Evaluation of Blood Alcohol Concentrations after Oral Administration of a Fixed Combination of Thyme Herb and Primrose Root Fluid Extract to Children with Acute Bronchitis.

BACKGROUND: The application of alcohol-containing medicinal products to children has been a subject of discussion for many years. A safety threshold of 0.125‰ blood alcohol concentration following the administration of a single dose has been recommended by the European Medicines Agency.The aim of this clinical study was to prove the safety of administering a fixed combination of thyme herb and primrose root fluid extract (Bronchicum(®) Elixir) containing 4.9% ethanol. METHODS: The herbal drug was administered for a period of 7-9 days to 16 children (ages 1-12 years) suffering from acute bronchitis for ≤ 48 h. After 3-5 days, a blood sample was taken 45 min (children ≥ 5 years: also 0 and 90 min) after application of the drug. The efficacy was assessed using the Bronchitis Severity Score. Global efficacy and tolerability were rated by the investigator and patients. RESULTS: All measured blood ethanol concentrations were below the threshold (mean value after 45 min: 0.0029 ± 0.0057‰ and after 90 min: 0.0051 ± 0.0078‰). The Bronchitis Severity Score decreased from 6.6 ± 1.0 to 0.9 ± 1.6 points. Global efficacy was assessed as "very good" and "good" in 60% (investigator) and 80% (patients) of cases. Global tolerability was rated as "very good" and "good" in more than 90% of cases. CONCLUSION: In conclusion, oral administration of the drug containing 4.9% ethanol to children (age 1-12 years) demonstrated a favourable risk/benefit ratio of the drug.

Tissue-specific neuro-glia interactions determine neurite differentiation in ganglion cells.

Guided formation and extension of axons versus dendrites is considered crucial for structuring the nervous system. In the chick visual system, retinal ganglion cells (RGCs) extend their axons into the tectum opticum, but not into glial somata containing retina layers. We addressed the question whether the different glia of retina and tectum opticum differentially affect axon growth. Glial cells were purified from retina and tectum opticum by complement-mediated cytolysis of non-glial cells. RGCs were purified by enzymatic delayering from flat mounted retina. RGCs were seeded onto retinal versus tectal glia monolayers. Subsequent neuritic differentiation was analysed by immunofluorescence microscopy and scanning electron microscopy. Qualitative and quantitative evaluation revealed that retinal glia somata inhibited axons. Time-lapse video recording indicated that axonal inhibition was based on the collapse of lamellipodia- and filopodia-rich growth cones of axons. In contrast to retinal glia, tectal glia supported axonal extension. Notably, retinal glia were not inhibitory for neurons in general, because in control experiments axon extension of dorsal root ganglia was not hampered. Therefore, the axon inhibition by retinal glia was neuron type-specific. In summary, the data demonstrate that homotopic (retinal) glia somata inhibit axonal outgrowth of RGCs, whereas heterotopic (tectal) glia of the synaptic target area support RGC axon extension. The data underscore the pivotal role of glia in structuring the developing nervous system.

Membrane recycling in the neuronal growth cone revealed by FM1-43 labeling.

Membrane dynamics within the chick ciliary neuronal growth cone were investigated by using the membrane-impermeant dye FM1-43. A depolarization-evoked endocytosis was observed that shared many properties with the synaptic vesicle recycling previously described at the presynaptic terminal. In addition, in the absence of depolarization a basal level of constitutive endocytotic activity was observed at approximately 30% of the rate of evoked endocytosis. This constitutive endocytosis accounted for large amounts of membrane retrieval: the equivalent of the entire growth cone surface area could be internalized within a 30 min period. Endosomes generated via constitutive and evoked processes were highly mobile and could move considerable distances both within the growth cone and out to the neurite. In addition to their different requirements for formation, evoked and constitutive endosomes displayed a significant difference in release properties. After a subsequent depolarization of labeled growth cones, evoked endosomes were released although constitutive endosomes were not released. Furthermore, treatment with latrotoxin released evoked endosomes, but not constitutive endosomes. Although the properties of evoked endosomes are highly reminiscent of synaptic vesicles, constitutive endosomes appear to be a separate pool resulting from a distinct and highly active process within the neuronal growth cone.

Cross-species collapse activity of polarized radial glia on retinal ganglion cell axons.

