LaPO 4 nanorods, it is found that drastic improvement of colloidal stability can be obtained through a transfer of particles from water towards ethylene glycol, thus enabling the investigation of liquid crystalline properties of these concentrated suspensions. Using polarization microscopy and small-angle x-ray scattering (SAXS), self-organization into nematic and columnar mesophases is observed enabling the determination of the whole phase diagram as a function of ionic strength and rod volume fraction. When an external alternative electric field is applied, a very efficient orientation of the nanorods in the liquid-crystalline suspension is obtained, which is associated with a signifi cant optical birefringence. These properties, combined with the high colloidal stability, are promising for the use of such high transparent and athermal material in electro-optical devices.

International audience; Spatial light modulators are often used to implement phase modulation. Since they are pixelated, the phase function is usually approximated by a regularly sampled piecewise constant function, and the periodicity of the pixel sampling generates annoying diffraction peaks. We theoretically investigate two pixelation techniques: the isophase method and a new nonperiodic method derived from the Voronoi tessellation technique.We show that, for a suitable choice of parameters, the diffraction peaks disappear and are replaced by a smoothly varying halo. We illustrate the potential of these two techniques for implementing a lens function and wavefront correction.

A multicentre validation study of the acute in vitro cytotoxicity of drugs involving six French laboratories from INSERM or pharmaceutical companies has been carried out. Thirty liquid or solid chemicals such as antibiotics, anticancer drugs and solvents were selected and incubated for 20 hr with normal rat hepatocytes and FaO hepatoma cells. Miniaturized and automated methods were defined for the evaluation of cytotoxic effects. Four endpoints were evaluated: the ratio of extracellular lactate dehydrogenase to total lactate dehydrogenase, total cellular protein content, reduction of a tetrazolium salt, and neutral red uptake. For each test IC(50) values were calculated. A good interlaboratory reproducibility was demonstrated. The neutral red assay was found to be the most sensitive and the least reproducible endpoint. More compounds were shown to be cytotoxic to hepatocytes than to hepatoma cells (18 v. 12). On the basis of the IC(50) values a few compounds were found to be much less cytotoxic than predicted from in vivo data, suggesting that a simple experimental protocol and non-specific cytotoxicity parameters are not sufficient to test certain drug families. However, such methods appear to provide a useful means of defining the concentration range of the drug that will be selected for further analysis using more specific tests.

An open study was carried out to examine the effect of moclobemide, a new antidepressant reversible inhibitor of MAO-A, on the pressor response induced by oral tyramine added to meals of different lipid and protein composition, and to correlate the blood pressure increase in the tyramine test with that obtained during an exercise test. Eight healthy volunteers of both sexes participated in the study. A tyramine sensitivity and an exercise test were performed beforehand. Subjects were included if, under fasting condition, their systolic blood pressure (SBP) increased by more than 30 mmHg after administration of 400 or 600 mg tyramine. Exercise tests were performed to determine the grade of effort that corresponded to a rise in SBP of 30 mmHg. Subjects received moclobemide 600 mg/d. Starting on Day 7, each subject consumed a standardized meal (52 g lipids, 43 g proteins, 86 g carbohydrates) just before taking moclobemide. Tyramine was added to these meals in daily increasing doses of 50, 100, 150...mg until an increase in SBPor = 30 mmHg was obtained. On moclobemide treatment, an average dose of 250 mg tyramine (range 150-400 mg) increased SBP by 36.6 mmHg. The time to reach peak SBP was longer (175 min) than in the fasting condition before the trial (40.6 min).(ABSTRACT TRUNCATED AT 250 WORDS)

The cytotoxicity of 30 chemicals was assessed in rat hepatocyte primary cultures using four methods: lactate dehydrogenase release, neutral red uptake, the MTT assay, and measurement of total protein content. Comparison of the data obtained in vitro (IC50 values) and in vivo (LD50 values) resulted in a significant correlation (p Assessment of the cytotoxicity of 54 additional chemicals would provide a more accurate predictability limit around l,500μg/ml and the estimated predictability confidence interval could be reduced to 90–100%.

