Search

Assessing the relationship between antimicrobial usage (AMU) and antimicrobial resistance (AMR) requires the accurate and precise utilisation of register data. Therefore, validation of register-based data is essential for evaluating the quality and, subsequently, the internal validity of studies based on the data.In this study, different smoothing methods for Veterinary Medicine Statistic Program database (VetStat)-records were validated by comparing these with farm-records. Comparison between measurements included accuracy as; completeness and correctness, and precision as; a relative difference of the error, correlation with Fisher's z transformation and reliability coefficient. The most valid methods of those examined were then used in re-analyses of the abundance of AMR genes in 10 finisher batches from a previous study.Improved accuracy was found when detailed smoothing methods were applied. Although the precision also increased, the effect was not as pronounced, as the usage estimate of all smoothing methods deviated moderately compared with the farm-registrations. Applying the most valid methods to the 10 finisher batches increased estimates of statistical model fit for aminoglycosides, lincosamides, tetracyclines and decreased estimates of statistical model fit for macrolides. The estimates of statistical model fit for sulfonamides and broad-spectrum penicillins remained the same.Through refined data transformation, VetStat-records can be used to calculate a daily amount of AMU per pig reflecting the true usage accurately and moderately precisely, which is the foundation for calculating lifetime AMU.

It is accepted that usage of antimicrobials (AMs) in food animals causes the emergence and spread of antimicrobial resistance (AMR) in this sector, while also contributing to the burden of AMR in humans. Curbing the increasing occurrence of AMR in food animals requires in-depth knowledge of the quantitative relationship between antimicrobial usage (AMU) and AMR to achieve desired resistance reductions from interventions targeting AMU. In the observational study, the relationships between lifetime AMU in 83 finisher batches from Danish farms and the AMR gene abundances of seven antimicrobial classes in their gut microbiomes were quantified using multi-variable linear regression models. These relationships and the national lifetime AMU in pigs were included in the predictive modelling that allowed for testing of scenarios with changed lifetime AMU for finishers produced in Denmark in 2014. A total of 50 farms from the observational study were included in validating the observational study and the predictive modelling. The results from the observational study showed that the relationship was linear, and that the parenteral usage of AMs had a high effect on specific AM-classes of resistance, whereas the peroral usage had a lower but broader effect on several classes. Three different scenarios of changed lifetime AMU were simulated in the predictive modelling. When all tetracycline usage ceased, the predicted interval reductions of aminoglycoside, lincosamide and tetracycline resistance were 4–42 %, 0–8 % and 9–18 %, respectively. When the peroral tetracycline usage of the 10 % highest users was replaced with peroral macrolide usage, the tetracycline resistance fell by 1–2 % and the macrolide and MLSb resistance increased by 5–8 %. When all extended-spectrum penicillin usage was replaced with parenteral lincosamide usage, the beta-lactam resistance fell by 2–7 %, but the lincosamide usage and resistance increased by 194 % and 10–45 %, respectively. The external validation provided results within the 95 % CI of the predictive modelling outcome at national level, while the external validation at farm level was less accurate. In conclusion, interventions targeting AMU will reduce AMR abundance, though differently depending on the targeted AM-class and provided the reduction of one AM-class usage is not replaced with usage of another AM-class. Predicting several classes of AMR gene abundance simultaneously will support stakeholders when deciding on interventions targeting AMU in the finisher production to avoid adverse and unforeseen effects on the AMR abundance. This study provides a sound predictive modelling framework for further development, including the dynamics of AMU on AMR in finishers at national level.

