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This study evaluated the pharmacokinetics of commercial gentamicin-impregnated collagen sponges (GICS) applied subcutaneously in dogs. In six healthy beagles, an 11 ×6â¯cm subcutaneous pocket was created, a folded 10×10â¯cm GICS was inserted, and saline was injected to mimic a seroma. Wound fluid samples were aspirated, and the gentamicin concentration was determined. Simultaneously, blood samples were collected to evaluate the corresponding systemic gentamicin concentration. All samples were collected before and 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, 18, 24, 36, 48, 72, 96, 120, and 168â¯hours after GICS placement. The local Cmax of gentamicin was reached after 0.5â¯hours (range, 0.5-1.0â¯hours) post-implantation in 5/6 dogs at a median concentration of 2053.3⯵g/mL (range, 918.0-2791.9⯵g/mL). Whitin 24â¯hours, the local concentration dropped below the MIC for Staphylococcus sp. (4⯵g/mL) in 5/6 dogs. Plasma Cmax was achieved at a median of 1.2â¯hours post-implantation (range, 1.0-2.0â¯hours) and reached a median concentration of 10.3⯵g/mL (range, 8.8-18.03⯵g/mL). After 6â¯hours, the gentamicin concentration in the plasma was below 4⯵g/mL in all dogs. The GICS provided a high local concentration of gentamicin in a short time with a local Cmax:MIC ratio of 513:1, largely sufficient to eliminate susceptible bacteria, including methicillin-resistant Staphylococcus pseudintermedius (MRSP) and Pseudomonas sp., in a clinical setting. The repeated administration of saline in the present study seemed to have induced a quicker gentamicin release from the GICS than described in previous studies that typically dealt with "drier" wounds.
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Plasma concentrations and pharmacokinetics of dexmedetomidine and buprenorphine after oral transmucosal (OTM) and intramuscular (i.m.) administration of their combination in healthy adult cats were compared. According to a crossover protocol (1-month washout), a combination of dexmedetomidine (40 µg/kg) and buprenorphine (20 µg/kg) was given OTM (buccal cavity) or i.m. (quadriceps muscle) in six female neutered cats. Plasma samples were collected through a jugular catheter during a 24-h period. Plasma dexmedetomidine and buprenorphine concentrations were determined by liquid chromatography-tandem mass spectrometry. Plasma concentration-time data were fitted to compartmental models. For dexmedetomidine and buprenorphine, the area under the plasma concentration-time curve (AUC) and the maximum plasma concentrations (Cmax ) were significantly lower following OTM than following i.m. administration. For buprenorphine, time to reach Cmax was also significantly longer after OTM administration than after i.m. injection. Data suggested that dexmedetomidine (40 µg/kg) combined with buprenorphine (20 µg/kg) is not as well absorbed from the buccal mucosa site as from the intramuscular injection site.
Assuntos
Buprenorfina/farmacocinética , Gatos/sangue , Dexmedetomidina/farmacocinética , Administração através da Mucosa , Animais , Buprenorfina/administração & dosagem , Dexmedetomidina/administração & dosagem , Interações Medicamentosas , Feminino , Injeções IntramuscularesRESUMO
A study was set up to investigate the influence of sodium salicylate on fever and acute phase reaction after lipopolysaccharide (LPS) injection in broiler chickens. An acute phase reaction was provoked through the intravenous injection of Escherichia coli LPS. Four oral doses of sodium salicylate were tested. Apart from body temperature, other inflammation indices, such as plasma corticosterone and ceruloplasmin, serum thromboxane B2 and zinc concentrations were monitored. Intravenous LPS induced a fever of about 1 degree C. A dose-dependent attenuation of the fever response of the chickens in the salicylate treated groups was observed. LPS-injected chickens also showed elevated plasma corticosterone and ceruloplasmin, while serum thromboxane and zinc concentrations decreased. Except for thromboxane B2, no linear relationship with increasing salicylate dose could be shown for the other blood variables. These data confirm that sodium salicylate is an effective antipyretic agent after injection of LPS in chickens, if used at an appropriate dosage. No dose-related change could be found for the other inflammation indices.
