Ultrastructure of the taste buds in the blind cave fish Astyanax jordani ("Anoptichthys") and the sighted river fish Astyanax mexicanus (Teleostei, Characidae).

This study describes the ultrastructure of the taste buds of the sighted river fish Astyanax mexicanus and of the blind cave fish Astyanax jordani (= Anoptichthys) (Teleostei, Characiformes, Characidae). In Astyanax and Anoptichthys, taste buds occur in the epithelia of the lips, oral cavity, and, in Anoptichthys, lower jaw. Both possess three types of taste buds: type I (elevated), type II (slightly elevated), and type III taste buds (not elevated or sunken). The taste buds are up to 60 microm high and up to 35 microm wide. The taste bud's sensory epithelium consists of 100--130 elongated cells: light cells, dense-cored-vesicles (dcv) -cells, dark cells, and degenerating cells. The dcv-cells are rich in dense-cored vesicles and are described for the first time in a teleostean taste bud. At the taste bud's base, there lie two to three basal cells. The basal cells of type I and type II taste buds have microvillus (spine)-like processes, in contrast to those of type III taste buds. The taste bud's nerve fiber plexus is situated between the bases of the elongated taste bud cells and the basal cells. Afferent synapses occur between dcv-cells and basal cells (presynaptic sides) and axons (postsynaptic side). Indistinct synapses occur between light cells and dark cells (presynaptic sides) and axons (postsynaptic side). The nerve fiber plexes of Anoptichthys type II and type III taste buds contain significantly more axon profiles than those of Astyanax. This may be associated with a compensatory improvement of the sense of taste in the blind, cave-dwelling fish.

Heterogeneity of fish taste bud ultrastructure as demonstrated in the holosteans Amia calva and Lepisosteus oculatus.

Taste buds are the peripheral sensory organs of the gustatory system. They occur in all taxa of vertebrates and are pear-shaped intra-epithelial organs of about 80 microm height and 50 microm width. Taste buds mainly consist of specialized epithelial cells, which synapse at their bases and therefore are secondary sensory cells. Taste buds have been described based on studies of teleostean species, but it turned out that the ultrastructure of teleostean taste buds may differ between distinct systematic groups and that this description is not representative of those taste buds in other main taxa of fishes, such as selachians, holosteans and dipnoans. Furthermore, it is not known how variable the micromorphologies of non-teleostean taste buds are. For this reason the taste buds of two holosteans, Lepisosteus oculatus and Amia calva, were investigated and compared. While in both species the taste buds are of the same shapes and sizes, the cellular components of their sensory epithelia differ: in Lepisosteus taste buds comprise two types of elongated light cells and one type of dark cells. In contrast, Amia taste buds contain only one type of light, but two types of dark elongated cells. Afferent synapses are common in the buds of both species, efferent synapses occur only in Lepisosteus taste buds. These differences show that even in the small group of holostean fishes the taste buds are differently organized. Consequently, a representative type of fish taste buds does not exist.

Fingerprinting taste buds: intermediate filaments and their implication for taste bud formation.

Intermediate filaments in taste organs of terrestrial (human and chick) as well as aquatic (Xenopus laevis) species were detected using immunohistochemistry and electron microscopy. During development, the potential importance of the interface between the taste bud primordium and non-gustatory adjacent tissues is evidenced by the distinct immunoreactivity of a subpopulation of taste bud cells for cytokeratins and vimentin. In human foetuses, the selective molecular marker for taste bud primordia, cytokeratin 20, is not detectable prior to the ingrowth of nerve fibres into the epithelium, which supports the hypothesis that nerve fibres are necessary for initiating taste bud development. Another intermediate filament protein, vimentin, occurs in derivatives of mesoderm, but usually not in epithelium. In humans, vimentin immunoreactivity is expressed mainly in border (marginal) epithelial cells of taste bud primordia, while in chick, vimentin expression occurs in most taste bud cells, whereas non-gustatory epithelium is vimentin immunonegative. Our chick data suggest a relationship between the degree of vimentin expression and taste bud cell proliferation especially during the perihatching period. It is suggested that surrounding epithelial cells (human) and mesenchymal cells (chick) may be contributing sources of developing taste buds. The dense perinuclear network of intermediate filaments especially in dark (i.e. non-sensory) taste disc cells of Xenopus indicates that vimentin filaments also might be associated with cells of non-gustatory function. These results indicate that the mechanisms of taste bud differentiation from source tissues may differ among vertebrates of different taxa.

Innervation of developing human taste buds. An immunohistochemical study.

