In vitro susceptibility of Listeria monocytogenes: comparison of the E test with the agar dilution test.

In vitro susceptibility testing was performed on 66 strains of Listeria monocytogenes. Recently obtained clinical isolates from Austria, France, Norway and Switzerland and 17 reference strains from three type culture collections were tested against ampicillin, gentamicin, trimethoprim-sulfamethoxazole, ampicillin-sulbactam, meropenem, and first-, second-, third- and fourth-generation cephalosporins. All isolates were susceptible to ampicillin, gentamicin, and trimethoprim-sulfamethoxazole, ampicillin-sulbactam, and meropenem and more than 86% of the minimal inhibitory concentration E test values were within plus/minus one dilution step of the agar dilution test. In vitro susceptibility of L. monocytogenes has not changed markedly during the last decades; the level of susceptibility of old reference strains did not differ from that of recently encountered clinical isolates. The E test seems to be a suitable method for Listeria.

Acute and prolonged toxicity to aquatic organisms of new and existing chemicals and pesticides.

The relation of acute to prolonged toxicity of existing and new chemicals and active ingredients of numerous pesticides in the aquatic environment have been compared with their physico-chemical properties. The data had been presented to the authorities in behalf of the pesticide registrations, the notification of new chemicals or research projects. The ecotoxicity ratios are expressed as acute to prolonged ratios (APR). They are determined for fish and crustacea with existing and new chemicals and pesticides. In the case of the pesticides the data are subsummarized into fungicides, herbicides, insecticides. The variability of the APR is evaluated. According to the statistic evaluation of the data it must be summarized, that the toxic effects are often extremely specific and depend not only on the substance, but also on the aquatic species, the duration of the exposure, and the endpoint of the test. In general, a factor of 100 between the EC/LC50 of acute toxicity and NOEC of prolonged toxicity is scientifically justified. The data indicate that there is no significant correlation between lipophilic properties (logPow) or water solubility and toxicity in aquatic organisms. For that reason structure-activity relationships based on lipophilic properties of the substance cannot lead to reasonable results. It must be stated, that the prolonged ecotoxicity can not be exactly extrapolated from acute toxicity data on the basis of physico-chemical properties.

Embryotoxic effects of thalidomide derivatives in the non-human primate callithrix jacchus. IV. Teratogenicity of micrograms/kg doses of the EM12 enantiomers.

The dose-response of the teratogenic potency of the thalidomide (Thd) derivative EM12 was evaluated in the common marmoset (Callithrix jacchus). The smallest daily dose found to be effective was 30 micrograms EM12/kg body wt. This is the lowest dose of a Thd derivative ever reported to induce severe skeletal abnormalities. Ten micrograms EM12/kg body wt may be considered the no-observed-adverse-effect-level (NOAEL) under the experimental conditions chosen. The teratogenic potencies of the two EM12 enantiomers were tested at 100 micrograms/kg body wt, the dose which just induces an almost 100% effect in the case of the racemate. The S(-)-EM12 was found to induce typical severe limb abnormalities such as amelia, phocomelia, and radius aplasia, and none of the exposed fetuses were devoid of skeletal defects. In contrast, only few and minor skeletal defects were observed after application of the R(+) enantiomer. Although a pronounced teratogenic potency of the R(+)-EM12 can now largely be excluded, these low-dose studies are not sufficient to completely rule out any teratogenic potential of this enantiomer, since racemisation to small amounts of the S(-) form may occur in vivo. Further studies with Thd derivatives which are unable to racemise are necessary to prove the assumed complete ineffectiveness of the R(+) enantiomers.

Developmental biology of the common marmoset: proposal for a "postnatal staging".

The published knowledge on neurobiological, psychological, and ethological aspects of development in Callithrix jacchus is still limited. We have collected published and unpublished data from several Callithrix colonies and pooled information on criteria for developmental progress and maturation using a questionnaire sent to numerous experts in the field. The data suggest that developmental stages can be defined not only for the embryonic and fetal but also for the postnatal period. Based on multifactorial definitions, using criteria selected from maturational changes in the motor and visual systems and behavioral features, we propose to subdivide postnatal development of the common marmoset into seven periods ("stages").

Direct in vivo biotinylation of erythrocytes as an assay for red cell survival studies.