Assuntos
Analgésicos não Narcóticos/uso terapêutico , Galinhas , Febre/veterinária , Doenças das Aves Domésticas/tratamento farmacológico , Salicilato de Sódio/uso terapêutico , Reação de Fase Aguda/tratamento farmacológico , Analgésicos não Narcóticos/administração & dosagem , Animais , Relação Dose-Resposta a Droga , Escherichia coli , Febre/induzido quimicamente , Febre/tratamento farmacológico , Lipopolissacarídeos , Masculino , Doenças das Aves Domésticas/induzido quimicamente , Salicilato de Sódio/administração & dosagemRESUMO
Four methods intended for screening muscle tissue for residues belonging to the tetracycline group were compared using artificially contaminated as well as incurred samples. Two agar diffusion methods were studied: one with Bacillus subtilis as a test strain, the second with Bacillus cereus. Two variants of each method were compared: thin plates for analysis of intact or minced meat, and thick plates for analysis of meat fluid. The thin plate variants could not be evaluated with artificially contaminated samples because it was impossible to prepare homogeneously spiked, undiluted meat. The thick plates were suited for doxycycline and chlortetracycline, but they did not detect oxytetracycline or tetracycline in spiked meat fluid. The results of these tests done on incurred meat were very good for doxycycline and satisfying or just failing for oxytetracycline, while the best detection capability was obtained when intact frozen meat was examined on thin plates seeded with B. cereus. Two commercially available screening tests were also evaluated. The Premi(R) test, an inhibitor test with Bacillus stearothermophilus as a test strain and an indicator for growth, was not suited for detection of tetracyclines up to the maximum residue limit. Tetrasensor(R), a receptor test specific for tetracyclines, proved a quick and simple test able to detect meat samples artificially contaminated with tetracycline, oxytetracycline, doxycycline or chlortetracycline, as well as meat incurred with oxytetracycline or doxycycline.
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Antibacterianos/análise , Resíduos de Drogas/análise , Contaminação de Alimentos/análise , Carne/análise , Tetraciclina/análise , Animais , Bovinos , Galinhas , Análise de Alimentos/métodos , Imunodifusão/métodos , Músculo Esquelético/químicaRESUMO
A novel, sensitive and specific method for the quantitative determination of ivermectin B(1a) in animal plasma using liquid chromatography combined with positive electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) is presented. Abamectin was used as the internal standard. Extraction of the samples was performed with a deproteinization step using acetonitrile. Chromatographic separation was achieved on a Nucleosil ODS 5 microm column, using gradient elution with 0.2% (v/v) acetic acid in water and 0.2% (v/v) acetic acid in acetonitrile. The method was validated according to the requirements defined by the European Community. Calibration curves using plasma fortified between 1 and 100 ng ml(-1) showed a good linear correlation (r > or = 0.9989, goodness-of-fit coefficient < or =8.1%). The trueness at 2 and 25 ng ml(-1) (n = 6) was +4.2 and -17.1%, respectively. The trueness and between-run precision for the analysis of quality control samples at 25 ng ml(-1) was -4.0 and 11.0%, respectively (n = 16). The limit of quantification of the method was 1.0 ng ml(-1), for which the trueness and precision also fell within acceptable limits. Using a signal-to-noise ratio of 3 : 1, the limit of detection was calculated to be 0.2 ng ml(-1). The specificity was demonstrated with respect to ivermectin B(1b). The method was successfully used for the quantitative determination of ivermectin B(1a) in plasma samples from treated bovines, demonstrating the usefulness of the developed method for application in the field of pharmacokinetics.
Assuntos
Anti-Helmínticos/sangue , Ivermectina/sangue , Animais , Bovinos , Cromatografia Líquida de Alta Pressão , Reprodutibilidade dos Testes , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Animal cell cultures are characterized by very complex nonlinear behaviors, difficult to simulate by analytical modeling. Artificial Neural Networks, while being black box models, possess learning and generalizing capacities that could lead to better results. We first trained a three-layer perceptron to simulate the kinetics of five important parameters (biomass, lactate, glucose, glutamine and ammonia concentrations) for a series of CHO K1(Chinese Hamster Ovary, type K1) batch cultures. We then tried to use the same trained model to simulate the behavior of recombinant CHO TF70R. This was achieved, but necessitated to synchronize the time-scales of the two cell lines to compensate for their different specific growth rates.