Morphological changes in developing human gustatory papillae during the 6th to the 23rd postovulatory week have been studied. The general innervation pattern of taste papillae and taste bud primordia was revealed immunohistochemically using antibodies against protein gene product 9.5 (PGP9.5), neurofilament H (NFH), neurofilament L (NFL), neurone-specific enolase (NSE), and tubulin. The autonomic and somatosensory nerve supply has been investigated using antibodies against substance P (SP), calcitonin gene-related peptide (CGRP), tyrosine hydroxylase (TH), neuropeptide Y (NPY), the neuronal form of nitric oxide synthase (n-NOS), and, enzyme histochemically, NADPH-diaphorase. Nerve fibers approach the basal membrane of the lingual epithelium around the 7th postovulatory week and invade the epithelium of papilla-like structures at the 8th week, but some also penetrate the basal membrane of the non-papillary epithelium. They are in close contact with slender epithelial cells that are considered to be the taste bud's progenitor cells. Early human taste buds situated at the anterior part of the tongue do not necessarily require a dermal (later fungiform) papilla. The NADPH-diaphorase reaction revealed positive results in dermal nerve fibers, but the immunohistochemical reaction against n-NOS was negative. Immunohistochemical detection of neuropeptides and vasoactive substances rendered negative results for developmental stages of 7-18 postovulatory weeks. By the 18th week, only SP was detected in dermal papillae, but not in the vicinity of taste buds' primordia. Thus, autonomic and somatosensory nerves seem not to play a key role in formation and maintenance of early human taste buds.

In vitro model for contact sensitization: II. Induction of IL-1beta mRNA in human blood-derived dendritic cells by contact sensitizers.

Epidermal mRNA for interleukin 1beta (IL-1beta) has been shown to be increased following exposure of mouse skin to sensitizing compounds. In addition, this early upregulation of IL-1beta was specific for contact sensitizers, while expression of IL-1beta was unaffected by irritants. Langerhans cells are the major source of IL-1beta within the epidermis in the induction phase of skin sensitization. Since the isolation of Langerhans cells from skin biopsies results only in low frequencies, we decided to use dendritic cells (DCs) generated from peripheral blood as Langerhans cell equivalents to investigate the ability of five contact sensitizers and one irritant to induce IL-1beta gene expression in vitro. For our studies we cultivated DCs in serum-free medium supplemented with granulocyte/macrophage-colony stimulation factor (GM-CSF) and interleukin 4 (IL-4). The DCs showed a typical dendritic morphology, a characteristic expression of surface markers and high stimulatory capacity for autologous T cells. 5-day-old DCs were incubated with subtoxic concentrations of the contact sensitizers pentadecyl-catechol, 2,4,6-trinitrobenezene sulfonic acid, 2,4-dinitrofluorobenzene, NiSO(4), K(2)Cr(2)O(7) and the irritant sodium dodecyl sulfate. IL-1beta mRNA expression was detected by using the reverse transcriptase-polymerase chain reaction (RT-PCR) technique and non-radioactive hybridization procedures. For all contact sensitizers, expression of IL-1beta mRNA increased, whereas treatment with the irritant SDS had no significant effect on IL-1beta expression. Thus we developed an in vitro system, which may be useful to evaluate allergic potentials of chemicals and products.

Scanning electron microscopical studies of developing gustatory papillae in humans.

Development and morphological changes of human gustatory papillae during postovulatory weeks 6-15 have been studied using scanning and transmission electron microscopy. The first papilla of the tongue appears around postovulatory week 6 in its caudal midline near the foramen caecum. In contrast, the dorsal epithelium of the anterior part of the tongue shows only small hillock- or papilla-like elevations from week 6 on, which comprise an aggregation of 5-20 epithelial cells. From week 7 on, most prominent fungiform papillae develop near the median sulcus and at the margins of the anterior part of the tongue. At their tops, the first primitive taste pores are found around week 10; these are often covered with processes of adjacent epithelial cells. Most pores, however, develop around weeks 14-15. The maturation of taste buds does not coincide with the appearance of taste pores, since taste bud cells are not fully differentiated in the observed period of time. Fungiform papillae are developed before filiform papillae, which do not occur within the first 15 weeks of gestation. Fungiform papillae tend to grow between weeks 8 and 15 of gestation, whereas the size of vallate papillae seems to be constant during this period.

Embryonic and early fetal development of human taste buds: a transmission electron microscopical study.