Deamination of cytosine arabinoside (ara-C) by cytidine deaminase is the main mode of inactivation of this drug which can be responsible for ara-C resistance. The present study was undertaken to determine the effect of tetrahydrouridine (THU; a potent inhibitor of cytidine deaminase) on ara-C transport and metabolism in human cells. A rapid transport of ara-C into freshly isolated hepatocytes and an increased intracellular accumulation of the unchanged drug were observed in the presence of 50 micrograms/ml THU. THU inhibited the intracellular deamination of ara-C by 80% and slowed elimination of the compound extracellularly. The intracellular ara-C concentrations achieved after incubation with 1 micrograms/ml ara-C plus 50 micrograms/ml THU are similar to those attained with ara-C (10 micrograms/ml) alone. Treatment of leukemic K562 cells with the combination of THU (50 micrograms/ml) and ara-C (1 micrograms/ml) led to an augmentation of intracellular ara-C triphosphate formation up to twofold.

High-performance liquid chromatography with UV detection (HPLC-UV) and gas chromatography with either nitrogen phosphorus (GC-NPD) or mass spectrometry (GC-MS) detection were used for the determination of ticlopidine in plasma. Solid-phase extraction of ticlopidine from plasma was performed using Extrelut columns without pH adjustment, and using hexane as the solvent of elution. With HPLC, a mobile phase of 0.01 M pH 7.8 phosphate buffer:acetonitrile (70:30) was passed through a mu Bondapack C-18 column at a rate of 1.3 mL/min. Ultraviolet detection at 235 nm was sensitive to plasma ticlopidine concentrations of 0.05 micrograms/mL. The GC-NPD and GC-MS were performed on a DB-17 fused-silica column using on-column injection. For GC-NPD and GC-MS, limits of quantification were found to be 0.020 and 0.005 micrograms/mL, respectively. Compared with HPLC-UV, the GC methods were found to be more reproducible, sensitive, and specific and therefore more suitable for pharmacokinetic applications.

In vitro metabolism of digoxin and its cleavage-related compounds was investigated using hepatocytes in primary culture and microsomal fractions both isolated from human livers. On these models, digoxin (DG3) and digoxigenin bisdigitoxoside (DG2) were not shown to be significantly metabolized in vitro in man. Therefore, it appeared that the stepwise cleavage of DG3 and DG2 sugars was not cytochrome P450 dependent. This enzymatic system probably plays a minor role in humans for this particular reaction. However, digoxigenin monodigitoxoside (DG1) and digoxigenin (DG0) which are known to be formed after intra-gastric hydrolysis of DG3, were extensively converted to polar compounds (mainly glucuronides). In addition, using human liver microsomes, a wide variability in UDP-glucuronyl transferase (UDPGT) activities responsible for DG1 glucuronidation was demonstrated. These results suggest that two main factors may contribute to the overall interindividual variability of digoxin biotransformation: 1), the individual intra-gastric pH which influences the sugar cleavage leading to DG1 and DG0; ii), a variability in the level of the hepatic UDPGT specific for digitalis compounds conjugation.