Direct in vivo labeling of erythrocytes with biotin is shown as a method for estimation of red cell survival as well as of enrichment of young or aged erythrocytes. Two succinimide esters (biotin-N-hydroxysuccinimide ester [BNHS], caproylamidobiotin-N-hydroxysuccinimide ester [C-BNHS] were used for biotin labeling of erythrocytes. With improved syntheses, pure BNHS (mp, 212 degrees-214 degrees C) and the spacered intermediate for C-BNHS, 6-(biotinylamide) hexanoate (mp, 225 degrees-226 degrees C) were obtained in an overall yield of 86%; the yield of C-BNHS (mp, 167 degrees-169 degrees C) was 68%. When three doses of 1 mg C-BNHS are injected intravenously into mice at 24-h intervals, all the red cells are biotin labeled. The rate of red cell production as well as the life span of red cells can be measured without any effect on erythropoiesis or damage by red cells in vitro. The survival curve seems to be linear, with 2.5%-3.3% disappearance of biotin-labeled red cells daily. In mice, in vivo biotin labeling avoids damaging red cells by in vitro procedures and does not influence the steady state of erythropoiesis by hypertransfusion. Therefore, in vivo biotin labeling is a very useful method for determining red cell survival time in small animals.

Ontogeny of monooxygenase activities in the marmoset monkey (Callithrix jacchus).

The pre- and postnatal development of monooxygenases in the liver and adrenal gland of marmoset monkeys (Callithrix jacchus) was investigated. Cytochrome P450 was detected in the fetal adrenal gland, but aldrin epoxidase, ethoxycoumarin O-deethylase, and ethoxyresorufin O-deethylase activities were below detection limits. Although fetal hepatic cytochrome P450 was not detected, low activities of aldrin epoxidase and ethoxycoumarin O-deethylase, but no ethoxyresorufin O-deethylase, could be detected in fetal liver. These enzymes attained adult marmosets activities when the offspring were approximately 2 months of age.

Ofloxacin in juvenile non-human primates and rats. Arthropathia and drug plasma concentrations.

Arthropathia in juvenile animals is the most important toxic effect induced by quinolones. We conducted pharmacokinetic and morphological studies with ofloxacin on non-human primates (Callithrix jacchus, Marmosets) and rats. In the marmoset, electron microscopy and the application of immuno-morphological methods proved to be suitable for the detection of specific alterations in cartilage (e.g. loss of proteoglycans and altered chondrocytes). Subsequently performed electron microscopic examinations in rats showed similar specific alterations of the femur cartilage surface after multiple oral applications of 600 mg ofloxacin/kg body wt. These results were correlated with pharmacokinetic data obtained for the same species. After single oral application of 100, 300 or 600 mg ofloxacin/kg body wt to 5 week-old rats peak plasma levels were achieved 15-45 min after administration indicating a rapid absorption of the drug. The following peak concentrations were measured for the three doses applied (mean +/- SD): 8.9 +/- 2.1, 22.6 +/- 7.5 mg/l and 33.5 +/- 9.8 mg/l, respectively. After 360 min the concentrations were 1.1 +/- 0.4, 5.9 +/- 2.5 and 15.9 +/- 5.1 mg/l, respectively. After subcutaneous injection of 100 mg ofloxacin/kg body wt the mean peak concentration was 27.7 +/- 2.6 mg/l after 45 min (0.5 +/- 0.2 mg/l after 360 min). In the marmoset higher plasma concentrations were measured with comparable doses. One, 3, and 6 h after the last of nine administrations of 200 mg ofloxacin/kg body wt, the mean (+/- SD) plasma concentrations were: 42.7 +/- 16.7, 40.6 +/- 9.5, and 26.5 +/- 3.6 mg ofloxacin/l plasma. Typical alterations of the joint cartilage of juvenile rats (e.g. opened chondrocyte cavities, swelling of rough endoplasmic reticulum and mitochondrial swelling in the chondrocytes) were induced by oral administration of ofloxacin at doses that were approximately 100 times higher than therapeutic ones, but led to peak plasma concentrations which were only approximately 10 times above the therapeutic level.

The enantiomers of the teratogenic thalidomide analogue EM 12. 2. Chemical stability, stereoselectivity of metabolism and renal excretion in the marmoset monkey.