BACKGROUND: Taste buds are assemblies of slender epithelial cells that receive chemical stimuli from the outer (oral) environment. In contrast to the large and well documented information on the morphology of taste buds in adult humans and animals, there are only a few reports on fetal ones, and ultrastructural studies of prenatal human taste buds are lacking completely. Therefore, the present investigation has been carried out to study the taste bud primordium, its morphological changes including synaptogenesis, cell differentiation, and taste pore formation from the time of the onset of taste bud formation around the 8th week until the 15th postovulatory week. METHODS: Taste bud primordia of 42 human embryonic/fetal tongues have been examined by means of transmission electron microscopy. RESULTS: Nerve fibers approach the lingual epithelium between the 6th and 7th postovulatory week. They penetrate the basal lamina during the 8th week and form synapses with poorly differentiated, elongated, epithelial cells. By the 12th week, more differentiated cell types are seen: 1) electron-dense cells resembling type III cells of the adult taste bud containing large numbers of dense-cored vesicles (80-150 nm in diameter); 2) electron-dark cells with well developed endoplasmic reticulum and many apical mitochondria, being candidates for type II cells. Basally, these cells have foot-like processes containing dense-cored vesicles (120-200 nm in diameter), but they do not synapse to nerve fibers. Type I cells, characterized by apically located dense secretory granules, are not observed. First shallow grooves above the taste bud primordium are found around the 10th week. Untypically differentiated apical cellular processes extend onto the surface. Most of the taste pores develop around the 14th to 15th week. In the taste pit, mucous material is not present during the first 15 weeks of gestation. Synapses between cells and afferent nerve fibers were found by the 8th week, reaching a maximum around the 12th to 13th week. CONCLUSIONS: The early presence of taste bud cells containing dense-cored vesicles suggests an at least dual function of embryonic/ fetal taste buds: First, from the 8th until the 14th week, non-gustatory, paracrine functions should be considered. After the 14th week of gestation, when typical taste pores are present, the taste buds possibly start their gustatory function. Differentiated marginal cells are possibly involved in the formation of the taste pore. The lack of type I cells producing the mucous material in the taste pit indicates that the taste bud has not achieved a fully developed function until the 15th week of gestation.

Light and electron microscopical demonstration of methylene blue accumulation sites in taste buds of fish and mouse after supravital dye injection.

Electron microscopical data regarding methylene blue staining of taste buds in the epithelia of the goldfish lip and the cirumvallate papilla of the mouse tongue after supravital dye application are presented for the first time. The ultrastructural details were compared with the corresponding light microscopical findings. The dye was applied in different concentrations by injection or in crystalline from directly to the surface of the tissues. Both methylene blue and tissue were simultaneously fixed by immersion in a paraformaldehyde-glutaraldehyde solution with the addition of phosphomolybdic acid. The ensuing dye precipitate was further stabilized by ammonium heptamolybdate. On the light microscopical level, the taste bud's receptive structures, i.e. the receptor area (fish) and the taste pit (mouse), exhibited the highest affinity for the dye. Additionally, the mucous material within the trenches around the circumvallate papillae in mice was intensely stained. On the electron microscopical level, the cationic phenothiazine dye bound to the receptor villi or to the mucus coating the receptive structures. In the case of higher dye concentrations, a staining of single taste bud cells took place starting apically and proceeding down to the base. Dye accumulations within the intercellular clefts between the epithelial cells or within other structures were observed only if the dye concentration was further increased. Since similar results were also obtained with the cationic phenazo dye Janus green, dye accumulation in the mucus covering the receptor villi may be representative of the general binding of organic cations, which are known to induce bitter taste sensations.

Basal lamina formation by porcine thyroid cells grown in collagen- and laminin-deficient medium.

Porcine thyroid cells isolated by dispase treatment were cultured in either (a) Matrigel, (b) agarose with the addition of different combinations of basic fibroblast growth factor and laminin, or (c) on agarose-coated dishes. The formation of follicles and the presence of a basal lamina was investigated by routine electron microscopy of Araldite-embedded material and by light and electron microscopical immunocytochemical detection of the basal lamina components, laminin and collagen type IV. After 10 days of culture in Matrigel or agarose, a basal lamina-like structure surrounded most follicles. Follicles of cells growing in agarose and overlaid with a medium containing thyrotropin and overlaid with a medium containing thyrotropin and fibroblast growth factor showed a fluorescent band at the basal side of the follicles after immunocytochemical staining with anti-laminin and anti-collagen antibodies. Routine electron microscopy showed that a basal lamina-like structure lined the outside of the follicle. This structure could be subdivided into a lamina lucida and a lamina densa. Electron microscopical immunogold labelling revealed that immunologically detectable laminin was confined to the lamina densa. These findings suggest that even in the absence of basal lamina components in the culture medium, thyroid cells are able to form follicles with a regular basal lamina when they are cultured in a three-dimensional environment.

