Generation of Mouse Small Intestinal Epithelial Cell Lines That Allow the Analysis of Specific Innate Immune Functions

Cell lines derived from the small intestine that reflect authentic properties of the originating intestinal epithelium are of high value for studies on mucosal immunology and host microbial homeostasis. A novel immortalization procedure was applied to generate continuously proliferating cell lines from murine E19 embryonic small intestinal tissue. The obtained cell lines form a tight and polarized epithelial cell layer, display characteristic tight junction, microvilli and surface protein expression and generate increasing transepithelial electrical resistance during in vitro culture. Significant up-regulation of Cxcl2 and Cxcl5 chemokine expression upon exposure to defined microbial innate immune stimuli and endogenous cytokines is observed. Cell lines were also generated from a transgenic interferon reporter (Mx2-Luciferase) mouse, allowing reporter technology-based quantification of the cellular response to type I and III interferon. Thus, the newly created cell lines mimic properties of the natural epithelium and can be used for diverse studies including testing of the absorption of drug candidates. The reproducibility of the method to create such cell lines from wild type and transgenic mice provides a new tool to study molecular and cellular processes of the epithelial barrier.     

Selective illumination of single photoreceptors in the house fly retina: local membrane turnover and uptake of extracellular horseradish peroxidase (HRP) and Lucifer Yellow

Summary Single photoreceptor cells in the compound eye of the housefly Musca domestica were selectively illuminated and subsequently compared electron-microscopically with the unilluminated photoreceptors in the immediate surroundings. The rhabdomeres of the illuminated cells remain largely unaffected, but the cells show an increase in the number of coated pits, various types of vesicles, and degradative organelles; some of the latter organelles are described for the first time in fly photoreceptors. Coated pits are found not only at the bases of the microvilli, but also in other parts of the plasma membrane. Degradative organelles, endoplasmic reticulum (ER) and mitochondria aggregate in the perinuclear region. The rough ER and smooth ER are more elaborate, the number of Golgi stacks, free ribosomes and polysomes is increased, and the shape and distribution of heterochromatin within the nuclei are altered. Illuminated photoreceptors also interdigitate extensively with their neighbouring secondary pigment cells. These structural changes in illuminated fly photoreceptor cells indicate an increase in membrane turnover and cellular metabolism. When applied to the eye, Lucifer Yellow spreads into the extracellular space and is taken up only by the illuminated photoreceptor cells. These cells show the same structural modifications as above. Horseradish peroxidase applied in the same way is observed in pinocytotic vesicles and degradative organelles of the illuminated cells. Hence, the light-induced uptake of extracellular compounds takes place in vivo at least partially as a result of an increase in pinocytosis.     

Intracerebral distribution pattern of radioactive morphine and morphine-like drugs after intraventricular and intrathecal injection

Summary The intracerebral distribution patterns of ¹⁴C-morphine, ³H-dihydromorphine, and ³H-fentanyl after intraventricular injection were studied autoradiographically in rats and rabbits. The extent of permeation into the ventricular wall was measured at different times after injection. The hydrophilic morphine and dihydromorphine could be demonstrated within the tissue up to 4 hours. They seemed to be retained within the gray matter and hindered in crossing fiber bundles. On the other hand, the lipophilic fentanyl was quickly removed from the brain but remained relatively longer demonstrable within the white matter. Also, after intrathecal injection of ¹⁴C-morphine a time dependent spread from the injection site was observed. The use of autoradiography in pharmacological experiments as described was found advantageous. Thus, it is possible to correlate directly, the time course of the pharmacological effect and the respective distribution pattern of the drug applied. This may lead to better information about the probable sites of drug action.     

