Wolfgang Kuhnel

Organization of the enteric nervous system in the human colon demonstrated by wholemount immunohistochemistry with special reference to the submucous plexus

To demonstrate the normal topography and structure of the enteric nervous system (ENS) in the human colon, the colonic wall of patients (n = 10, mean age 66.3 years), who underwent abdominal surgery unrelated to intestinal motility disorders, was submitted to wholemount immunohistochemistry. The specimens were stretched out and separated into the tunica muscularis, the outer and inner portion of the tela submucosa and the tunica mucosa. Prior to the application of the neuronal marker Protein Gene Product (PGP) 9.5, the laminar preparations were pretreated with the maceration agent KOH. The plexus myentericus was composed of prominent ganglia and interconnecting nerve fiber strands (NFS) forming a polygonal network, which was denser in the descending than in the ascending colon. Nerve cells were observed within the ganglia as well as in primary, secondary and tertiary NFS. The latter ramified into the adjacent smooth muscle layers, which contained the aganglionated plexus muscularis longitudinalis and circularis. The submucous plexus comprised three nerve networks of different topography and architecture: the delicate plexus submucosus extremus consisted of parallel orientated NFS with isolated nerve cells and small ganglia and was located at the outermost border of the tela submucosa adjacent to the circular muscle layer. The plexus submucosus externus was closely associated with the plexus submucosus extremus and composed of larger ganglia and thicker NFS. The plexus submucosus internus was situated adjacent to the lamina muscularis mucosae and formed a network with denser meshes but smaller ganglia and NFS than the plexus submucosus externus. The NFS of the aganglionated plexus muscularis mucosae followed the course of the smooth muscle cells of the lamina muscularis mucosae. The honeycomb-like network of the plexus mucosus was located within the lamina propria mucosae and divided into a subglandular and a periglandular portion. Single and accumulated nerve cells were observed within the plexus mucosus as a regular feature. The findings confirm the complex structural organisation of the ENS encountered in larger mammals, in particular the subdivision of the submucous plexus into three different compartments. PGP 9.5-immunohistochemistry applied to wholemount preparations comprehensively visualized the architecture of the intramural nerve plexus in human colonic specimens. In addition to conventional cross-sections, this technique allows a subtle assessment and classification of structural alterations of the ENS in patients with colorectal motor disorders.

Immunohistochemical visualization of the enteric nervous system using antibodies against Protein gene product (PGP) 9.5

Both the investigation of the enteric nervous system and the diagnosis of its pathological changes require reliable staining methods. In order to assess the potential of protein gene product (PGP) 9.5 as a marker for the enteric nervous system, we examined its immunoreaction in whole-mount preparations of the guinea pig and porcine small intestine, using a rabbit polyclonal antiserum. The immunohistochemical technique reveals the fundamental architectural features of the ganglionic and aganglionic plexuses. Furthermore, it enables a reproducible and differentiated visualization of the enteric nerve cells to be made, so that the various nerve cell types can be morphologically identified.

Development of a biocomposite to fill out articular cartilage lesions. Light, scanning and transmission electron microscopy of sheep chondrocytes cultured on a collagen I/III sponge.

The regenerative capacity of hyaline articular cartilage is limited. Thus, lesions of this tissue are a proarthrotic factor, and up to now the conservative treatment of cartilage lesions and arthrosis does not yield satisfying results. Therefore, autologous transplantation of articular chondrocytes is being investigated in a variety of different assays. The aim of our study was to create a mechanically stable cell-matrix implant with viable and active chondrocytes which could serve to fill out articular lesions created in the knees of sheep. For this purpose, articular cartilage was collected from knee lesions, chondrocytes were liberated enzymatically and seeded in culture flasks and cultured till confluency. Cells were then trypsinized and grown on a type I/III collagen matrix (Chondro-Gide, Geistlich Biomaterials, Wolhusen, Switzerland) for 3, 6 and 10 days before being fixed and embedded for electron microscopy by routine methods. Scanning electron microscopy was performed after dehydration in acetone, critical point drying and sputter-coating with gold-paladium. Light microscopically, clusters of chondrocytes can be seen on the surface of the matrix with a few cells growing into the matrix. Transmission electron microscopic photographs yield a rather differentiated chondrocyte-like appearance, which is evidence of a matrix-induced redifferentiation after dedifferentiation during the growth period in the culture flasks. Scanning electron microscopic results show large, flattened chondrocytes without signs of differentiation on plastic, whereas chondrocytes grown on the Chondro-Gide sponge show a more roundish aspect wrapping firmly around the collagen fibrils, exhibiting numerous contacts with the matrix. This cell-matrix biocomposite can now serve to fill out articular cartilage lesions created in the knees of sheep.