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Directed control of cell metabolism by a modification of the physicochemical conditions (presence of Na-butyrate and modification of the temperature) was used to modulate the productivity of human recombinant tissular plasminogen activator (t-PA) expressed under control of SV40 promoter in Chinese Hamster Ovary (CHO) cell lines. We showed that both by adding Na-butyrate or lowering temperature from 37 degrees C to 32 degrees C there is an increase in the amount of t-PA excreted, while cell growth is significantly reduced. The treatments also increased the intracellular amount of t-PA. We measured the distribution of cell cycle phases by cytometry and used a modification of the equations of Kromenaker and Srienc (1991, 1994 a, b) to analyse the intracellular t-PA production rate in the different cell cycle phases. Intracellular t-PA was shown to accumulate in G1 phase in all conditions (at 37 degrees C, at 32 degrees C and in presence of butyrate). Moreover, we have shown that the distribution of the time cells treated by butyrate are maintained in the G1cell cycle phase is significantly increased. t-PA produced in the different cell culture conditions tested was analysed by zymogram and western blotting: neither butyrate, neither the shift of temperature changed significantly the overall quality of the protein. The N-glycan patterns of recombinant human t-PA was also analysed with carbohydrate-specific lectins. Butyrate caused a transitory increase in N-linked complex high-mannose oligosaccharides without any effect on the sialic acid content of t-PA.
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Gentamicin is a broad-spectrum aminoglycoside antibiotic widely used in veterinary medicine for the treatment of serious infections. The purpose of this study was to develop and validate a method to determine gentamicin residues in edible tissues of swine and calf. Extraction of gentamicin was performed using a liquid extraction with phosphate buffer containing trichloroacetic acid, followed by a solid-phase clean-up procedure on a CBA weak cation-exchange column. Tobramycin was used as the internal standard. After drying of the eluate, the residue was redissolved and further analyzed by reversed-phase liquid chromatography/electrospray ionization tandem mass spectrometry (MS/MS). Chromatographic separation of the internal standard tobramycin and the gentamicin components was achieved on a Nucleosil (5 microm) column using a mixture of 10 mM pentafluoropropionic acid in water and acetonitrile as the mobile phase. The gentamicin components C1a, C2 + C2a and C1 could be identified with the MS/MS detection, and subsequently quantified. The method was validated according to the requirements of the EC at the maximum residue limit (MRL) (100 ng g(-1) for muscle and fat, 200 ng g(-1) for liver and 1000 ng g(-1) for kidney), half the MRL and double the MRL levels. Calibration graphs were prepared for all tissues and good linearity was achieved over the concentration ranges tested (r > 0.99 and goodness of fit <10%). Limits of quantification of 25.0 ng g(-1) were obtained for the determination of gentamicin in muscle, fat, liver and kidney tissues of swine and calf, which correspond in all cases to at least half the MRLs. Limits of detection ranged between 0.5 and 2.5 ng g(-1) for the tissues. The within-day and between-day precisions (RSD) and the results for accuracy fell within the ranges specified. The method was successfully used for the determination of gentamicin in tissue samples of swines and calves medicated with gentamicin by intramuscular injection.