Metabolism of mitoxantrone was studied in primary cultures of hepatocytes freshly obtained from rat, rabbit and humans. Metabolic pattern was evaluated by a high performance liquid Chromatographic method which specifically resolved mitoxantrone from its mono- and dicarboxylic acid derivatives. Studies were carried out over 48 hr and at [ 14 C]mitoxantrone concentrations ranging between 1 and 20 μM. In all species studied, metabolism occurred: extracellular unchanged mitoxantrone concentrations represented around 50, 25 and 20% of total extracellular radiolabel at 48 hr in rat, rabbit and humans, respectively. Although minor interspecies variability was observed in total amount of drug biotransformed by hepatocytes, large variability in the metabolic pattern occurred between the different species: hence, in rats the main derivatives were two polar compounds and only trace amounts of the mono- and dicarboxylic acid metabolites were present. In both rabbits and humans however, these polar derivatives represented minor metabolic pathways and the main metabolites were the mono- and dicarboxylic acid derivatives. While the percentage of total biotransformation was similar in these two latter species, the monocarboxylic acid derivative was the main metabolite in rabbits while the dicarboxylic acid was predominant in humans. Only small interindividual differences (N = 4) were observed in the metabolism of mitoxantrone by human hepatocytes in primary culture. These data demonstated that: (i) in all species, mitoxantrone was biotransformed into metabolites which rapidly effluxed in the extracellular compartment, (ii) there were low interspecies differences between rat, rabbit and humans in terms of total biotransformed drug, but (iii) large interspecies variability was demonstrated in the qualitative (rat versus both rabbit and human) and relative (rabbit versus man) patterns of the metabolites. Furthermore, the metabolism of mitoxantrone was linear over a wide range of concentrations (i.e. 1–20 μM). In conclusion, rabbit appears to be the animal species most closely related to humans in terms of mitoxantrone metabolism.

The pharmacokinetic behaviour of flunitrazepam and its main active metabolite, N-desmethyl flunitrazepam, was investigated in 12 patients with liver disease (cirrhosis or hepatitis) compared to 6 healthy volunteers. A gas-liquid chromatographic method allowing for simultaneous determination of flunitrazepam and N-desmethyl flunitrazepam in plasma samples was developed. The accuracy and the precision near the quantification limit of ca. 1 ng/ml were better than 5%. Plasma levels of flunitrazepam were not significantly altered by hepatic failure, whereas plasma levels of N-desmethyl flunitrazepam were lower in patients than in healthy subjects. Pharmacokinetic parameters did not differ significantly between healthy subjects and liver disease patients: the oral clearance was 3.5 +/- 0.8, 3.5 +/- 1.9 and 4.0 +/- 1.2 ml/min/kg, respectively in healthy subjects, patients with hepatitis and patients with cirrhosis. The apparent elimination half-life was 22 +/- 5 h in healthy subjects, 25 +/- 10 h in patients with hepatitis and 20 +/- 6 h in patients with cirrhosis. However, the expected increase of the drug free fraction during liver disease could decrease the therapeutic and toxic ranges of flunitrazepam in these patients.

Over past decades, numerous in vitro and/or ex vivo models have been developped to investigate drug metabolism. In the order of complexity we found the isolated perfused liver, hepatocytes in co-culture with epithelial cells, hepatocytes in suspension and in primary culture and subcellular hepatic microsomal fractions. Because they can be easily prepared from both animals (pharmacological and toxicological species) and humans (whole livers as well as biopsies obtained during surgery) hepatocytes in primary culture provide the most powerfull model to better elucidate drug behavior at an early stage of preclinical develompent such as: the characterization of main biotransformation reactions, the identification of phase I and phase II isozymes involved in such reactions, the evaluation of inter-species differences allowing the selection of a second toxicological animal species more closely related to man on the basis of metabolic profiles, the detection of the inducing and/or inhibitory effects of a drug on metabolic enzymes, the prediction of drug interactions, the estimation of inter-individual variability in biotransformation reactions. The use of hepatocytes, and in particular those obtained from humans, at an early stage of drug development allows the obtention of more predictive preclinical data and a better knowledge of drug behavior in humans before the first administration of the drug in healthy volunteers.