Topography of the enteric nervous system in Peyer's patches of the porcine small intestine

The mechanisms of intercommunication between the immune and nervous systems are not fully understood. In the case of the intestine, the enteric nervous system is involved in the regulation of immune responses. It was therefore decided to employ immunohistochemical techniques to investigate the structural organization of the enteric nervous system in Peyers patches of the porcine small intestine. Using antibodies against various nervous system-specific markers (protein gene product 9.5, neuron-specific enolase, neurofilament 200, S-100 protein and the glial fibrillary acidic protein), an intimate and specific structural association could be demonstrated between enteric nerves and the compartments of Peyers patches: follicles, interfollicular regions and domes. Peyers patches have a close topographical relationship to the two submucosal plexuses. Enteric nerves are located around the follicle in the interfollicular area — the so-called “traffic area”-and in the dome area, which plays an important role in the uptake and presentation of antigens.

Effects of hyaluronic acid on the morphology and proliferation of human chondrocytes in primary cell culture

Hyaline articular cartilage is a specialised connective tissue with weight bearing and adsorbing functions. Injury or loss of which often leads to impaired joint function and severe pain. Since the self-renewing abilities of hyaline articular cartilage are limited, there is major interest in the development of bioengineered cartilaginous implants. A cell-matrix-biocomposite composed of a collagen I/III scaffold seeded with autologous chondrocytes is currently being used in clinical trials; however, in order to optimise culture conditions, we cultured human condrocytes and seeded them on type I/III collagen membranes and on Thermanox plastic coverslips with media containing 0 to 500 microg/ml Hyaluronic Acid. After 4 days, the cells were either fixed or BrdU incorporation procedures begun. HE staining clearly demonstrated that cells grown in HA form three dimensional clusters and produce secretory vesicles as opposed to the monolayer control cells with noticeably fewer secretory vesicles. BrdU incorporation revealed a noticeable increase in cell proliferation in cells grown in 100 microg/ml; however, no comparable increase in 500 micorg/ml but rather a slight depression in proliferation. Immunohistochemistry for collagen II and aggrecan revealed an obvious increase in deposition of these two substances with increased HA administration as compared to the control; however, again, the higher concentration of HA, 500 microg/ml, did not result in a further increase in production. These results suggest that HA at 100 microg/ml not only influences chondrocytes to differentiate and produce more Collagen II and aggrecan, but also increases proliferation. We, therefore, propose that the addition of HA at low to middle dosages in condrocyte culturing might help improve condrocyte redifferentation and thus, the bioengineered cartilage.

Alterations in intercellular junctions of the uterine epithelium during the preimplantation phase in the rabbit.

SummaryIn the rabbit, the pseudopregnant uterus has been used as a model for studying alterations characteristic of the preimplantation phase. Alterations in intercellular junctions of the uterine epithelium were investigated during early pseudopregnancy (day 0 to day 6) by means of the freeze-fracture technique.In the uterine epithelium of oestrous females the zonula occludens belongs to the “tight” type of tight junctions. During pseudopregnancy an impressive proliferation of tight junctional belts can be observed. The basal strands proliferate, forming loops perpendicular to the luminal surface, whereas the more or less parallel arrangement of the luminal strands is maintained. At day 4 of pseudopregnancy macular tight junctions begin to develop on the lower portions of the lateral plasmalemma and are extensive by day 6 post hCG.Small gap junctions are infrequent between cells of the uterine epithelium and show no significant changes during the preimplantation phase.The physiological significance of the present morphological observations is discussed in the light of changes occurring during the preimplantation period.