Assuntos
Antibacterianos/análise , Cromatografia Líquida de Alta Pressão/métodos , Gentamicinas/análise , Espectrometria de Massas por Ionização por Electrospray/métodos , Animais , Antibacterianos/isolamento & purificação , Antibacterianos/farmacocinética , Calibragem , Bovinos , Gentamicinas/isolamento & purificação , Gentamicinas/farmacocinética , Estrutura Molecular , Padrões de Referência , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Espectrometria de Massas por Ionização por Electrospray/instrumentação , Suínos , Tobramicina/análiseRESUMO
This work presents the development and the validation of an LC-MS-MS method with atmospheric pressure chemical ionization for the quantitative determination of levamisole, an anthelmintic for veterinary use, in porcine tissue samples. A liquid-liquid back extraction procedure using hexane-isoamylalcohol (95:5, v/v) as extraction solvent was followed by a solid-phase extraction procedure using an SCX column to clean up the tissue samples. Methyllevamisole was used as the internal standard. Chromatographic separation was achieved on a LiChrospher 60 RP-select B (5 microm) column using a mixture of 0.1 M ammonium acetate in water and acetonitrile as the mobile phase. The mass spectrometer was operated in MS-MS full scanning mode. The method was validated for the analysis of various porcine tissues: muscle, kidney, liver, fat and skin plus fat, according to the requirements defined by the European Community. Calibration graphs were prepared for all tissues and good linearity was achieved over the concentration ranges tested (r>0.99 and goodness of fit <10%). Limits of quantification of 5.0 ng/g were obtained for the analysis of levamisole in muscle, kidney, fat and skin plus fat tissues, and of 50.0 ng/g for liver analysis, which correspond in all cases to half the MRLs (maximum residue limits). Limits of detection ranged between 2 and 4 ng/g tissue. The within-day and between-day precisions (RSD, %) and the results for accuracy fell within the ranges specified. The method has been successfully used for the quantitative determination of levamisole in tissue samples from pigs medicated via drinking water. Moreover the product ion spectra of the levamisole peak in spiked and incurred tissue samples were in close agreement (based on ion ratio measurements) with those of standard solutions, indicating the worthiness of the described method for pure qualitative purposes.
Assuntos
Anti-Helmínticos/farmacocinética , Cromatografia Líquida de Alta Pressão/métodos , Levamisol/farmacocinética , Espectrometria de Massas/métodos , Animais , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Suínos , Distribuição TecidualRESUMO
Experiments, earlier performed in our laboratory, showedthe stimulating effect of butyric acid on monoclonalantibody production by hybridoma cells. Itssimulaneous inhibitory effect on cell growth canhowever compensate for this, so that no increase ofmonoclonal antibody titer might be obtained. We showin this article an experiment with addition of butyricacid in the middle of the growth phase of a batchculture, as a strategy to take real profit of such anaddition by a significant increase of final monoclonalantibody concentration. Indeed, in this way asignificant cell density could be obtained before theaddition of butyric acid, while the remaining culturetime was still sufficiently long for its action,resulting in a two fold increase of final monoclonalantibody titer. The experiment was carried out in a 2 L bioreactor, showing the real practical interest ofsuch an addition for the large scale production ofproteins. Furthermore, analysis of the produced IgG bySDS-PAGE and Western blot did not reveal structuralchanges after stimulation by butyric acid. An originalpoint of our study is the characterization of the cellbehaviour, by flow cytometry and other relatedtechniques, leading to a better insight in the effectof the butyric acid addition on cell growth andmonoclonal antibody production. Although there existsa lot of knowledge about the effects of butyrate oncells in the field of molecular biology, our article isat our knowledge one of the first to show some of itseffects on cell behaviour in bioreactor culture,carried out under perfectly defined and controlledconditions, and with the aim to stimulate monoclonalantibody production.
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The intracellular pH (pH(i)) is an important factor in the regulation of different cellular processes. It might therefore be used as a marker of the physiological state of cells cultivated in a bioreactor environment. We developed and validated therefore a methodology that permits a reproducible and reliable pH(i) measurement under such bioreactor culture conditions, contrary to earlier reported measurements, carried out on cells resuspended in buffers under nongrowth conditions. The hybridoma cells were sampled from the culture, stained with the pH-sensitive dye BCECF-AM (BCECF = 2',7'-bis-carboxyethyl-5,6-carboxyfluorescein), and analyzed by flow cytometry. Such a measurement is perfectible to changes of the cells between the moment of sampling and of final analysis on the flow cytometer. All intermittent steps were for this reason studied in detail, either to determine the optimal conditions to be used or to characterize their influence on the final pH(i) value measured. Additional experiments were carried out, showing the representativeness of the measured pH(i) value for the pH(i) the cells possess really in the culture at the moment of sampling.