Surveillance of porcine reproductive and respiratory syndrome (PRRS) in negative sow farms is usually performed by testing for the presence of antibodies against PRRS virus in serum with a commercial ELISA test. The objective of this study was to evaluate the feasibility of pooling serum samples for detection of PRRS virus antibodies by ELISA. The effect of pool size on the sensitivity and specificity of the ELISA test was evaluated by testing true positive samples and false positive samples, respectively, diluted in negative sera. The effect of three different cut-off values for the interpretation of the diagnostic test (0.4, 0.3 and 0.2) was evaluated as well. Furthermore, the obtained sensitivity and specificity estimates were used to calculate the herd sensitivity and herd specificity of surveillance protocols in different scenarios. The results showed that pooling serum samples to detect PRRSV antibodies resulted in a decrease in sensitivity and an increase in specificity, compared to testing individual samples, while the reduction of the s/p cut-off value recommended by the manufacturer (0.4) had the opposite effect. We describe an approach that can increase the herd sensitivity of a surveillance protocol for breeding herds, while maintaining high herd specificity and low testing costs. This can be achieved by sampling a larger number of animals and running the samples in pools. Therefore, the conventional monitoring protocols based on ELISA on individual samples can be improved by using pooled-sample testing.

handled in an identical manner, using two other pigs. Each pig was placed in a separate room to prevent cross-contamination. Blood was collected on day 7 and day 14 after infection and tested by PCR and ELISA. To test whether the hands of personnel contaminated with PRRSV – positive meat juice could infect naive pigs, 4 ml aliquots of juice from meat samples on day 0 and day 1 after thawing were placed on to the palm of the left hand (S. A. D.) and spread across its surface. Immediately following contact with the juice (0 minutes), the contaminated palm was made to contact a PRRSV-naive pig. The pig was then placed in a separate pen, the hand were washed and the palm and fingers of the left hand swabbed using sterile swabs (Dacron; Fisher Scientific). The process was then repeated twice more using different pigs and either 15 or 30 minutes between contamination and contact. For the purpose of controls, the process was repeated using sterile saline. The pigs were housed in separate pens in different rooms to avoid cross-contamination, blood sampled on day 7 and day 14 after contact with the saline, and the samples were tested by PCR and ELISA as previously described. Throughout the study, strict biosecurity protocols were practiced to minimise the risk of accidental transmission of PRRSV between pigs, according to previously published protocols (Otake and others 2002, Dee and others 2004). PRRSV RNA concentrations detected in the meat samples are summarised in Table 1. There were no significant differences in the mean concentration of PRRSV RNA over time (P=0·72). Viable PRRSV was detected in samples from all eight sampling days as confirmed by positive PCR and ELISA results from the eight bioassay pigs. Naive pigs contacting the hands of personnel at 0 and 30 minutes following contamination with juice from PRRSV-positive meat samples became infected, while the pig exposed to contaminated hands 15 minutes following contact with meat juice remained negative. All swabs taken from hands after washing and all samples from negative control animals were PRRSV negative. The results of this study support previously published information documenting the survival of infectious PRRSV in muscle tissue when stored at 4°C (Bloemraad and others 1994). Furthermore, in the absence of sanitation, the hands of personnel may harbour viable virus (Otake and others 2002). Justification for the study was based on observations of the introduction of non-processed meat from PRRSV-infected populations in PRRSV-naive sow herds. While initially presumed to be rare occurrence, follow-up discussions with swine practitioners from four states throughout the USA (Minnesota, Indiana, North Carolina, and Texas), as well as Mexico and South America, indicated that the practice of introducing fresh or frozen pork is not uncommon at the commercial production level. Evaluation of the survival of porcine reproductive and respiratory syndrome virus in non-processed pig meat