Inhibition of incorrect axonal outgrowth has been shown to be a crucial guidance mechanism during the development of the nervous system. Within the visual system of chick and rat, extension of retinal ganglion cell axons is essentially restricted to distinct layers of the retina and distinct brain regions such as the tectum opticum. In addition, populations of ganglion cells from defined retina locations project topographically to defined tectal areas, their growth possibly being inhibited by radial glia in incorrect tectal regions. In the current study, we aimed to analyse potential inhibitory activity of retinal glia during outgrowth of ganglion cell axons of embryonic chick and rat. The response of ganglion cell axons originating from different retina locations when exposed to purified retinal radial glia cell membranes were monitored in collapse assays by time lapse video recording. The interaction of axons growing on purified glial somata or glial endfeet was analysed in outgrowth assays. Our results indicate that (1) nasal and temporal chick growth cones are equally induced to collapse by cell membranes from retinal radial glia: 75% nasal and 72% temporal. (2) The collapse inducing component of radial glia can be inactivated by defined heat treatment, reducing collapsing activity to 6% nasal and 5% temporal. (3) Rat growth cones respond in a similar way to chick radial glia. (4) Rat axons grow perfectly on endfeet but not on somata of radial glia of the chick. In summary, the data suggest that radial glia are functionally polarized with permissive endfeet and inhibitory somata based on heat-labile proteins. Glia polarization is likely to inhibit aberrant growth of ganglion cell axons into outer retina layers. However, retinal radial glia are unlikely to participate in preordering axons within the retina and therefore do not affect the topographic projection. Finally, the inhibitory function of radial glia is conserved between birds and mammals and represents possibly a fundamental mechanism for structuring the central nervous system.

Different cell surface areas of polarized radial glia having opposite effects on axonal outgrowth.

During neuronal development neurites are likely to be specifically guided to their targets. Within the chicken retina, ganglion cell axons are extended exclusively into the optic fibre layer, but not into the outer retina. We investigated, whether radial glial cells having endfeet at the optic fibre layer and somata in the outer retina, might be involved in neurite guidance. In order to analyse distinct cell surface areas, endfeet and somata of these glial cells were purified. Glial endfeet were isolated from flat mounted retina by a specific detachment procedure. Glial somata were purified by negative selection using a monoclonal antibody/complement mediated cytolysis of all non-glial cells. Retinal tissue strips were explanted either onto pure glial endfeet or onto glial somata. As revealed by scanning and fluorescence microscopy, essentially no ganglion cell axons were evident on glial somata, whereas axonal outgrowth was abundant on glial endfeet. However, when glial somata were heat treated and employed thereafter as the substratum, axon extension was significantly increased. Time-lapse video recording studies indicated that purified cell membranes of glial somata but not of endfeet induced collapse of growth cones. Collapsing activity was destroyed by heat treatment of glial membranes. The collapsing activity of retinal glia was found to be specific for retinal ganglion cell neurites, because growth cones from dorsal root ganglia remained unaffected. Employing four different kinase inhibitors revealed that the investigated protein kinase types were unlikely to be involved in the collapse reaction. The data show for the first time that radial glial cells are functionally polarized having permissive endfeet and inhibitory somata with regard to outgrowing axons. This finding underscores the pivotal role of radial glia in structuring developing nervous systems.

Axonal versus dendritic outgrowth is differentially affected by radial glia in discrete layers of the retina.

Formation of neural cell polarity defined by oriented extension of axons and dendrites is a crucial event during the development of the nervous system. Ganglion cells of the chicken retina extend axons exclusively into the inner retina, whereas their dendrites grow into the outer retina. To analyze guidance cues for specific neurite extension, novel in vitro systems were established. Ganglion cells were purified by enzymatically facilitated detachment of the ganglion cell layer. A newly developed retrograde labeling technique and the expression analysis of the cell type-specific 2A1 antigen were used to monitor ganglion cell purification. In highly purified ganglion cells explanted onto retinal cryosections (cryoculture), axon formation was induced when the cells were positioned on the inner retina. In contrast, on outer layers of the developing retina dendritic outgrowth was prevalent. Because radial glia have been demonstrated to be instructive in neuritogenesis, distinct glial cell compartments located in inner and outer retina, respectively, were isolated for functional assays. Glial end feet were purified by a physical detachment technique. Glial somata were purified by complement mediated cytolysis of all nonglial cells. When ganglion cells were cultured on different glial compartments, axon formation occurred on end feet but not on glial somata. In striking contrast, on glial somata dendrites were formed. The data support the notion that ganglion cell polarity is affected by the retinal microenvironment, which in turn is possibly influenced by radial glia, being themselves polarized.

Maturation of peroxisomes in differentiating human hepatoblastoma cells (HepG2): possible involvement of the peroxisome proliferator-activated receptor alpha (PPAR alpha).

