A differentiated porcine bronchial epithelial cell culture model for studying human adenovirus tropism and virulence.

The species specificity of human adenoviruses (HAdV) almost precludes studying virulence and tropism in animal models, e.g. rodent models, or derived tissue and cell culture models. However, replication of HAdV type 5 (HAdV-C5) has been shown after intravenous injection in swine. In order to study adenovirus replication in airway tissue propagation of bronchial epithelial cells from porcine lungs was established. These primary cells proved to be fully permissive for HAdV-C5 infection in submerged culture, demonstrating efficient HAdV genome replication, infectious viral particle release (1.07×10(8) TCID(50)/ml±6.63×10(7)) and development of cytopathic effect (CPE). Differentiation of porcine bronchial epithelial cells was achieved at the air-liquid interface on collagen I coated 0.4µm polyester membranes. Morphology, expression of tubulin and occludin, the development of tight-junctions and cilia were similar to human bronchial epithelial cells. Infection with HAdV-C5 from the basolateral side resulted in release of infectious virus progeny (2.05×10(7) TCID(50)/ml±2.39×10(7)) to the apical surface as described recently in human bronchial epithelial cells, although complete CPE was not observed. Differentiated porcine bronchial epithelial cells hold promise as a novel method for studying the virulence and pathophysiology of pneumonia associated HAdV types.

The Na+/H+ exchanger isoform 2 is the predominant NHE isoform in murine colonic crypts and its lack causes NHE3 upregulation.

The Na(+)/H(+) exchanger isoform NHE2 is highly expressed in the intestinal tract, but its physiological role has remained obscure. The aim of this study was to define its expression, location, and regulatory properties in murine colon and to look for the compensatory changes in NHE2 (-/-) colon that allow normal histology and absorptive function. To this end, we measured murine proximal colonic surface and crypt cell NHE1, NHE2, and NHE3 expression levels, transport rates in response to acid, hyperosmolarity and cAMP in murine proximal colonic crypts, as well as changes in transcript levels and acid-activated NHE activity in NHE2 (-/-) crypts. We found that NHE2 was expressed most abundantly in crypts, NHE1 equally in crypts and surface cells, and NHE3 much stronger in surface cells. NHE2, like NHE1, was activated by low intracellular pH (pH(i)), hyperosmolarity, and cAMP, whereas NHE3 was activated only by low pH(i). Crypts isolated from NHE2 (-/-) mice displayed increased acid-activated NHE1- and NHE3-attributable Na(+)/H(+) exchange activity, no change in NHE1 expression, and NHE3 expression levels twice as high as in normal littermates. No change in cellular ultrastructure was found in NHE2 (-/-) colon. Our results demonstrate high NHE2 expression in the crypts and suggest a role for NHE2 in cryptal pH(i) and volume homeostasis.

Temporal and regional profile of neuronal and glial cellular injury after induction of kaolin hydrocephalus.

AIM: To study regional and temporal pattern of glial and neuronal reaction to induction of kaolin hydrocephalus in adult rats. Enzyme- and immunohistochemistry was performed in 20 adult rats with kaolin-hydrocephalus after 2, 4, 6 and 8 weeks to glial glutamatergic transmission activity (GLDH) and neuronal nitrous oxide synthetic activity (nNOS). Also, glial fibrillary acidic protein (GFAP), 68 kd neurofilament protein (NF68) and synaptophysin (SYN38) and basic fibroblastic growth-factor (bFGF) were stained. Results were quantified by imaging analysis (SCION IMAGE) and expressed as relative immunopositive area. After 2 weeks, nNos-activity increased in cortical and hippocampal neurones (CA1 and CA3) and GLDH-activity also showed increases, most significant in periventricular white matter (25.7 +/- 3.8 vs. 15.5 +/- 4.9; p < 0.001) and hippocampus (p < 0.01). After 4 or 6 weeks, global cortical GLDH-activity showed further marked increases (25.7 +/- 3.9 vs. 11.3 +/- 1.5; p < 0.05), while sustained structural changes have occurred: GFAP decreased in periventricular (3.3 +/- 0.5 vs. 6.3 +/- 1.2; p < 0.01), hippocampal and cortical astrocytes (0.9 +/- 0.34 vs. 5.0 +/- 0.7%; p < 0.01), whereas NF68 in cortical efferent neurones increased (6.5 +/- 1.5% vs. 4.7 +/- 0.1; p < 0.01) followed by a decrease in cortical and hippocampal (CA1) SYN 38 (p < 0.05). Acute glial and neuronal reactions were almost functional and in chronic stages sustained structural changes predominated. Since neuronal reactions were pronounced in selective vulnerable areas glial reaction was not restricted to periventricular astrocytes.