Elektronenmikroskopische Untersuchungen an den Hüllen der Oozyten und Eier des Flussbarsches Perca fluviatilis

Zusammenfassung Die Hüllen von Oozyten und reifen Eiern des Fußbarsches wurden licht- und elektronenmikroskopisch untersucht. In Oozyten vor der Bildung der Zona radiata sind die meisten der keulenförmigen Mikrovilli gerade auf die Follikelzellen ausgerichtet. Gelegentlich verlaufen Mikrovilli auch parallel zur Oozytenoberfläche. Die Zona radiata reiferer Eizellen besteht aus zwei Schichten, der elektronendichten Zona radiata externa und der weniger dichten Zona radiata interna. Während die erstere aus einem hochorganisierten Material besteht, ist die letztere aus einer Substanz von geringerer Dichte zusammengesetzt. In der Matrix der Zona radiata interna liegen unregelmäßig angeordnete flaschenbürstenähnliche Einschlüsse. Nach Kontrastierung mit l%iger Phosphorwolframsäure und 0,5% igem Uranylazetat sind feine Fibrillen in der Matrix nachweisbar. Die Zona radiata externa ist außen von einer Gallerthülle umgeben. Sie ist etwa fünfmal so stark wie die gesamte Zona, radiata. Diese Gallerthülle besteht aus homogenem Material von geringerer Dichte. Nach Anfärbung mit 0,2%-igem Rutheniumrot färbt sich diese Schicht intensiv rot. Daraus läßt sich auf das Vorhandensein von sauren Mukopolysacchariden schließen. In der Gallerthülle liegen elektronendichte Körnchen, in denen sich bei hoher Auflösung eine hochorganisierte, kristalloide Struktur nachweisen läßt. Die Gallerthülle wird von den zytoplasmatischen Fortsätzen der kegelförmigen Follikelzellen durchdrungen. Diese Fortsätze gehen in Ausläufer über, die in die Porenkanäle der Zona radiata eindringen. In einigen Fällen umhüllen die Ausläufer der Follikelzellen die Mikrovilli der Oozyte vollständig. Normalerweise liegen die Mikrovilli und die Ausläufer aber deutlich voneinander getrennt. Die Mikrovilli enden entweder direkt an der Zellmembran der Follikelzellen oder sie dringen tief in Membraneinstülpungen ein. Mikrovilli und die Ausläufer der Follikelzellen befinden sich in den Porenkanälen fast reifer Eier. Kurz vor der Ovulation werden sie wahrscheinlich zurückgezogen. Die Porenkanäle reifer Barscheier sind im äußeren Drittel der Zona radiata interna verschlossen.

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Summary The envelope of oocytes and mature eggs of the perch was examined by light and electron microscope. In oocytes before the formation of the zona radiata most of the clubshaped microvilli project straightly towards the follicular cells. Some, however, run parallel to the oocyte surface. The zona radiata of more mature oocytes consists of the electron dense zona radiata externa and the less electron dense zona radiata interna. While the former consists of a highly organized material, the latter is composed of a substance of low density, with the exception of a few brush-shaped inclusions which are scattered irregularely within the matrix of the zona radiata interna. After treatment with 1% phosphotungstic acid and 0.5% uranyl acetate tiny fibres become visible. External to the zona radiata externa the oocytes are enveloped by a jelly layer, about five times as thick as the zona radiata. The jelly layer appears to consist of a rather homogeneous material of low density. After treatment with a solution of 0,2% ruthenium red the jelly layer is stained intensely red, suggesting the presence of acidic mucopolysaccharides. Within the matrix of the jelly layer are embedded electron dense granules of unknown function. At high resolution they reveal a well organized, crystalloid structure. The jelly layer is penetrated by cytoplasmic processes of the cone-shaped follicular cells. These processes develop extensions which enter the pore canals of the zona radiata. In some cases these extensions envelope the microvilli of the oocytes completely. But normally the microvilli and the extensions are clearly separated. The microvilli make either direct contact with the membrane of the follicular processes or penetrate deeply into membrane interdigitations. Microvilli together with follicular extensions are present in the pore canals of almost mature eggs. They seem to be withdrawn just before ovulation. The pore canals of the zona radiata are partly closed in mature eggs.

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Ich verdanke die Anregung zu dieser Arbeit einer Diskussion, die ich mit Herrn Prof. Dr. E. A. Arndt, Rostock, anläßlich seines Vortrages am 21. Februar 1966 in Kiel führte.