This study introduces a new laboratory model of whole blood platelet aggregation stimulated by endogenously generated thrombin, and explores this aspect in haemophilia A in which impaired thrombin generation is a major hallmark. The method was established to measure platelet aggregation initiated by tissue factor evaluated by means of impedance aggregometry. Citrated whole blood from healthy volunteers and haemophilia A patients with the addition of inhibitors of the contact pathway and fibrin polymerization was evaluated. In healthy persons, a second wave of platelet aggregation was found to coincide with the thrombin burst and to be abolished by thrombin inhibitors. In this system, platelet aggregation in severe haemophilia A (n = 10) was found to be significantly decreased as compared with healthy individuals (912 ± 294 vs. 1917 ± 793 AU × min, P = 0.003), most probably due to the weak level of thrombin generation. For the first time, analysis of platelet aggregation as induced by endogenously generated thrombin was demonstrated. The new method makes it possible to explore the influence of the coagulation system on platelet function. In contrast to the general understanding, the data suggest that the impaired thrombin generation in haemophilia may affect platelet activation. Future studies will address whether our results may contribute to understanding differences in bleeding phenotypes and response to haemostatic substitution observed among patients. This study introduces a new laboratory model of whole blood platelet aggregation stimulated by endogenously generated thrombin, and explores this aspect in haemophilia A in which impaired thrombin generation is a major hallmark. The method was established to measure platelet aggregation initiated by tissue factor evaluated by means of impedance aggregometry. Citrated whole blood from healthy volunteers and haemophilia A patients with the addition of inhibitors of the contact pathway and fibrin polymerization was evaluated. In healthy persons, a second wave of platelet aggregation was found to coincide with the thrombin burst and to be abolished by thrombin inhibitors. In this system, platelet aggregation in severe haemophilia A (n = 10) was found to be significantly decreased as compared with healthy individuals (912 ± 294 vs. 1917 ± 793 AU × min, P = 0.003), most probably due to the weak level of thrombin generation. For the first time, analysis of platelet aggregation as induced by endogenously generated thrombin was demonstrated. The new method makes it possible to explore the influence of the coagulation system on platelet function. In contrast to the general understanding, the data suggest that the impaired thrombin generation in haemophilia may affect platelet activation. Future studies will address whether our results may contribute to understanding differences in bleeding phenotypes and response to haemostatic substitution observed among patients.

ObjectivesReliable methods for monitoring antimicrobial resistance (AMR) in livestock and other reservoirs are essential to understand the trends, transmission and importance of agricultural resistance. Quantification of AMR is mostly done using culture-based techniques, but metagenomic read mapping shows promise for quantitative resistance monitoring.MethodsWe evaluated the ability of: (i) MIC determination for Escherichia coli; (ii) cfu counting of E. coli; (iii) cfu counting of aerobic bacteria; and (iv) metagenomic shotgun sequencing to predict expected tetracycline resistance based on known antimicrobial consumption in 10 Danish integrated slaughter pig herds. In addition, we evaluated whether fresh or manure floor samples constitute suitable proxies for intestinal sampling, using cfu counting, qPCR and metagenomic shotgun sequencing.ResultsMetagenomic read-mapping outperformed cultivation-based techniques in terms of predicting expected tetracycline resistance based on antimicrobial consumption. Our metagenomic approach had sufficient resolution to detect antimicrobial-induced changes to individual resistance gene abundances. Pen floor manure samples were found to represent rectal samples well when analysed using metagenomics, as they contain the same DNA with the exception of a few contaminating taxa that proliferate in the extraintestinal environment.ConclusionsWe present a workflow, from sampling to interpretation, showing how resistance monitoring can be carried out in swine herds using a metagenomic approach. We propose metagenomic sequencing should be part of routine livestock resistance monitoring programmes and potentially of integrated One Health monitoring in all reservoirs.

Zusammenfassung Die exokrine Pankreassekretion bei jungen Schweinen nach Futterung von Gerste mit oder ohne Zusatz von β-Glukanase In einem Versuch mit jungen Mastschweinen wurde der Einflus eines Zusatzes von β-Glukanase zu einem auf Gerste basierenden Grundfutter auf die exokrine Pankreassekretion untersucht. Um eine kontinuierliche Entnahme von Pankreassaft zu ermoglichen, wurde an vier mannlichen Schweinen im Alter von 6 Wochen (Korpergewicht 10.4 ± 1.1 kg) eine Katheterisation des Pan-kreasganges vorgenommen. Die Tiere erhielten ein Grundfutter bestehend aus Gerste mit vollem Schalenanteil (75.8 %), Soja-schrot (15 %) und Fischmehl (4 %) ohne oder mit Zusatz von β-Glukanase (0.25 g/kg Futter). Die Futterung der Schweine erfolgte dreimal taglich um 8, 15 und 22 Uhr mit jeweils 120 g Futter pro Mahlzeit. Pankreassaft wurde dreimal uber die Dauer von jeweils 24 Stunden gesammelt. Zur Beurteilung der Pankreassekretion wurde sowohl die Sekretion uber 24 Stunden als auch die Sekretion in den drei postprandialen Perioden herangezogen. Unabhangig vom β-Glukanase-Zusatz im Futter blieb das Muster der Pankreassekretion im Hinblick auf das Sekretionsvolumen und auf den Gehalt an Enzymen unverandert. Die Ausscheidung von Pankreassaft zeigte einen zweiphasigen Verlauf, wobei nach der Futterung nur ein einziger Peak mit Enzymaktivitat beobachtet wurde. Die Anwesenheit von β-Glukanase im Futter hatte keinen Einflus auf das ausgeschiedene Gesamtvolumen (p > 0.05). Die Tiere, die das Futter mit β-Glukanase bekamen, zeigten uber 24 Stunden eine geringere Proteinsekretion (p < 0.05). Die Gesamtaktivitat von Chymo-trypsin im Pankreassaft lag bei diesen Tieren jedoch hoher (p < 0.05) und war jeweils signifikant hoher nach den Mahlzeiten am Morgen und am Nachmittag (p < 0.05). Die erhohte Chymotrypsinsekretion konnte darauf hindeuten, das die enzymatische Spaltung von β-Glukan durch β-Glukanase eine bessere Verfugbarkeit des im Futter enthaltenen Proteins zur Folge hat.