The rate of metabolism as well as the metabolic profiles of the enantiomers and the racemate of 2-(2,6-dioxopiperidine-3-yl)-phthalimidine (EM 12), a teratogenic thalidomide analogue, have been investigated in vitro and in the marmoset monkey in vivo. The half-life of racemic EM 12 and of the two enantiomers in vitro (phosphate buffer pH 7.4, 37 degrees C) were all in the same range (12 h). Two major hydrolysis products were found which were formed via amide cleavage of the piperidinedione ring of the molecule (EM 27 and EM 356). Their concentrations were similar. In contrast, EM 356 was the main metabolite of EM 12 present in the urine of marmoset monkeys following single i.p. or p.o. doses of 5 mg/kg b.w. About twice as much EM 356 was produced after administration of R-EM 12 than after administration of S-EM 12. The concentration ratio of the metabolites obtained after p.o. and i.p. administration of the substances were in the same range. Separation of the EM 12 enantiomers present in urine suggests that considerable racemisation took place in vivo, although at a slower rate than in vitro: about 25% of the respective optical antipodes were present 5 h after administration of the R-enantiomer and 7.5 h after administration of the S-enantiomer (compared to 1.5 h in vitro). Our results indicate that racemic EM 12 as well as its enantiomers are chemically and metabolically more stable than thalidomide; however, extensive racemisation occurs both in vivo and in vitro. The metabolism and renal excretion of the enantiomers of EM 12 in the marmoset monkey were shown to be stereoselective.

Identification of the main gestagen metabolite in marmoset (Callithrix jacchus) urine by NMR and MS spectroscopy.

Hydroxypregnanolone is the most abundant progesterone metabolite in the urine of marmoset monkeys (Callithrix jacchus). The substance is excreted as conjugate. The concentration of this steroid may be monitored by high performance, thin layer chromatography and postchromatographic derivatization. Hydroxypregnanolone was purified and subsequently identified by NMR spectroscopy and gas chromatography/mass spectroscopy. The exact chemical structure is 5 alpha-pregnane-3 alpha, 7 alpha-diol-20-one.

Embryotoxic effects of thalidomide-derivatives in the non-human primate Callithrix jacchus. I. Effects of 3-(1,3-dihydro-1-oxo-2H-isoindol-2-yl)-2,6-dioxopiperidine (EM12) on skeletal development.

The response of pregnant marmosets (Callithrix jacchus) to the thalidomide derivative EM 12 was evaluated. EM 12 was selected for these studies because it is more active than thalidomide and is much more stable for hydrolysis. Skeletal gross structural abnormalities were observed when EM 12 was given to marmosets for 3-7 days during the period between days 49 and 60 post ovulation. Using the treatment schedule finally adapted in our laboratory, i.e. treatment during days 51-57 post ovulation, doses of 5 (or 10) mg EM 12/kg body wt induced the typical limb abnormalities known from man with an 80-100% certainty. In some animals we could observe the typical pattern of abnormalities even with doses as low as 1 mg EM 12/kg body wt. Abnormalities of the skeleton induced during this sensitive period are described. None of these (except some bifurcations of ribs) were seen in any of the ten litters (23 fetuses) serving as controls during the period of the study.

Embryotoxic effects of thalidomide derivatives in the non-human primate Callithrix jacchus. II. Elucidation of the susceptible period and of the variability of embryonic stages.

Data are presented on the normal intra- and inter-litter variability of the embryonic stages in the common marmoset (Callithrix jacchus) on day 56 of pregnancy. The embryology of the marmoset is described with respect to the susceptible period for the teratogenic action of thalidomide.

Embryotoxic effects of thalidomide derivatives on the non-human primate Callithrix jacchus; 3. Teratogenic potency of the EM 12 enantiomers.

The teratogenic potencies of the enantiomers of 2-(2,6-dioxopiperidine-3-yl)-phthalimidine ( = EM 12), a teratogenic thalidomide analogue, were investigated in Callithrix jacchus, a primate very sensitive to the teratogenic action of this thalidomide analogue. The results indicate that the S-(-)-form of EM 12 is clearly more teratogenic than the R-(+)-form. The interpretation of the studies designed to evaluate stereo-selective differences in the teratogenicity of the enantiomers becomes difficult, since both enantiomers racemise in vivo with appreciable rates (Schmahl et al. 1988a, b). Therefore, it cannot be concluded as yet that the R-(+)-form lacks all teratogenic potential.