Assuntos
Reatores Biológicos , Hibridomas/metabolismo , Concentração de Íons de Hidrogênio , Animais , Soluções Tampão , Calibragem , Sobrevivência Celular , Células Cultivadas , Temperatura Baixa , Meios de Cultura , Citometria de Fluxo , Fluoresceínas , Corantes Fluorescentes , Hibridomas/citologia , Cinética , Camundongos , Reprodutibilidade dos Testes , Coloração e Rotulagem/métodos , Fatores de TempoRESUMO
In this paper, we propose an alternative strategy to the ones proposed before (Oh et al., 1993; Øyaas et al., 1994a) to get real increases of global final antibody titer and production at hyperosmotic stress, by reducing the detrimental effect of such a stress on cell growth, and conserving the stimulating effect on antibody production. It consists of cultivating the cells in continuous culture and increasing the osmolality stepwise. In this way, the cells could progressively adapt to the higher osmolality at each step and antibody titers could be nearly doubled at 370 and 400 mOsm kg-1, compared to the standard osmolality of 335 mOsm kg-1. Surprisingly, the stimulation of antibody production was not confirmed for higher osmolalities, 425 and 450 mOsm kg- 1, despite the minor negative effect on cell growth. Intracellular IgG analysis by flow cytometry revealed at these osmolalities a significant population of non-producing cells. However, even when taking into account this non-producing population, a stimulating effect on antibody production could not be shown at these highest osmolalities. It seems to us that osmolality has a significant effect on the appearance of these non-producing cells, since they were not observed in continuous cultures at standard osmolality, of comparable duration and at an even higher dilution rate. The appearance of the non-producing cells coincides furthermore with modifications of the synthesised antibody, as shown by electrophoretic techniques. It is however not really clear if these two observations reflect actually the same phenomenon. Hyperosmolality affects the cell behaviour in continuous culture in multiple ways, independently of the growth rate, counting all at least partially for the observed stimulation of antibody production: acceleration of the amino acid, and in particular the glutamine metabolism, increase of the cell volume, increase of the intracellular pH and accumulation of cells in the G1 cell cycle phase.
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We show in this paper the results obtained when studying the behavior of hybridoma cells by monitoring of the pHi during batch and continuous bioreactor cultures. A first set of experiments, consisting of a batch culture and a continuous culture at variable dilution rate, was set up under normal physiological operating conditions. Significant pHi variations were measured during these cultures. For the batch culture, maximal pHi values around 7.60 were associated with the middle of the growth phase, while lower values were found in the culture beginning and during the decay phase, respectively 7.47 and 7.40. For the continuous culture, pHi increased with increasing dilution rate, ranging between 7.40 and 7. 60 for dilution rates between 0.010 and 0.040 h-1. These pHi variations were found for both cultures to be linked to variations of the specific growth rate of the cells. The observed link between pHi and cell growth provided us a general framework for the study of the effect of suboptimal operating conditions on cell behavior, all having a particular interest in animal cell culture technology. First, our results indicate that a decrease of the medium pH of its normal value of 7.00 to 6.70 did not necessarily result in cytoplasmic acidification, at least not after prolonged exposition times in continuous culture, and this despite a pronounced growth inhibitory effect. This effect can therefore rather be explained by the combination of the increased demand for maintenance energy associated with the higher DeltapH gradient maintained across the cell membrane and the decreased supply of energy, in particular via the glucose metabolism. Second, our results indicate that also increased ammonium ion concentrations do not lower pHi. This observation, together with the results of batch cultures carried out at a more alkaline pH, indicates that the form NH3, and not the form NH4+, has a negative effect on cell growth of our hybridoma cell line. Third, in the case of an osmolality increase, a significant pHi increase was observed, with mean values of 7.35 at the lower osmolalities 335 and 370 mOsm/kg and 7.45 at the higher osmolalities of 400, 425, and 450 mOsm/kg. This higher pHi might account at least partially for the increased monoclonal antibody production observed at hyperosmolality and coincided furthermore with a faster glutamine consumption rate.