No human study has investigated the possible impact of breastfeeding on semen quality and levels of reproductive hormones, but a recent study of another hypothesis indicated an association with oligozoospermia. We investigated the association between breastfeeding, semen quality and levels of reproductive hormones. From a Danish pregnancy cohort established in 1984-1987, 347 sons were selected according to maternal smoking during pregnancy and followed up with questionnaires, semen analysis and blood sampling in 2005-2006. Complete data were available for 269 men aged 18-21 years. Breastfeeding was not statistically significantly associated with sperm concentration, total sperm count, sperm motility or morphology, oligozoospermia, follicle-stimulating hormone, inhibin B, luteinizing hormone, sex hormone-binding globulin (SHBG), the calculated level of free testosterone, free oestradiol, the free testosterone/free oestradiol ratio or the follicle-stimulating hormone/inhibin B ratio. Total testosterone and total oestradiol was 16% (p = 0.01) and 14% (p = 0.06), respectively, lower among men never breastfed in comparison with men breastfed exclusively for 1 month or longer. When taking SHBG into account, neither free testosterone nor free oestradiol was different between the two groups. This study shows no association between breastfeeding and sperm quality or reproductive hormones and a strong association is unlikely. A larger study would be needed to detect more subtle effects.

The results of this study demonstrate that there are large differences in the amount of pancreatic juice secreted and in the chemical and enzymatic composition of pancreatic juice when the pouch and the catheter methods were used, and these differences must be taken into consideration in future studies with either method.A study was performed to compare the two most commonly used methods to collect pancreatic juice from growing pigs; namely, the pouch method (PM) and the catheter method (CM). In the first part of the study, three barrows (initial weight 37 kg) were fitted with a pancreatic pouch re-entrant cannula. An isolated pouch was prepared in which the pancreatic duct enters the duodenum. In the second part of the study, also with three barrows (initial weight 32 kg), a catheter was inserted into the pancreatic duct.At several points during the 24-h collection, the hourly rate of pancreatic juice secretion in CM pigs was larger (p0.05) than for PM pigs. CM pigs also had a higher (p0.05) daily volume of secretion, 4.09 vs 2.63 L/24 h for PM pigs. The pH of pancreatic juice collected from CM pigs was consistently higher (p0.01) throughout the 24-h collection. In contrast, the concentration and daily output of bicarbonate did not differ between CM and PM pigs. The concentration of protein in pancreatic juice from PM pigs (7.21 g/L) was higher (p0.001) than for CM pigs (4.08 g/L). Specific amylase and lipase and total amylase activities were greater (p0.01) in pancreatic juice collected from PM pigs. Specific and total carboxyl ester hydrolase and colipase activities were substantially (p0.01) larger in pancreatic juice collected from CM pigs. A major difference between the methods was that trypsin and chymotrypsin were fully active in pancreatic juice from PM pigs, whereas virtually no trypsin or chymotrypsin activity was detected in pancreatic juice from CM pigs. Specific and total chymotrypsin activities did not differ between PM and CM pigs. Both specific and total trypsin activities were substantially higher in pancreatic juice from CM pigs: 3682 U/L and 12,752 U/24 h, respectively, vs 1031 U/L and 2639 U/24 h, respectively, in pancreatic juice from PM pigs.

Four differently modified forms of acyl-CoA-binding protein (ACBP) were identified in ACBP purified from bovine liver. The majority of the purified ACBP was focused at pH 5.9 in isoelectric focusing and could be shown to be N-acetylated ACBP without any further modifications. Two minor peaks were focused at pH 5.25 and 4.85 respectively. Mass spectrometry and sequence determination showed that the pI 5.25 form was acetylated at Lys18 and that the pI 4.85 form was malonylated in the same position. Furthermore, it could be shown that non-enzymic glycosylation occurred during purification. The acetylated and malonylated variants of ACBP were only found in adult cattle.