[Injuries of the iliac and femoral vessels--a life-threatening emergency situation in hip prosthesis surgery]. / Die Verletzung der Iliacal- und Femoralgefässe--eine lebensbedrohliche Notfallsituation in der Hüftprothesenchirurgie.

The pattern of operation injuries of the arterial vessels which may occur during hip surgery is illustrated by this summary of our own cases. Injuries of the femoral vessels seem to be preventable if the Hohmann-retractor is carefully placed on the anterior margin of the acetabular rim under digital control. Furthermore drilling damage of the iliac vessel appears to be avoidable. However, the replacement manoeuvre of the artificial acetabulum can be associated with massive bleeding despite greatest care. Under these circumstances immediate surgical intervention through an inguinal retroperitoneal approach is mandatory.

Histochemical localization of 3 beta-hydroxysteroid dehydrogenase in marmoset ovaries during pro- and diestrus, with special reference to substrate specificity.

We applied qualitative cytochemical procedures to investigate and compare the distribution of 3 beta-hydroxysteroid dehydrogenases (HSDH) in pro- and diestrus ovaries of sexually mature marmosets (Callithrix jacchus) using dehydroepiandrosterone or etiocholane-3 beta-ol-17-one as the substrate. During proestrus dehydroepiandrosterone dehydrogenase (3 beta-5 alpha-HSDH) activity was found in the theca of tertiary follicles and in atretic granulosa cells. In granulosa cells at advanced stages of degeneration, HSDH activity was distinctly higher than in thecal cells. The activity of etiocholane-3 beta-ol-17-one dehydrogenase (3 beta-5 beta-HSDH) exhibited a gradient in preovulatory follicles, ranging from high levels in granulosa cells adjacent to the basement membrane to low levels in cells bordering on the antrum and in cumulus oophorus cells. During diestrus 3 beta-5 alpha-HSDH activity was only detected in the corpora lutea; the level of 3 beta-5 beta-HSDH activity was unchanged in the theca of tertiary follicles and was high in the cells of the corpora lutea. HSDH activity was no longer detectable in atretic granulosa cells using either dehydroepiandrosterone or etiocholane-3 beta-ol-17-one as the substrate. Comparison of the distribution of HSDH during proestrus and diestrus revealed that steroidogenesis in marmoset ovaries occurs in follicular elements during diestrus and almost exclusively in the corpora lutea during diestrus. From this phase-dependent localization, it is possible to determine the stage of the estrous cycle. Furthermore, our findings indicate that the localization of HSDH is dependent on the conformational structure of the substrate used.

Determination of ovulation and pregnancy in the marmoset (Callithrix jacchus) by monitoring of urinary hydroxypregnanolone excretion.

In Callithrix jacchus the urinary excretion of hydroxypregnanolone (5 alpha-pregnane-3 alpha, 7 alpha-diol-20-one) shows a luteal rise during the ovulatory cycle. This progesterone metabolite can therefore be used as an indication of ovulation, implantation, and subsequently for the persistence of the pregnancy. The excretion can be monitored by high performance thin-layer chromatography (HPTLC) and postchromatographic derivatization after enzymatic hydrolysis of the conjugate. Profiles of urinary hydroxypregnanolone levels are presented and correlated with luteinizing hormone excretion. The method is noninvasive and therefore suited for long-term studies in these monkeys.

[Injection methods for the demonstration of the coronary arterial vasculature (author's transl)]. / Verschiedene morphologische Darstellungen des Koronargefäss-systems des Hundes

Four different methods for the demonstration of the coronary arterial vasculature are examined. Among the techniques studied corrosion casts (epoxid- and acrylic resin) permit the best qualitative and quantitative evaluation of superficial and deep arteries and arterioles. Down to the smallest infusible vessels of 15-25 mum there is practically no shrinkage. X-ray arteriography with an aqueous solution of barium sulphate is less suitable because X-rays allow no satisfactory stereoscopic view. The demonstration of arterioles and collaterals is limited to vessels larger than 50 mum because of the low resolution of the X-ray tube. Silicone rubber does not only fill the arteries, but also the capillary bed and the venous system. Therefore deeper parts of the vasculature can only be studied after slicing the heart. The substantial shrinkage of the material makes quantitative measurements difficult.