Efficiency of various dissociation methods for the preparation of thyroid single cell suspensions.

For comparison of the physiological potential of single thyroid cells versus cells integrated into follicles it would be ideal to work with suspensions consisting exclusively of single cells instead of a mixture of single cells and follicle fragments. In this study, various techniques for the isolation of single cells have been tested for their effect on cell viability, the ultrastructure of the isolated cells, the percentage of single cells and the ability of these cells to form follicles in culture. In addition, the cells were characterized for the preservation of their morphology and the ability to respond to TSH by comparing their immunocytochemical staining pattern with anti-vimentin and anti-ras p21 antibody to that of the intact thyroid tissue. Dispase treatment of thyroid tissues alone produced suspensions with a relatively small proportion of single cells. These cells stained with anti-vimentin and anti-ras p21 antibody to a similar percentage as thyroid cells in the intact gland. A combination of dispase treatment with either filtration or trypsin treatment severely compromised the viability of the cells. A high proportion of single cells with a good viability could be obtained either by centrifugation of dispase treated tissues or by culturing of dispase treated tissues as monolayers and subsequent detachment from the culture vessels with trypsin. Whereas the immunological staining with anti-vimentin and anti-ras oncogene antibody in the centrifuged cells resembled that of intact tissue, cells cultured as monolayers reacted differently. The differences in the immunological staining were still observed when the cells which had been grown as monolayers were stimulated with TSH. Differential centrifugation appeared to be the ideal method for the isolation of unaltered and viable single cells but is a rather laborious method to obtain larger amounts of single thyroid cells.

The plastome-encoded zfpA gene of a moss contains procaryotic as well as eucaryotic promoter consensus sequences and its RNA abundance is modulated by cytokinin.

Plastid DNA of the moss Physcomitrella patens has been sequenced. An open reading frame (ORF 315) was identified downstream from rbcL, between trnR-CCG and psaI. This ORF shares homology with zfpA, a putative regulatory gene in Pisum sativum. The moss ORF is preceded by a Shine-Dalgarno sequence, two plastid promoter consensus sequences, and three TATA boxes. A specific probe detected three transcripts of low abundance in the wild-type moss and a cytokinin-sensitive chloroplast mutant. Steady state levels of zfpA transcripts were different in the two genotypes. In mutant protonemata treated with cytokinin, steady state levels of the largest transcript decreased significantly.

Applications for risk-adjusted outcome measures.

This paper reports on the development and application of multiple risk-adjusted measures of hospital performance (mortality, readmission, complications). The indices are based on patient-level data so they can be aggregated at any level (hospital, specialty, physician), are easy to use and interpret by hospitals, and provide an inexpensive method for evaluating hospital performance using existing databases. This paper focuses on the development of practical applications of these measures in the quality improvement process.

Electron microscopic demonstration of lectin binding sites in the taste buds of the European catfish Silurus glanis (Teleostei).

Taste buds in the European catfish Silurus glanis were examined with electron microscopic lectin histochemistry. For detection of carbohydrate residues in sensory cells and adjacent epithelial cells, gold-, ferritin- and biotin-labeled lectins were used. A post-embedding procedure carried out on tissue sections embedded in LR-White was applied to differentiate between the sensory cells: The lectins from Helix pomatia (HPA) and Triticum vulgare (WGA) bound to N-acetyl-galactosamine and to N-acetylglucosamine residues occurring especially in vesicles of dark sensory cells. This indicates a secretory function of these cells. Most light sensory cells--with some exceptions, probably immature cells--, are HPA-negative. The mucus of the receptor field and at the top of the adjacent epithelial cells was strongly HPA-positive. Pre-embedding studies were performed in order to obtain information about the reaction of the mucus with lectins under supravital conditions. The mucus of the taste bud receptor field exhibited intensive binding to WGA, but not to the other lectins tested. Most lectins bound predominantly to the surface mucus of the nonsensory epithelium and to the marginal cells close to the receptor field. The strong lectin binding to mucins and the relatively weak lectin binding to cell surface membranes in pre-embedding studies suggest that the mucus possibly serves as a barrier which is passed selectively only by a small amount of lectins or lectin-carbohydrate complexes. Lectin-carbohydrate interactions may play a role in recognition phenomena on the plasmalemmata of the taste bud sensory cells. Recognition processes directed to bacteria or viruses should be considered as well.

Lectin histochemistry on mucous substances of the taste buds and adjacent epithelia of different vertebrates.