We studied the time structure of pancreatic secretion in two experiments involving seven 6- to 7-wk-old intact male pigs, surgically fitted with a jugular vein catheter for blood sampling, pancreatic catheter, and a duodenal T-cannula for chronic pancreatic juice sampling for 72 h at 30- to 60-min intervals. Pigs were kept in metabolic cages in a regimen of 12 h of light alternating with 12 h of darkness and were fed at 0800, 1500, and 2200 daily a standard diet based on barley, soybean meal, and fish meal. Beginning 4 d after surgical recovery, three 24-h collections of pancreatic juice and blood sampling were begun either at 0700 or 0800 every 2nd d for 5 d. Pancreatic secretion exhibited a pattern characterized by distinct meal-related secretions of the first phase (postprandial), containing large amounts of protein and enzymes (trypsin and chymotrypsin), and by non-food-stimulated secretions of the second phase with less protein and enzymes. During the dark span, the first phase was practically absent; the response of the pancreatic secretion to the 2200 meal was not very pronounced. Apart from the anticipated circadian rhythm demonstrable by single cosinor analysis on a group basis, a prominent 8-h component was almost invariably statistically significant. Moreover, an approximately 3.43-h component was also prominent. These data indicate that pancreatic secretions are circadian periodic and that their response to a standard meal is also circadian-stage dependent. The circadian components may have been free-running because the pigs were adjusting themselves to the changing phase and that resulted in the period being different from exactly 24 h.

The effect of age and weaning on the activities of digestive enzymes with emphasis on the lipolytic enzymes before and after weaning was investigated. The activities of amylase, chymotrypsin, trypsin, carboxyl ester hydrolase, pancreatic lipase, and colipase in pancreatic tissue and the activity of gastric lipase in the cardiac mucosa of the stomach in 45 pigs were response variables. The activity of trypsin was not affected by weaning and the rate of increase was similar during the whole experiment. The activities of chymotrypsin and amylase decreased at weaning (P < .05). After weaning the activity of chymotrypsin increased more slowly than before weaning (P < .001), whereas the rate of increase of amylase activity remained unchanged. Lipase, colipase, and carboxyl ester hydrolase activities decreased at weaning (P < .001), whereas gastric lipase activity increased at weaning (P < .01). The development of lipase, colipase, and carboxyl ester hydrolase activity decreased postweaning (P < .01), whereas gastric lipase activity increased before weaning and remained constant after weaning. Pancreatic lipase had a considerably higher capacity for hydrolyzing tributyrin, and the total activity of pancreatic lipase was up to 600 times higher than that of gastric lipase. The lipolytic enzymes displayed a non-parallel pattern of development, and we suggest that this reflects the importance of these enzymes during the suckling and postweaning phases, respectively. However, the significance of gastric lipase for the digestion of fat in pigs remains to be elucidated.

It is shown that the usual dispersion formula used in all calculations of the real part of the anomalous scattering factors, f', is only valid within the dipole approximation. A new correction term which is positive and of the order of Etot/mc2, with Etot the total binding energy of the electrons in the atom, is needed. Based on experimental data, the K-shell contribution to the new correction for f' is found to be larger than 0.013 for Si and 0.073 for Ge. Formulae expressing a total K-shell photoelectric cross sections in terms of the dipole approximation values are given. It is concluded that at present no theoretical calculations give f' better than to within an accuracy of the order -Etot/mc2.

A corn field partially under attack by armyworms was photographed in 1973 with 70 mm Aerochrome Infrared Type 2443 film and a Wratten 12 filter at scales of 1/15,840 and 1/6,336 during the early silking stage. In 1974, in the absence of armyworm defoliation, the field was again overflown at the same plant stage, and with the same camera/film/filter configuration used in 1973. Defoliation was not detectable on otherwise healthy corn plants (i.e., no drought stress); but defoliation was detected on plants already under stress due to drought.

Since 1975 the antitumor activity of new agents introduced by the Division of Cancer Treatment (DCT) has been detected or confirmed in animal screening systems consisting of a panel of six spontaneous allografted murine tumors, and three human tumor xenografts [25], A plan of clinical development has been formulated which includes, as a minimum requirement, testing all new agents in a clinical panel that matches in disease orientation those preclinical screening systems [50]. Accordingly, Phase II studies with every drug are expected to be completed in patients with breast, colon, and lung carcinomas, malignant melanoma, acute leukemia, and lymphoma. It is hoped that such a plan will obviate incomplete clinical evaluations which were all too prevalent in the past [64].