The purpose of this study was to compare 4 methods for the reduction of aerosol transmission of Porcine reproductive and respiratory syndrome virus (PRRSV): high-efficiency particulate air (HEPA) filtration, 2x-low-cost filtration, bag filtration, and use of a filter tested against particles derived from dioctylphthalate (DOP). The HEPA-filtration system used a prefilter screen, a bag filter (Eurovent [EU] 8 rating), and a HEPA filter (EU13 rating). The low-cost-filtration system contained mosquito netting (prefilter), 2 fiberglass furnace filters, and 2 electrostatic furnace filters. Bag filtration involved the use of a filter rated EU8 and a minimum efficiency reporting value (MERV) of 14. The 95%-DOP, 0.3-microm-filtration system involved a pleat-in-pleat V-bank disposable filter with a 95% efficiency rating for particles 0.3 microm or greater in diameter and ratings of EU9 and MERV 15. No form of intervention was used in the control group. The experimental facilities consisted of 2 chambers connected by a 1.3-m-long duct containing the treatments. Recipient pigs, housed in chamber 2, were exposed to artificial aerosols created by a mechanically operated mister containing modified live PRRSV vaccine located in chamber 1. Aerosol transmission of PRRSV occurred in 0 of the 10 HEPA-filtration replicates, 2 of the 10 bag-filtration replicates, 4 of the 10 low-cost-filtration replicates, 0 of the 10 95%-DOP, 0.3-microm-filtration replicates, and all 10 of the control replicates. Using a similar approach, we further evaluated the HEPA- and 95%-DOP, 0.3-microm-filtration systems. Infection was not observed in any of the 76 HEPA-filtration replicates but was observed in 2 of the 76 95%-DOP, 0.3-microm replicates and 42 of the 50 control replicates. Although the difference between the 95%-DOP, 0.3-microm and control replicates was significant (P0.0005), so was the level of failure of the 95%-DOP, 0.3-microm system (P = 0.02). In conclusion, under the conditions of this study, some methods of air filtration were significantly better than others in reducing aerosol transmission of PRRSV, and HEPA filtration was the only system that completely prevented transmission.

International audience; We have made an attempt to identify the true material stress-strain curves of some amorphous polymers (PC, PMMA, CR39 (diethylene glycol bis(allyl carbonate)) from nanoindentation experiments by using non-self similar tip shapes, i.e. tips promoting an increasing strain with an increasing indentation depth. We firstly found that, for a defined strain rate, the relationship linking the contact pressure to the ratio between the contact depth and the contact radius is intrinsic to the material, i.e. it doesn't depend on the tip shape used. From this relationship, for each material, we calculated the evolution of the average stress with the average strain beneath the tip, by using the theoretical background developed for elasto-perfectly plastic materials. By comparing to compression test results, we concluded that this approach works well for PC and PMMA only for an average strain below 12%. This approach is inapplicable for the CR39 material even at low strains. We assumed that this is mainly due to the occurrence of a significant strain hardening, that doesn't allow us to neglect the strain gradient existing beneath the tip.

Liverbeads, cryopreserved hepatocytes entrapped within an alginate matrix, were examined for their relevance in the comet assay. It was estimated by their capacity to activate the indirectly acting mutagens, cyclophosphamide (CP), benzo[a]pyrene (BP), dimethylbenzanthracene (DMBA) and 2-acetylaminofluorene (2-AAF), into DNA reactive metabolites. The comet assay performed in alkaline condition is a sensitive method for detecting strand breaks at the level of individual cells and allows use of quiescent cells. Experimental conditions as treatment time, cell density, beads dissociation and viability were investigated. Significant statistical positive results assessed by the tail extent moment (TEM) were observed with both human and rat Liverbeads after 12h duration incubation compared to metabolic non-competent cells, HeLa S3. Due to the maintenance of specific functions assessed by the observed capacity to metabolize xenobiotics, Liverbeads represent a suitable tool system, easy to handle, for the detection of promutagens using the comet assay.