Post-natal development of the rat testis: steroid hormone receptor distribution and extracellular matrix deposition.

The control of testicular development and differentiation depends on hormones and a variety of cell-cell interactions mediated mainly by paracrine factors. In the second and third weeks of post-natal development important changes take place in the rat testis, e.g. the tubular lumen starts to open on post-natal day 10, the blood-testis barrier starts to form on day 15, and Sertoli cell proliferation ceases on day 15. In the present study the expression in different testicular compartments of the androgen receptor (AR), progesterone receptor (PR), and extracellular matrix proteins such as laminin, entactin-1 (nidogen-1) and fibronectin, during post-natal development was examined using immunohistochemistry and semiquantitative image analysis. An intratubular AR peak on days 14-17, an increase in intratubular PR expression on days 14-16, and an increase in peritubular entactin-1 expression during the second and third weeks post-partum are demonstrated. These results suggest that a variety of changes occur at the cellular level during this period when certain milestones of testicular development occur, substantiating the hypothesis of a particular role for paracrine interactions during the development of the rat testis.

Epithelial cell turnover--extracellular matrix relationship in the small intestine of human adults.

In the human small intestine, proliferation, migration, differentiation and death of epithelial cells take place in separated compartments along the crypt-villus axis. It has been shown in different cell systems that these basic biological activities are influenced by extracellular matrix proteins. To investigate possible relationships in the epithelium of the human adult small intestine we examined immunohistochemically the distribution of type IV collagen, laminin, fibronectin and tenascin, and compared the sites of their expression with the various cell activities. Epithelial cell proliferation and cell death have been detected by an antibody against Ki-67 and the TUNEL-assay, respectively. The results show that Ki-67 staining is restricted to the crypts and TUNEL-positive cells are only present in the upper villus region. Type IV collagen is uniformly present in the epithelial basement membrane along the crypt-villus axis providing a scaffold for other components of the extracellular matrix. Laminin appears to be associated with epithelial cell differentiation, since it is strongly expressed in the villus basement membrane but only weakly underneath the crypt epithelium. Although fibronectin displays a staining pattern similar to that of laminin, it might rather be responsible for cell adhesion. Strong indications have been found that tenascin could be related to epithelial cell death since it was particularly expressed at the villus tip, where the cells undergo apoptosis.

Membrana limitans interna and epiretinal membrane lying on macular holes. Some morphological observations.

The structure of the membrana limitans interna (MLI) in the region of the macula has been investigated by electron microscopy in (a) 2 enucleated human adult eyes and (b) 38 surgically removed samples associated with an epiretinal membrane (ERM). In the enucleated eye, the glia cells were vitrad bordered either by the lamina rara or, directly, by the lamina densa. Both extended into a coarse network whereby the lamina densa, through repeated branches and anastomoses, delimited large meshes, the lamina rara formed their contents. High magnification revealed that both meshes and contents of this network were composed by a further, finer network. It is suggested that strips and small openings of the finer network are homologous to the cords and intercordal spaces, respectively, which have been indicated as the common, basic structures of most of the basement membranes. The MLI excised with an ERM had the same structure. In some of the ERM associated with a macular hole, myofibroblasts prevailed among the cells. They showed indented nucleus, stress fibers abuting on the plasma membrane or in apparent continuity with bundles of extracellular filaments (microtendons), gap junctions. The cells lay on or were surrounded by a discontinuous basement membrane.

Regeneration of a transected peripheral nerve by transplantation of spinal cord encapsulated in a vein.

The objective was to investigate the regeneration of a transected peripheral nerve after transplantation of fragmented embryonic (E14-15) spinal cord cells which were encapsulated within a vein cavity. After 3 months transplantation, axonal regeneration was observed by staining with HE and antibody to neurofilament subtypes in six of 10 rats. In all six animals compound muscle action potentials to electrical stimulation could be recorded and indicated incomplete reinnervation of the fibular and tibial nerve, respectively. A chronic inflammation process around the transplant and a negative result of staining neurofilaments within the vein cavity and the transected nerve were found in animals lacking electrophysiological response to stimulation.

In the mammalian eye type VI collagen tetramers form three morphologically different aggregates.