We have studied the alterations of peroxisomes in the human hepatoblastoma cell line HepG2, induced to differentiate by long-term cultivation (20 days without passaging) using morphological and biochemical techniques as well as mRNA analysis. Ultrastructural studies revealed alterations in shape and size of peroxisomes, with significant increases in mean diameter and formation of small clusters exhibiting heterogeneous staining for catalase after 20 days in culture. These alterations of peroxisomes correspond to the changes described during the maturation process from prenatal to adult human hepatocytes. As revealed by Northern and Western blotting there was marked elevation of the mRNA (190%) and protein (180%) of the peroxisomal branched-chain acyl-CoA oxidase. This protein is the key regulatory enzyme for the side chain oxidation of cholesterol for bile acid synthesis, a pathway associated with mature hepatocytes. Concomitantly a marked increase of bile canaliculi was noted by light and electron microscopy. This differentiation process was confirmed also by the increase of albumin synthesis (mRNA: 160%; protein: 190%) which is generally used as a differentiation marker of hepatocytes in culture. Interestingly, the mRNA for peroxisome proliferator-activated receptor alpha (PPAR alpha) increased drastically by almost 390% and its corresponding protein by 150%, suggesting its involvement in maturation of the peroxisomal compartment in differentiating HepG2 cells. In contrast to the wellknown increases during the drug-induced peroxisome proliferation of cytochrome P450 4A, multifunctional enzyme 1, palmitoyl-CoA oxidase and the 70-kDa peroxisomal membrane protein, those proteins were either not altered or only slightly elevated during the differentiation process, suggesting that peroxisome proliferation and maturation are two distinct and differentially regulated processes.

Photoreceptor differentiation analyzed by the novel monoclonal antibody 1G1.

In vertebrates, photoreceptor development has become a key model system to study mechanisms of cell differentiation. A still unresolved question is why photoreceptor maturation is retarded over an extended period of embryogenesis though photoreceptors are among the first cells born in the retina. We have generated the novel monoclonal antibody 1G1 which binds to outer photoreceptor segments of adult retinae of various species including chicken and rat. In the developing chicken retina presumptive photoreceptor cells were labeled by MAb 1G1 at embryonic day 10 (E10). Retinal cell cultures revealed that the corresponding antigen is expressed on the cell surface of rods and cones likewise. Metabolic labeling with bromodeoxyuridine in vitro indicated that 1G1 antigen expression is restricted to postmitotic cells. Comparison of single cell cultures starting from different developmental stages showed that antigen expression can be induced prematurely, if cells are released from their native tissue environment. In order to analyze potential regulatory cell interactions, retinal cells were cultured on cryosections of the eye (cryoculture). The percentage of 1G1+ cells which contacted the pigment epithelium, was significantly lower in comparison to cells located on retinal tissue. The data are consistent with the notion that the pigment epithelial cells which contact retinal photoreceptors in vivo, could be partially inhibitory and consequently delay photoreceptor differentiation.

In vivo observations of timecourse and distribution of morphological dynamics in Xenopus retinotectal axon arbors.

Changes in neuronal structure can contribute to the plasticity of neuronal connections in the developing and mature nervous system. However, the expectation that they would occur slowly precluded many from considering structural changes as a mechanism underlying synaptic plasticity that occurs over a period of minutes to hours. We took time-lapse confocal images of retinotectal axon arbors to determine the timecourse, magnitude, and distribution of changes in axon arbor structure within living Xenopus tadpoles. Images of axons were collected at intervals of 3 min, 30 min, and 2 h over total observation periods up to 8 h. Branch additions and retractions in arbors imaged at 3 or 30 min intervals were confined to shorter branches. Sites of additions and retractions were distributed throughout the arbor. The average lifetime of branches was about 10 min. Branches of up to 10 microns could be added to the arbor within a single 3 min observation interval. Observations of arbors at 3 min intervals showed rapid changes in the structure of branchtips, including transitions from lamellar growth cones to more streamlined tips, growth cone collaps, and re-extension. Simple branchtips were motile and appeared capable of exploratory behavior when viewed in time-lapse movies. In arbors imaged at 2-h intervals over a total of 8 h, morphological changes included longer branches, tens of microns in length. An average of 50% of the total branch length in the arbor was remodeled within 8 h. The data indicate that the elaboration of the arbor occurs by the random addition of branches throughout the arbor, followed by the selective stabilization of a small fraction of the new branches and the retraction of the majority of branches. Stabilized branches can then elongate and support the addition of more branches. These data show that structural changes in presynaptic axons can occur very rapidly even in complex arbors and can therefore play a role in forms of neuronal plasticity that operate on a timescale of minutes.

Axonal guidance in the chicken retina.