The induction of DNA damage by four known promutagens (cyclophosphamide (CP), benzo( a )pyrene (BP), dimethylbenz( a )anthracene and 2-acetylaminofluorene (2AAF) was investigated on Hep G2 using the alkaline single cell electroporesis (SCGE) test, most often referred as the “comet assay”. After a 3-day incubation, lysed cells embedded in agarose were electrophoresed under alkaline conditions, dyed with a SYBR®gold fluorogen and analysed by the Komet® software. Among the comet parameters provided by the image analysis program, statistical analysis did not identify any in particular that could best represent the DNA damages. All promutagens, when compared with the control, caused a statistically significant increase in DNA migration as determined by different parameters such as Olive tail moment, tail extent moment, tail/head or tail length. The data demonstrated the ability and the sensitivity of the comet assay when performed on Hep G2 in the detection of DNA damage induced by promutagens, and its suitability in mutagenicity testing in in vitro short-term assays.

The French Drug Agency is responsible for the control and delivery of batch release certificates. In the case of an influenza pandemic, the use of inactivated vaccines, produced according to well-established procedures and controlled according to the European Pharmacopea and FDA requirements, will be strictly dependent on the necessary delays for production and controls. Mutual recognition between the National Control Laboratories in Europe might help in shortening the delays. If new, inactivated vaccines are produced either on cell cultures or by using genetically modified organisms, and if live attenuated vaccines are needed, it would be suitable to organize ad hoc working groups and international collaborative studies in fields of both research and regulation.

13 pages; International audience; It is possible to reduce the diffraction peaks of a Spatial Light Modulator (SLM) by breaking the periodicity of the pixels shape. We propose a theoretical investigation of a SLM that would be based on a Voronoi diagram, obtained by deforming a regular grid, and show that for a specific deformation parameter the diffraction peaks disappear and are replaced with a speckle-like diffraction halo. We also develop a simple model to determine the shape and the level of this halo.

The pharmacokinetics of mexiletine and its metabolite hydroxy-methyl-mexiletine have been investigated following single-dose and during multiple-dose administration of a sustained-release form of mexiletine to six post-myocardial infarct patients. Comparison of single-dose and washout pharmacokinetics, after short-term multiple-dose administration, showed significant (p < 0.005), but not systematic, modifications in mexiletine apparent clearance for three patients. Furthermore, for these patients, simulation with both sets of parameters indicated that the steady-state was achieved before washout experiment in two cases. The fraction of mexiletine metabolized to hydroxy-methyl-mexiletine was lower for multiple-dose administration (about 18 per cent) than for the single dose (about 42 per cent). The hydroxy-methyl-mexiletine elimination rate constant was about four times that of mexiletine. Mexiletine clearance could be accounted for by other metabolic pathways. In one patient, hydroxy-methyl-mexiletine was undetectable even during multiple-dose administration, despite a significant increase in mexiletine clearance. However, the observed changes in mexiletine disposition had no therapeutic implications and active plasma levels were achieved by the third day of administration and maintained in the therapeutic range (0.75 to 2 micrograms ml-1) in all patients after a twice daily dosage regimen.

The metabolism of 2-n-propyl-2-pentenoate (delta 2-VPA) was evaluated in human hepatic microsomal fractions. Two biotransformation pathways have been particularly investigated. In the presence of the cytochrome P-450 co-factor, NADPH, the main metabolites recovered were delta 3-VPA, delta 2,4-VPA and VPA. The glucuronidation of delta 2-VPA was also studied on various hepatic microsomal fractions using Brij 35 as activator and UDP-glucuronic acid as co-factor. A large interindividual variability occurred in this metabolic pathway. Km and Vmax were 0.85 mmol/l and 1.75 nmol.min-1.mg-1, respectively, for delta 2-VPA and 1.11 mmol/l and 5.71 nmol.min-1.mg-1 for VPA, respectively. The good correlation (r = 0.82; p less than 0.001) observed between the glucuronidation of VPA and delta 2-VPA as well as the mutual inhibition of each other's glucuronidation strongly suggests that (a) common single UDP-glucuronosyltransferase isoenzyme(s) was (were) involved in this glucuronidation step. The glucuronidation of specific substrates for various UDP-glucuronosyltransferase isoenzymes showed a good relationship between the glucuronidations of delta 2-VPA and morphine, a substrate for UDP-glucuronosyltransferase-2B. Moreover, morphine competitively inhibits delta 2-VPA glucuronidation. It seems the same isoenzyme or, at least, (a) very closely related isoenzyme(s) belonging to UDP-glucuronosyltransferase-2 isoenzyme, is involved in delta 2-VPA glucuronidation.