The action of a new non-N-methyl-D-aspartate (NMDA) receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), on synaptic transmission in area CA1 of the rat hippocampus has been examined. Intracellular and extracellular recordings showed CNQX to be a potent antagonist of synaptic potentials evoked by stimulation of the Schaffer collateral-commissural fibre system. One to 2 microM CNQX was sufficient to reduce the excitatory postsynaptic potential (EPSP) by 50%. CNQX is therefore about 100 times more potent than previously available non-NMDA receptor antagonists. In the presence of CNQX, a small depolarizing potential could still be evoked. This potential was sensitive to the NMDA-receptor blocker, 2-amino-5-phosphonovaleric acid (APV), increased in size on depolarizing the neurone and also increased in size on removing Mg2+ from the perfusing medium. This residual EPSP therefore has characteristics which are consistent with its mediation via the NMDA receptor-coupled ionophore. These results indicate a dual composition of the monosynaptic excitatory potential in area CA1.

Most drugs which act on the central nervous system (CNS) do so by interfering with chemical transmission between nerve cells. This may be achieved in many ways, e.g., receptor blockade, stimulation or depression of presynaptic transmitter synthesis, blockade of transmitter uptake or inactivation, and so on. Most of the centrally active drugs which have been in use for a long time were originally discovered on an empirical basis. Examples are naturally occurring alkaloids (many of which had been known for generations to be psychoactive, analgesic, and so on) and more or less randomly synthesized molecules (e.g., barbiturates, neuroleptics). Many of these agents have proved to be very useful tools to the basic scientist in his efforts to investigate and understand the workings of the nervous system. With this understanding of transmitters, receptors, and synaptic mechanisms, it has in turn been possible to transform the art of drug molecule design into more of a science. Ultimately, it becomes possible to predict the pharmacological profile of a new molecule before it is synthesized. In this way, drugs can be designed which have a sharper profile of action, so increasing specificity and eliminating unwanted side effects. There are, however, other considerations to be made before a substance which performs well in experimental models can be considered to have a future in the clinic. It must be able to cross the blood-brain barrier, either directly or as a precursor; its biological half-time should not be inappropriately short or long; it should not have side effects (e.g., on visceral organs) which are unrelated to its central action.

Electrophysiological recordings from mouse neurones in tissue culture have been used to investigate how agents which interact with the benzodiazepine receptor modulate neuronal responses to gamma-aminobutyric acid (GABA) and its mimetics, 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP) and piperidine-4-sulphonic acid (P4S). Experiments were performed in a physiological medium, pH 7.35 at 34-36 degrees C. gamma-Aminobutyric acid, THIP and P4S were applied by iontophoresis to neuronal somata. Responses were assessed by current-clamp or voltage-clamp recordings. Midazolam (an agonist at the benzodiazepine receptor) and the beta-carboline, methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM; an inverse agonist at the BZ receptor), were applied by pressure ejection from blunt pipettes. The potency order of the agonists was GABA greater than P4S greater than THIP. Midazolam (10(-7)-10(-5) M) potentiated responses to all three agonists to a similar extent with a shift to the left of the dose-response curve. The drug DMCM (10(-6)-10(-5) M) decreased the responses to all three agonists to a similar extent. The DMCM-induced depression was of a non-competitive nature. It has previously been proposed that THIP is a partial agonist and P4S an antagonist at the GABA receptor coupled to the benzodiazepine receptor, or that the benzodiazepine-receptor-coupled and electrophysiological GABA receptors are distinct. In the present study, responses to the three agonists were modulated to a comparable extent following manipulation of the benzodiazepine receptor. It is therefore unnecessary to invoke the above explanations to account for these results.

Phase I trials in 1979 include some drugs representing totally new structures, new schedules of old compounds undergoing reevaluation, and second generation compounds. The rational development of analogs based on structure-activity relationships and on overcoming pharmacologic or toxicologic problems of parent compounds requires much future emphasis; two such examples (pentamethylmelamine and AZQ) are cited here. For all drugs, a plan of clinical development should ensure a more thorough initial evaluation as well as validation of concepts and systems that have prompted their introduction into the clinic. Establishment of clinical usefulness for the new structures, and particularly for three compounds herein reintroduced after a long period of oblivion, would constitute tangible proof of methodological and technological advances that have taken place in the development and clinical evaluation of anticancer drugs.