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Untersuchungen mit Unterstützung durch die Deutsche Forschungsgemeinschaft. Herrn Prof. Dr. O. Moritz, Institut für Pharmakognosie, danke ich für die Möglichkeit, das Elektronenmikroskop seines Institutes zu benutzen. Frau R. Bardenhewer, geb. Schmidtke, danke ich für die Hilfe bei der Präparation und für die Anfertigung der photographischen Vergrößerungen.     

Modification of tumor cells by a low dose of Newcastle disease virus

Summary Augmented tumor-specific T cell responses were observed against the high metastatic murine lymphoma variant ESb when using as immunogen ESb tumor cells that had been modified by infection with a low dose of Newcastle disease virus (NDV). Such virus-modified inactivated tumor cells (ESb-NDV) were potent tumor vaccines when applied postoperatively for active specific immunotherapy of ESb metastases. We demonstrate here that immune spleen cells from mice immunized with ESb-NDV contain enhanced immune capacity in both the CD4⁺, CD8^– and the CD4^–, CD8⁺ T cell compartments to mount a secondary-tumor-specific cytotoxic T cell response in comparison with immune cells from mice immunized with ESb. ESb-NDV immune CD4⁺, CD8^– helper T cells also produced more interleukin 2 after antigen stimulation than the corresponding ESb immune cells. There was no participation of either CD4⁺ or CD8⁺ virus-specific cells in the augmented response. The specificity of the T cells for the tumor-associated antigen remaind unchanged. Thus, there is the paradox that the virus-mediated augmentation of the tumor-specific T cell response in this system involves increased T helper activity but does not involve the recognition of viral epitopes as potential new helper determinants.Abbreviations CTL cytolytic T lymphocytes - IL-2 interleukin 2 - rIL-2 recombinant IL-2 - mAb monoclonal antibody - NDV Newcastle disease virus - SSC syngeneic spleen cell     

Synaptic destabilization by neuronal Nogo-A

Formation and maintenance of a neuronal network is based on a balance between plasticity and stability of synaptic connections. Several molecules have been found to regulate the maintenance of excitatory synapses but nothing is known about the molecular mechanisms involved in synaptic stabilization versus disassembly at inhibitory synapses. Here, we demonstrate that Nogo-A, which is well known to be present in myelin and inhibit growth in the adult CNS, is present in inhibitory presynaptic terminals in cerebellar Purkinje cells at the time of Purkinje cell-Deep Cerebellar Nuclei (DCN) inhibitory synapse formation and is then downregulated during synapse maturation. We addressed the role of neuronal Nogo-A in synapse maturation by generating several mouse lines overexpressing Nogo-A, starting at postnatal ages and throughout adult life, specifically in cerebellar Purkinje cells and their terminals. The overexpression of Nogo-A induced a progressive disassembly, retraction and loss of the inhibitory Purkinje cell terminals. This led to deficits in motor learning and coordination in the transgenic mice. Prior to synapse disassembly, the overexpression of neuronal Nogo-A led to the downregulation of the synaptic scaffold proteins spectrin, spectrin-E and β-catenin in the postsynaptic neurons. Our data suggest that neuronal Nogo-A might play a role in the maintenance of inhibitory synapses by modulating the expression of synaptic anchoring molecules. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Regulation of Adhesion by Flexible Ectodomains of IgCAMs

To perform their diverse biological functions the adhesion activities of the cell adhesion molecules of the immunoglobulin superfamily (IgCAMs) might be regulated by local clustering, proteolytical shedding of their ectodomains or rapid recycling to and from the plasma membrane. Another form of regulation of adhesion might be obtained through flexible ectodomains of IgCAMs which adopt distinct conformations and which in turn modulate their adhesion activity. Here, we discuss variations in the conformation of the extracellular domains of CEACAM1 and CAR that might influence their binding and signaling activities. Furthermore, we concentrate on alternative splicing of single domains and short segments in the extracellular regions of L1 subfamily members that might affect the organization of the N-terminal located Ig-like domains. In particular, we discuss variations of the linker sequence between Ig-like domains 2 and 3 (D2 and D3) that is required for the horseshoe conformation.