[3H]Navelbine (NVB) was administered orally to two patients. Drug levels in biological fluids were monitored by radioimmunoassay (RIA) and direct radioactivity (RA) determinations. NVB absorption was rapid: maximum plasma concentrations appeared in the first 2 h after oral administrations. Pharmacokinetic parameters estimated from RIA data were in complete accordance with those obtained from i.v. injections. Bioavailability (i.v./po) estimated from RIA and RA data averaged 40.6 and 93.0%, respectively. NVB urine excretion was low. Fecal excretion remained its main elimination route. Moreover, large differences were observed in area under NVB plasma concentration-time curve (AUC) values obtained by the two methods, implying intense drug bio-transformations.

Vinblastine and Vincristine pharmacokinetics, including tissue distribution, metabolism and biliary excretion, were investigated, using both "in vitro" and "in vivo" models, after i.v. injections in rats. Plasma kinetic curves were best fitted to a two-compartment open model. The average terminal half-lives of VLB and VCR were 14.3 h and 7.5 h, respectively. The systemic clearance and apparent distribution volume for VLB, respectively 1.49 l/h/kg and 11.46 l/kg, were significantly greater than those of VCR, 0.12 l/h/kg and 0.41 l/kg. VCR was found to be widely distributed in tissues after i.v. injections in rats. The highest drug accumulation site was the intestine (122.0 ng/g wet tissue at 24 h). Liver and kidneys also retained high proportions of drug (respectively, 47.0 ng/g and 44.4 ng/g at 24 h). Biliary excretion was more rapid for VCR (42.7% of total radioactivity excreted over 24 h) than VLB (28.2% of total dose over 24 h). For both molecules, the percentage of radioactivity excreted in bile over 30-48 h ranged between 40-50% of total dose. At high doses, either biliary excretion rate or cumulated excretion was reduced. High performance liquid chromatography analysis of bile samples revealed four biotransformation products for VLB and three for VCR. When incubated in freshly isolated rat hepatocytes, VLB penetrated more rapidly and intensely into the cells (more than 90% of total dose taken up over 20 min) than VCR (only about 40% accumulated), probably through a passive diffusion mechanism followed by tight cellular binding. "In vitro" metabolism patterns were similar to those found "in vivo", except for the most polar metabolites observed "in vitro". Two anti-Vinca monoclonal antibodies with different specificities were used to test VCR metabolite immunoreactivities. The results suggested that some structural modifications occurred in the catharantine moiety of the molecule but that the dimeric structure seemed to be well conserved after biotransformation.

The authors monitored the plasma levels of clobazam (CLO) and its principal metabolite, N-desmethylclobazam (NCLO) during chronic treatment of more than 400 epileptic patients receiving different co-medications, such as phenytoin (PH), carbamazepine (CBZ), sodium valproate (VPA) and phenobarbital (PB). This study investigated the influence of age and antiepileptic drugs on plasma levels of CLO and NCLO. Plasma concentrations measured 3 hr after morning administration of CLO varied from 30 to 700 [formula; see text] for CLO, and from 160 to 7000 [formula; see text] for NCLO. Plasma levels of CLO were higher in patients aged 20-30 years. NCLO concentrations increased with age up to 20 years. Coadministered antiepileptic compounds significantly decreased maximal plasma levels of CLO. Moreover, PH and CBZ a significantly increased the plasma levels of NCLO. Results on the influence of CBZ on CLO kinetics were confirmed in a group of ten patients receiving PB and VPA and later PB, VPA and CBZ as CLO associated drugs. The influence of VPA on the pharmacokinetics parameters of CLO was also evaluated in a patient in the latter group.