During retina development, ganglion cells extend their axons exclusively into the innermost tissue layer, but not into outer retina layers. In order to elucidate guiding mechanisms for axons, tissue strips of embryonic chicken retinae were explanted onto retinal cryosections (cryoculture). Ganglion cell axons originating from the explant grew preferentially on the innermost retina layer of cryosections, whereas outer tissue layers were avoided, very much as in vivo. Stereotropism, interaction with laminin of the basal lamina and axonal fasciculation did not significantly affect oriented axonal outgrowth, so that stereotropism as a guidance mechanism could be excluded. Ganglion cell axons were not directed by physical barriers, e.g. microstructured silicon oxide chips. Similarly, UV induced protein inactivation revealed that laminin present in the inner retina did not provide a guidance cue. Even in the absence of ganglion cell axons in retinal cryosections due to prior optic nerve transection in ovo, the growth preference for the innermost retina layer was maintained in cryocultures. However, oriented elongation of axons along the innermost retina layer was lost when radial glial endfeet were selectively eliminated in retinal cryosections. In addition, glial endfeet provided an excellent growth substratum when pure preparations of endfeet were employed in explant cultures. The preference for glial endfeet positioned at the inner retina surface was accompanied by the avoidance of outer retina layers, most likely because of inhibitory components in this region. This assumption is corroborated by the finding that addition of exogenous growth-promoting laminin to cryosections did not abolish the inhibition. Laminin on glass surfaces provided an excellent substratum. Axonal outgrowth was also seriously hampered on specifically purified cells of the outer retina. Most notable, however, in cryocultures aberrant innervation of outer retina layers could be induced by prior heat or protease treatment of cryosections, which pointed to proteins as potential inhibitory components. In summary the data substantiate the hypothesis that within the retina, ganglion cell axons are guided by a dual mechanism based on a permissive and an inhibitory zone. Radial glia is likely to be instructive in this process.

Age at marriage, sex-ratios, and ethnic heterogamy.

"This paper focuses on the effects of age at marriage and the sex-ratio on patterns of ethnic homogamy among Israeli women. We hypothesize that later marriages are more likely than early marriages to be heterogamous as the 'marriage market' shifts from school to the work-place. By the same token, when facing severe marriage squeezes women will be forced to out-marry. Employing data from the 1983 census, we model mate selection of women from Afro-Asian and Euro-American origin in various birth-cohorts. The results do not fully support our hypotheses: we find that in and of itself, age at marriage does not enhance ethnic heterogamy."

Graded distribution of the neural 2A10 antigen in the developing chicken retina.

During retinal histogenesis, post-mitotic cells become located in different tissue layers, where they differentiate into distinct cell types. In an attempt to elucidate mechanisms of cell differentiation, we have employed hybridoma technology in conjunction with various in vitro techniques. Here, we present monoclonal antibody 2A10, which binds specifically to the cell surface of neurons and outgrowing neurites. Within the retina 2A10 antigen expression is developmentally regulated being most pronounced during the period of tissue layer formation. Elevated antigen expression is limited to post-mitotic neurons as revealed by labeling with bromodeoxyuridine. Retinal ganglion cells, which are the first neurons to develop, appear not to influence the overall developmental regulation of the antigen in the retina, since elimination of these cells by virtue of optic nerve transection in ovo did not alter the antigen expression. The antigen is distributed in a graded fashion in the radial axis of the retina. Maximal immunoreactivity was found at the inner surface of the retina (optic fiber layer), whereas only minute reactivity was detected in the outermost layer. This graded distribution could possibly be involved in a topographic system providing positional information for differentiating neurons. Operationally, MAb 2A10 is a useful marker for retinal neurons, and provides a tool for establishing pure Müller glia cultures by complement-mediated cytolysis of retinal neurons.

[Glucose tolerance in miniature swine]. / Untersuchungen zur Glukosetoleranz an Miniaturschweinen.

Miniature pigs of the "Mini-Lewe" breed were used to study the behavior of glucose and insulin. Firstly, the blood glucose levels within definite intervals of life were determined, thereafter glucose and insulin data were collected during a day and for a period of 60 min (taking 5 blood samples). In intravenous glucose tolerance tests animals with varying glucose utilization were established.

[Dopamine-induced secondary cardiomyopathy (author's transl)]. / Dopamin-induzierte sekundäre Cardiomyopathie.

When myocardial fibre necrosis was produced experimentally in rats by high doses of dopamine, the main lesions were found in the left ventricular and septal myocardium. They could also be demonstrated in the left atrium. This report, which concerns clinical observations, is suggestive of a secondary cardiomyopathy in humans due to dopamine. The following pathogenetic mechanism is envisaged. The effects of dopamine are due to myocardial depletion of high energy phosphates following maximal stimulation and the increased, though inadequate adaptation of coronary resistance. The release of norepinephrine is also important. Aggravating factors may include the disturbed relationships of pulmonary ventilaltion to perfusion and the opening and dilatation of arterio-venous shunts in the lung, with subsequent hypoxaemia.