In the present study carbohydrate residues in taste buds (TBs) and adjacent epithelial formations of a teleostean fish, a frog and the rabbit were detected by means of lectin histochemistry. Biotinylated lectins from Pisum sativum (PSA), Arachis hypogaea (PNA), Dolichos biflorus (DBA), Triticum vulgaris (WGA and succinylated WGA), Glycine max (SBA) and Ulex europaeus (UEA I) have been applied. The lectins were bound to an avidin-biotin-peroxidase-complex (ABC) and visualized by diaminobenzidine/H2O2. Most intensive reactivity was observed at the taste disc cells of the frog with DBA, S-WGA and SBA. PNA did not bind to the TBs of any of the animals tested. As shown in SBA preparations, sialic acid is present in a nonacylated and an acylated form in the mucosa of the frog's tongue. The TBs of the fish possess all the sugars we looked for except for the disaccharide D-galactose-(1-3)-beta-D-N-acetyl-galactosamine (Gal/GalNAc) and sialic acid. The TBs of the rabbit contain GalNAc, as detected with DBA, but not with SBA; and fucose (Fuc), mannose (Man) and N-acetyl-glucosamine (GlcNAc). As revealed by preincubation of the tissue sections with neuraminidase in TB cells of the rabbit, sialic acid masks Gal/GalNAc and GalNAc. These lectin-binding characteristics show that in the TBs of some selected representatives which belong to different vertebrate classes exist different mucous substances. These substances possess different binding characteristics to specific sugars, and this is possibly of particular interest to chemoreception phenomena.

Histamine content and release of isolated rat gastric mucosal cells.

Enzymatically dispersed rat gastric cells were subdivided (Percoll) in fractions (F1, F2, F3) with different number of parietal cells (PC) and the intracellular histamine content was estimated (ng/10(6) cells): F1 (9% PC): 17 +/- 4 (SEM), F2 (26% PC): 80 +/- 9 and F3 (77% PC): 134 +/- 15. Histidinedecarboxylase showed the same pattern of distribution, F1 low, F2 medium, F3 high activity. Incubated F3 cells constantly released histamine (19 +/- 3 ng/10(6) cells/h), a process which could be stimulated by carbachol, forskolin and hexoprenaline. The data suggest: rat gastric histaminocytes copurify with PC, background stimulation by histamine should be considered in isolated cell systems, vagal stimulation may release histamine within the gastric mucosa.

[Tracheopexy with support frames. An experimental and clinical study]. / Die Tracheopexie mit Stützgerüsten. Eine experimentelle und klinische Studie.

The use of acrylic frames to support a weak trachea has been successful in 62,9% of the treated cases (in 22 of 35 operations). The use of ceramic frames has been successful in 92,3% of the cases (in 12 of 13 operations). On the basis of experimental studies, we reconstructed in this manner two rigid stenoses of the trachea by a two-stage method. The first stage consists in expanding the stenosis by means of an endotracheal support of silicone rubber tube; the second stage, in suturing ceramic frames to the trachea. Both operations by this method have been successful. Our methods and results are discussed.

[Roentgen microanalysis demonstration of zinc in the gustatory system of the bullhead, Ameiurus nebulosus (Teleostei)]. / Röntgenmikroanalytische Darstellung von Zink im Geschmackssystem des Zwergwelses, Ameriurus nebulosus (Teleostei).

The trace metal Zinc (Zn) seems to be essential for the normal functioning of the gustatory sense. In this study it was tried to localize Zn within the peripheral and central parts of the bullhead's gustatory system by the use of scanning electron microscopy and x-ray microanalysis. In freeze dried preparations of the bullhead's barbel taste buds (and the taste buds of rabbits) Zn is found in randomly distributed granules, which cannot be related to distinct taste bud regions. Furthermore, Zn occurs in sub-units of the central gustatory nuclei, the vagal and facial lobe of the rhombencephalon. Therefore Zn appears to be essential for intact peripheral as well as central gustatory processes, at least in lower vertebrates.

[Final procedure after colectomy with special consideration of ileorectal anastomosis (author's transl)]. / Definitive Versorgung nach Kolektomie unter besonderer Berück-sichtigung der ileorektalen Anastomose.

The indication for ileorectal anastomosis after colectomy is most likely given - with all reservations - in cases of ulcerative colitis and polyposis coli, not so often in Crohn's disease. Our own studies of dwarf pigs have shown that no qualitative changes take place in the histochemical characteristics either of ileum mucosa or in that of the rectum, following ileorectostomy. Late results of shelling out the rectal mucosa, and the tendencies towards constructing a reservoir of terminal ileum in the pelvis, remain to be seen. While keeping the risks at a justifiable level it should be our aim to enable our often still young patients to lead a normal life without social handicaps.