Struktur und Cytochemie der Harderschen Drüse von Kaninchen

Summary1.The Harderian gland in rabbits, representing the type of a tubulo-alveolar gland, is located on the medial and posterior aspect of the eyeball and consists of two different parts, a small white lobe and a larger red one. The secretory cells in the tubular endpieces of both lobes are lipids containing cells. The lipid droplets can be stained with Sudan IV and Sudan black B. The luminal surface of both cell types is characterized by an alcianophilia at pH 2,5.2.The tubules of both lobes have a single layer of columnar epithelium. The lipid vacuoles in the cells of the red lobe are large, these of the white lobe small. The multilocular cytoplasm of all cells contains many free ribosomes and high amounts of mitochondria lying very closely together. All cells exhibit numerous and large Golgi-zones but only few ergastoplasm membranes.3.The lateral surfaces of the secretory cells are connected by elaborate junctional complexes (Zonulae occludentes, zonulae adhaerentes, desmosomes). These lateral surfaces are increased by intercellular canaliculi.4.Before being released into the glandular lumen, the limiting membranes of adjacent lipid droplets fuse, thus forming a large lipid vacuole. Extrusion generally is characterized by the coalescence of the limiting membrane with the plasmalemma, the formation of an opening at the cell surface and the discharge of the secretory lipid material. In the course of another mechanism of extrusion, the fat vacuoles are transported to the apical part of the cell where consequently the plasmamembrane bulges into the lumen. Eventually the fat vacuole is pinched off surrounded by a thin cytoplasmic envelope.5.Terminal fibers of the autonomic nervous system penetrate the basal membrane and can be found closely attached to the secretory or myoepithelial cells, partly by forming large swellings, which may be deeply embedded into the cytoplasm of the innervated cell. These terminal parts of the axons contain groups of synaptic and dense-cored vesicles, mitochondria and neurotubuli. Specific pre- and postsynaptic membranes have not been observed. The possible function of the harderian gland is discussed.Zusammenfassung1.Die Glandula harderiana des Kaninchens ist eine tubulo-alveoläre Drüse. Sie liegt an der medialen und hinteren Wand der Orbita und besteht aus zwei, makroskopisch unterscheidbaren Anteilen: Ein kleinerer oberer Teil ist von weißer Farbe, der größere untere ist rot. Die sezernierenden Zellen in den tubulösen Endstücken beider Lappen enthalten Fette, die sich mit Scharlachrot und Sudanschwarz B anfärben. Die luminale Oberfläche aller Zelltypen färbt sich mit Alcianblau bei pH 2,5 an.2.Die Drüsentubuli beider Lappen sind von einem einschichtigen kubischen bis zylindrischen Epithel ausgekleidet. Die Drüsenzellen sind im roten Anteil großblasig, im weißen Lappen sehr fein vakuolisiert. Das Cytoplasma dieser multiloculären Zellen enthält freie Ribosomen und eine erstaunlich große Zahl von Mitochondrien, die meistens eng aneinander lagern. Alle Zellen enthalten ferner mehrere Golgi-Stapel, aber nur spärliche Ergastoplasmamembranen.3.Die exokrinen Zellen werden an den seitlichen Kontaktflächen durch ein differenziertes Schlußleistennetz (Zonulae occludentes, Zonulae adhaerentes, Desmosomen) verbunden. Die sezernierende Oberfläche wird durch die Ausbildung interzellulärer Sekretkapillaren vergrößert.4.Bevor die Fettsubstanzen in die Lichtungen der Drüsenschläuche extrudiert werden, kommt es zur Verschmelzung benachbart liegender Pettvakuolen. Die Extrusion wird abgeschlossen durch das Verschmelzen der Hüllmembran der Fettvakuolen mit der Plasmamembran, durch Ausbildung einer Öffnung in der Plasmamembran und durch das Ausfließen der Fettsubstanzen. Ein anderer Extrusionsmechanismus besteht darin, daß apikal gelegene Fettvakuolen die Zellmembran weit in die Lichtung hinein vorwölben und anschließend mit einer Hüllmembran abgenabelt werden.5.Terminale Nervenfasern mit bekannter Innenstruktur durchbrechen die Basalmembran und lagern sich den Drüsen- oder Myoepithelzellen eng an, teilweise in Vertiefungen ihrer Oberfläche eingebettet. Diese terminalen Axone enthalten synaptische Vesikel, Bläschen mit einem massendichten Granulum, Mitochondrien und Neurotubuli. Spezialisierte prä- und postsynaptische Membranen kommen nicht vor. Die Frage nach der funktionellen Bedeutung der Harderschen Drüse wird diskutiert.