The organization of the aggregates occurring in the stroma: (1) of the murine and human cornea after incubation in an ATP acidic solution; (2) of surgically excised epiretinal membranes (ERM); and (3) of the trabecular meshwork of monkey eyes was investigated morphologically and immunocytochemically on thin section electron microscopy. Morphology. The aggregates in the cornea appeared as cross-banded fibrils. The bands were uniformly electron dense (single banded form); they were separated from each other by interbands consisting of a bundle of filaments emerging in cross section as small areas of randomly assembled dot-like structures. In the ERM, most of the aggregates stood out as heteromorphic cross-banded bodies showing dense bands with electron denser borders (double banded form) and interbands composed of longitudinally oriented, parallel sheets or laminae of amorphous material enclosing thin, similarly oriented filaments. These extended, thinner and double in number (since interlacing with similar components of the opposite sheet), into the pale central zone of the dense band. The aggregates of the trabecular meshwork were heteromorphic, had uniformly dense bands (single banded form as in the cornea), but their interbands displayed longitudinal sheets (as the ERM aggregates). Immunocytochemistry revealed type VI collagen in the three eye aggregates with gold particles preferentially localized at the interbands. The specificity of the antibodies used was tested by Western blot analysis of type VI collagen samples extracted from human placenta and on homogenates of human cornea. In conclusion, the results indicate that the tetramers of type VI collagen may aggregate differently into structures with distinct supramolecular arrangements. These are illustrated in schematic drawings.

Re-evaluation of epoxy resin sections for light and electron microscopic immunostaining.

Epoxy resins provide optimal tissue morphology at both the light and the electron microscopic level and therefore enable correlative studies on semithin and thin sections from the same tissue block. Here we report on an approach to retain these advantages for immunolabeling studies by adapting and combining well-known techniques, i.e., surface etching with sodium ethoxide and heat-mediated antigen retrieval. We propose a simple procedure for immunostaining semithin and thin epoxy resin sections. To check its applicability, well characterized, commercially available antibodies (against E-cadherin, alpha-catenin, and beta-catenin) were used on sections of human small intestine. By light microscopy, the immunostaining efficiency was compared on cryo-, paraffin, and epoxy semithin sections processed in parallel. The most detailed results were obtained on semithin sections, where the labeling precisely delineated the lateral plasma membrane of the enterocytes. At the electron microscopic level the procedure did not damage the structures and allowed an efficient, reproducible immunogold labeling extending homogeneously over exceptionally wide tissue areas. The three antibodies specifically labeled the zonula adherens of the junctional complex between epithelial cells and, in agreement with light microscopic observations, the lateral plasma membrane.

Glucocorticoid receptor distribution in rat testis during postnatal development and effects of dexamethasone on immature peritubular cells in vitro.

In this study, the occurrence of the glucocorticoid receptor in the rat testis during early stages of postnatal development and its potential functional significance were investigated. Quantitative analyses of immunohistochemically labelled paraffin sections revealed that the receptor was present during all stages of postnatal development in the nuclei of interstitial cells such as Leydig cells, macrophages and fibroblasts, and endothelial cells of blood vessels. The labelling index increased initially, with maximum levels reached within the second week of postnatal development, and decreased thereafter. Within the seminiferous tubules, the glucocorticoid receptor could be detected in the nuclei of germ cells as well as Sertoli cells, reaching the highest levels in 3-week-old rats, mainly due to immature germ cell staining. In contrast, approximately 50% of the peritubular cell nuclei were stained throughout postnatal development. In vitro experiments on immature and immortalized peritubular cells demonstrated a dose-dependent and significant decrease in proliferation and fibronectin secretion after administration of dexamethasone. The data of this study suggest that glucocorticoids have a consistently repressive effect on peritubular cells throughout postnatal development. In summary, labelling of germ cells, especially in immature rats, might indicate an inhibition of spermatogenesis by corticosteroids.

Simultaneous apocrine and merocrine secretion in the rat coagulating gland.