Assuntos
Reatores Biológicos , Hibridomas/citologia , Hibridomas/metabolismo , Líquido Intracelular/química , Animais , Contagem de Células/efeitos dos fármacos , Técnicas de Cultura de Células/métodos , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Relação Dose-Resposta a Droga , Espaço Extracelular/química , Espaço Extracelular/metabolismo , Hibridomas/fisiologia , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Líquido Intracelular/metabolismo , Camundongos , Concentração Osmolar , Compostos de Amônio Quaternário/farmacologia , Fatores de TempoRESUMO
Endothelial cells are involved in important pathological situations. They could be the target for infectious processes as for example in Cowdriosis, an important disease in cattle due to the rickettsia Cowdria ruminantium prevalent in the south of the Sahara. They are also connected to angiogenic processes related to tumor invasion.Our results indicate that AIDS related Kaposi sarcoma cells may be of endothelial origin. We conclude from our data the mobility of those cells, related to the expression of the metalloproteinases (especially the 92 kD form of the enzyme), is an important factor in Kaposi saroma dissemination and is the main factor limiting the scale up of Cowdriosis vaccine production in Bovine Umbilical Endothelial Cell line. We showed that PMA and TNF increased the 92 kD Metallaproteinase and that TGFß, produced in an inactive form in cultures of Human Umbilical Vein Endothelial Cells, is a potential inhibitor of Kaposi sarcoma spreading, and could also be useful in improving our process for Cowdria ruminantium vaccine production, since it reduces the sensitivity of the cells to mechanical stress without affecting significantly the overall infectious process.
RESUMO
This study evaluates the suitability of flow cytometry with the fluorochrome BCECF for measuring the intracellular pH (pHi) of cultured cells, and monitors the changes in pHi in murine hybridoma in batch culture and chick embryo fibroblast in monolayer culture (5th passage). The technique produced highly reproducible, repeatable results. The theoretical sensitivity from the calibration curve was 0.0004 pH units. But analysis of the standard deviation of the histogram of the green/red fluorescence ratios indicated a mean sensitivity of 0.08 (0.07-0.09) pH units. Interference due to cell size, fluorochrome incorporation and esterases were minimized by establishing a calibration curve with the cells whose pHi was to be measured using the 525/610 nm fluorescence ratio after excitation at 488 nm. The pHi of exponentially growing, batch cultured hybridomas was 7.50 at the start of culture. pHi increased during the exponential growth phase and dropped towards cell death. The pHi of the chick fibroblasts in monolayer culture was 7.30.
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Citometria de Fluxo , Fluoresceínas , Animais , Linhagem Celular , Embrião de Galinha , Fibroblastos/metabolismo , Corantes Fluorescentes , Hibridomas/metabolismo , Concentração de Íons de Hidrogênio , Camundongos , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
The unfolding by guanidine hydrochloride of the toxic fragment of a Bacillus thuringiensis toxin belonging to the CryIC class reveals a two-step denaturation under both acid and alkaline conditions. This demonstrates the existence of two structural domains as building blocks for this toxin. Protease digests performed on a CryIA(b) and CryIC B. thuringiensis toxin, under native and partially denatured conditions, confirm this conclusion. Whereas the native CryIC toxin is completely protease resistant, the CryIA(b) toxin, earlier described as consisting of two structural domains [Convents, D., Houssier, C., Lasters, I. & Lauwereys, M. (1990) J. Biol. Chem. 265, 1369-1375], is cleaved by three proteases, resulting in at least two common fragments. This suggests that this toxin is built up of two globular units linked by a protease-susceptible linker. The detection of a stable intermediate along the denaturation curve allows us to study and compare the consecutive unfolding of the structural domains for both toxins. By addition of a protease, under conditions where such an unfolding intermediate exists, a single denaturation phase can be assigned to a specific part of the protein. These experiments lead to the conclusion that the domain whose stability is highly dependent on pH corresponds to the N-terminal half of both toxins.