Vindesine (VDS) pharmacokinetics, including tissue distribution, metabolism and elimination, were investigated in rats using both in vivo and in vitro models. VDS was found to be intensively distributed in tissues after i.v. injection in rat. The most important drug accumulation site was the spleen (615.0 ng/g at 24 h). Liver and kidneys also retained VDS in significant amounts (respectively 170.1 +/- 11.0 ng/g and 145.0 +/- 17.0 ng/g at 24 h). Urine excretion of drug over 7 days was low (10.1 +/- 1.8% of total dose) and consisted mainly of unchanged drug (more than 85%). The major excretion route for VDS was the feces (69.6 +/- 2.5% of total dose) via the bile (50% of total dose excreted in 72 h). High performance liquid chromatography analysis (HPLC) of collected bile samples revealed the excretion of three VDS biotransformation products. These results were confirmed in vitro using freshly isolated rat hepatocytes in suspension. Rapid and high VDS uptake by liver cells, probably through a passive diffusion mechanism followed by a tight cellular binding, was demonstrated. Moreover, VDS was intensively converted, in vitro, into four metabolites which were rapidly excreted into the extracellular medium. In contrast, the intracellular medium contained almost exclusively unchanged drug, presumably fixed to tubulin proteins. Two anti-VDS monoclonal antibodies with different specificities were used to test metabolite immunoreactivities. The results suggested that some structural modifications occurred in the catharantine moiety of the molecule but that the VDS dimeric structure seemed well conserved after biotransformation.

Methotrexate (MTX) and 7-hydroxy-methotrexate (7-OH-MTX) plasma concentration-time curves (AUC) have been analyzed in 24 patients after different routes of MTX administration. After an i.v. bolus (50 mg/m2), the AUC for 7-OH-MTX is correlated with that for MTX and inversely correlated with the MTX plasma clearance. When MTX is administered with plasma steady level standardization, using the test-dose protocol, at a level of 10(-5) M over 36 hr (10(-5), 36 hr), 7-OH-MTX-AUC is still correlated with the i.v. bolus pharmacokinetic parameters. The dose prediction using the classical test-dose protocol provides a less efficient MTX dose adjustment at 5 X 10(-4) M over 8 hr (5.10(-4), 8 hr) and the hydroxylation process is no more correlated with the i.v. parameters. On the opposite, upon 6 successive infusions with 10(-5), 36 hr or 5.10(-4), 8 hr protocols, the plasma concentrations of 7-OH-MTX are not significantly modified. This suggests that the hydroxylation process is not inducible.

This paper describes a general approach to compute steady-state dosage regimens. The method deals with individual linear compartmental mammillary pharmacokinetic models and requires estimated values of model parameters. It is based on the linearity principle and computes dosage regimens to achieve and maintain specified steay-state plasma concentrations, e.g. mean value or minimum effective and maximum safe levels. An interactive computer program performing the calculations has been developed.

The effects of isosorbide dinitrate (ISDN) and its 2- and 5-mononitrate metabolites (2-ISMN and 5-ISMN) against platelet aggregation and thromboxane release were investigated by analysis of platelet aggregation curves. ISDN, 2-ISMN and 5-ISMN (isosorbide nitrates, ISN) inhibited both ADP- and epinephrine (EPI)-induced platelet aggregation. ISN affected specifically the extent of ADP-induced aggregation and the velocity of EPI-induced effects. 2-ISMN was more potent against platelet aggregation compared to ISDN and 5-ISMN. The isosorbide nitrates were poor inhibitors of both arachidonic acid-induced aggregation and platelet TxB2 release. The differential inhibition by the three isosorbide nitrates of endogenous TxB2 release during ADP-induced aggregation further indicates that 2-ISMN is a significantly more potent platelet inhibitor than either ISDN or 5-ISMN. These studies suggest a role of the metabolites in modulating the pharmacological effects of ISDN on platelet activity.