The coagulating gland of the rat synthesizes two prevalent secretory proteins (transglutaminase and 115 K) that are discharched in a different manner, one being secreted in an apocrine fashion (transglutaminase) and the other one in a merocrine way (115 K). Differences in the intra- cellular pathway and the release of either protein were studied using immunofluorescence on semithin sections, immunoelectron microscopy of preembedding-processed chopper sections and postembedding-processed ultrathin sections of rat coagulating gland. Immunohistochemical staining using an anti-transglutaminase antibody resulted in dense labeling of the cytoplasm of secretory cells and their apical blebs, whereas the cisternae of the rough endoplasmic reticulum and the Golgi apparatus were completely unlabeled. When, on the contrary, the anti-115 K antiserum was used, dense labeling of the cisternae of the rough endoplasmic reticulum, the Golgi apparatus, and the secretory granules was seen. Intraluminal secretion was also labeled, but the secretory blebs remained unlabeled. Our findings show that, in the coagulating gland of the male rat, the two secretory proteins studied are processed in parallel, but at completely different intracellular pathways. They are released via different extrusion mechanisms. Transglutaminase is synthesized outside the endoplasmic reticulum, reaches the apical cell pole by free flow in the cytoplasm, and is released via apocrine blebs, the membranes of which appear to be derived from the apical plasma membrane. The protein 115 K, on the other hand, follows the classic route, being synthesized within the cisternae of rough endoplasmic reticulum, subsequently glycosylated in the Golgi apparatus, and released in a merocrine fashion. The mutual exclusion of the two secretory pathways and the regulation of the alternative release mechanism are still unresolved issues.

Paracrine interaction in testicular somatic cells.

Initiation of spermatogenesis is regulated by signals derived from intratubular Sertoli cells as well as extratubular Leydig cells, both being systemic targets of hypophyseal gonadotropins. In addition to Leydig and Sertoli cells, a number of other cell types are present in the testis viz. peritubular cells, macrophages and vascular components. The specific paracrine functions of these cells are only partially understood. The peritubular and Sertoli cells form the structural scaffold of the germinal epithelium and are responsible for intratubular pressure, release and transport of spermatozoa and the formation of the blood-testis barrier. We have performed ex vivo and in vitro studies on the ultrastructure of peritubular and Sertoli cells and the distribution of steroid hormone receptors, cytoskeletal and extracellular matrix proteins using rat testes from different stages of postnatal development. Morphological observations were related to in vitro findings of gene expression on the respective hormones and structural proteins. In the developing rat testis, the peritubular cells showed a strong and consistent expression of fibronectin, entactin, laminin as well as the glucocorticoid, androgen, estrogen and partially also the progesterone receptor, while the Sertoli cells were devoid of glucocorticoid receptor and entactin. The glucocorticoid receptor was present in around 20% of the intratubular germ cells (in the 2nd postnatal week) and in 50% of the peritubular cells. In Leydig cells also, the expression reached its climax in the 3rd weak and declined thereafter. This is perhaps pointing to a differentiation-inhibiting role of glucocorticoids in gonocyte differentiation. In the 3rd developmental week, the androgen receptor was present in about 15% of all gonocytes and later in 50% of peritubular cells and about 40% of interstitial cells. The estrogen receptor was absent in peritubular cells of the adult testis. The progesterone receptor was present in about 30% of the peritubular and 25% of the Leydig cells. Taking into account the significant increase in seminiferous tubules following postnatal developmental day 18, the peritubular cells seem to exert an androgen dependent growth stimulus to the seminiferous cords perhaps via the Sertoli cells. In vitro studies of peritubular and Sertoli cells cultured either alone or in co-culture showed by RT-PCR the expression of the androgen and the glucocorticoid receptors in both cell types, as well as fibronectin. Secretion of fibronectin occurred in a clear-cut time-dependent increase in monocultures of peritubular cells (on day 3 of culture). In co-cultures of Sertoli and peritubular cells, fibronectin bio- synthesis was down-regulated. The paracrine interplay between extracellular matrix and hormonal signals joining peritubular and Sertoli cells is essential in the differentiation of the seminiferous tubules.

[Removal of the internal limiting membrane in macular holes. Clinical and morphological findings]. / Entfernung der Membrana limitans interna bei Makulalöchern. Klinische und morphologische Befunde.

UNLABELLED: The recommended treatment for full-thickness macular holes is removal of the posterior hyaloid and sometimes the epiretinal membrane from the retina during vitrectomy in order to release the assumed intravitreous traction. We have employed a technique involving the additional removal of the membrana limitans interna (MLI) from the retina in the vicinity of the macular hole. We report on our clinical results and ultrastructural findings. MATERIALS AND METHODS: Between December 1995 and July 1996, we performed vitrectomies on 39 eyes of 37 patients with full-thickness macular hole. After removal of the attached posterior hyaloid, a specially developed forceps was used to remove a circular area of the MLI approximately three to four disc diameters in size. At the conclusion of the operation, 20% C3F8 gas was injected and the patient instructed to stay in a prone position for 8 days. RESULTS: Intraoperatively, "rhexis" of the MLI only rarely produced bleeding or recognizable retinal edema. Complete closure of the hole was observed postoperatively in 36 of the 39 eyes (92%). A visual improvement of at least two lines was achieved in 77% of eyes with successful closure. Pigment irregularities or edematous changes could not be detected either clinically or by fluorescein angiography in any of the 39 eyes. Electron microscopy was performed on 23 of the membranes. The salient feature was the MLI. Canals leading from the inner to the outer surface of the MLI contained Müller cell processes with clear signs of necrosis or degeneration. On the vitreous side, the MLI usually exhibited myofibroblasts. CONCLUSIONS: The MLI was successfully removed in all 39 eyes with a full-thickness macular hole. This procedure led to very good anatomic and functional results. It remains for future studies to determine the pathogenic significance of the necrotic processes detected by electron microscopy in the MLI canals.