Endocytosis, storage, and release of IgE by human platelets: Differences in patients with type I allergy and nonatopic subjects

Platelets of atopic individuals differ in alpha-granular contents and in the amount of biologically active mediators released compared with platelets of nonatopic subjects. Because platelets carry the low-affinity IgE receptor (CD23), they may contribute to long-lasting IgE sensitivity by serving as a storage pool for IgE. We compared 45 atopic individuals with immediate-type allergies and 25 nonatopic control subjects with respect to storage and release of IgE by their platelets. Platelets of atopic individuals were characterized by a 10-fold higher median IgE content compared with those of nonatopic control subjects. The platelet IgE content correlated with the serum IgE level in the four atopic individuals with seasonal allergies who were followed up monthly over 1 year. Platelet stimulation with platelet activating factor, but not with thrombin or adenosine diphosphate, resulted in a release of 65% of the stored IgE. Conversely, platelet stimulation with monoclonal IgE/kappa resulted in the release of the chemokine RANTES. Platelet alpha-granules were identified as the main storage compartment for IgE by postembedding immunocytochemistry. Although more than half of the alpha-granules showed gold labeling for IgE, additional labeling was found on the external face of the plasma membrane and within the open canalicular system, indicating endocytosis and exocytosis of IgE. Moreover, the detection of CD23 not only on the plasma membrane but also on membranes of the alpha-granules further supports the existence of an exchange of IgE between the blood plasma and an internal storage compartment. Endocytosis could be confirmed by the uptake of an IgE myeloma protein coupled to colloidal gold. We conclude that platelets of atopic individuals may contribute to allergic inflammation by serving as a storage pool for IgE and by their increased capacity to liberate further mediators of allergy in response to IgE stimulation.

The antitumor potency of progesterone antagonists is due to their differentiation potential

A new therapy for the progesterone receptor positive mammary carcinoma may be the treatment with progesterone antagonists. This new class of antihormones causes a strong inhibition of tumor growth comparable to the potency of ovariectomy in a panel of experimental mammary carcinomas. The mechanisms of the strong tumor-inhibiting action of progesterone antagonists on experimental mammary carcinomas mainly depends on a progesterone receptor mediated process leading to induction of terminal differentiation and a blockade of the cell cycle. To further characterize the antitumor mechanism of progesterone antagonists we analyzed the effects of Onapristone and ZK 112.993 on DMBA- and MNU-mammary tumors of the rat and MXT-tumors of the mouse after different therapy intervals. These hormone-dependent mammary tumors normally display intraductal growth in papillary, cribiform or solid formation, whereas after treatment periods of 2-6 weeks with progesterone antagonists they displayed dysplastic ductal and acinous formations, usually filled with secretory material. Whereas tumor size, mitotic index, and the grade of tumor malignancy decreased distinctly, the volume fraction of glandular structures in the tumors as well as the appearance of apoptosis increased 3-fold compared to the controls. In addition, the mammary glands of progesterone antagonist treated animals showed the morphological features of differentiation with the appearance of secretory activity. Interestingly, the staining pattern of some of the lectins used, especially UEA 1 binding pattern, fits to the concept of differentiation since recent studies revealed a higher degree of fucosylation only in benign lesions of human breast cancers. Therefore, these data underline the concept of a differentiation potential of progesterone antagonists on progesterone receptor positive mammary carcinomas.

Endometrial Secretion and Early Mammalian Development

The maternal organism provides essential conditions for preimplantation development of the mammalian embryo by means of tubal and uterine proliferation and secretion. The uterine mucosa of a sexually mature rabbit shows the typical picture of an oestrous endometrium, moderate proliferation of the cavum and glandular epithelium (Fig. l a). The surface of the endometrium carries a single-layered, cubic to cylindrical epithelium, areas of which are covered with cilia. The cavum epithelium sinks down into short, narrow crypts, the uterine glands, in the tunica propria. There are no signs of typical secretory activity (Fig. 1 b, c).