Differential distribution of the mitochondrial heat-shock protein 60 in rat gastrointestinal tract.

Immunohistochemical studies on various parts of the rat gastrointestinal tract by means of an antibody against the mitochondrial chaperonin, heat-shock protein 60 (hsp60), has revealed a cell-specific distribution pattern. The active form of hsp60 is a heptameric complex that is involved in the import and refolding of nuclear-encoded proteins destined for the mitochondrial matrix. This chaperonin is detectable in highly replicating cells, e. g., keratinizing cells of the esophagus and short-living epithelial cells of the intestine. In the stomach, some of the oxyntic cells contain hsp60-positive mitochondria lying near intracellular canaliculi. Neuronal cells of the enteric nervous system present intense positive staining for hsp60 in some areas. All other non-epithelial cells of the digestive tract show weak or no hsp60 immunoreactivity. The presence of hsp60 in mitochondria seems to reflect two different forms of mitochondrial renewal: (1) total reformation of mitochondria and their content after mitotic division and (2) regeneration of these organelles following high activity, e. g., ATP synthesis.

Parenteral versus enteral nutrition: morphological changes in human adult intestinal mucosa.

In animal experiments total parenteral nutrition induces an atrophy of the small intestinal mucosa. In humans morphological data are few and controversial. Therefore, the aim of this study was to investigate the effect of parenteral nutrition on the intestinal mucosa of human adults. For this purpose samples of the proximal jejunum of a) patients with chronic pancreatitis receiving total parenteral nutrition as presurgical treatment, b) enterally nourished patients without (controls) and c) with chronic pancreatitis were compared using light and scanning electron microscopy. Statistical differences were assessed applying computer-assisted morphometry. The results demonstrated that the thickness of the jejunal mucosa decreased already in enterally nourished patients with chronic pancreatitis. However, after total parenteral nutrition the decrease (atrophy) was enhanced due to a strong reduction in villus height albeit the crypt length increased. In addition, scanning electron microscopy revealed distinctive changes in mucosal surface pattern, whereby finger-like villi were replaced by leaf-like villi and by long, winding bifurcating ridges. Cell shedding was absent. In conclusion, total parenteral nutrition in humans induces 1) an atrophy and 2) a remodelling of the intestinal mucosa (epithelium and lamina propria) with a decrease in the absorbing surface. These alterations involve both cell proliferation and cell shedding. The response of the mucosa to parenteral nutrition is immediate and the effect of the treatment in bringing about morphological alterations is more efficacious at the beginning than in the successive period. The basic disorder (chronic pancreatitis) of the patients nourished parenterally contributes to mucosal atrophy, but not to remodelling.

Modulation of apoptosis by starvation: morphological and biochemical study of rat intestinal mucosa.

Morphology at light and electron microscopic levels, expression and activation of transglutaminase and DNA fragmentation at internucleosomal sites were used as markers to study the effect of starvation on the apoptosis of small intestinal epithelial cells. The cells entering apoptotic programme in well-fed animals undergo many morphological changes in apical cytoplasm involving alterations in actin cytoskeleton organisation which may cause a discharge of microvilli. Some free floating cells in the intestinal lumen show characteristics of apoptotic cell death, e.g. shrinkage of cell and peripheral condensation of chromatin, while mitochondria and lysosomes remain unchanged. Apoptotic bodies are also seen in scanning electron micrographs. During progressive starvation, epithelial cells do not enter the apoptotic cell death programme. Biochemical markers for apoptosis such as increased transglutaminase activity and DNA fragmentation are clearly discernible in normally fed animals. The percentage of cells labelled immunohistochemically by antibody against transglutaminase decreased during starvation while DNA fragmentation was absent. The exact mechanism for suppressing apoptosis in intestinal cells under starvation is not known. However, the data presented here support the existence of such a regulatory process.