Sarcoglycan complex in masseter and sternocleidomastoid muscles of baboons: an immunohistochemical study.

The sarcoglycan complex consists of a group of single-pass transmembrane glycoproteins that are essential to maintain the integrity of muscle membranes. Any mutation in each sarcoglycan gene causes a series of recessive autosomal dystrophin-positive muscular dystrophies. Negative fibres for sarcoglycans have never been found in healthy humans and animals. In this study, we have investigated whether the social ranking has an influence on the expression of sarcoglycans in the skeletal muscles of healthy baboons. Biopsies of masseter and sternocleidomastoid muscles were processed for confocal immunohistochemical detection of sarcoglycans. Our findings showed that baboons from different social rankings exhibited different sarcoglycan expression profiles. While in dominant baboons almost all muscles were stained for sarcoglycans, only 55% of muscle fibres showed a significant staining. This different expression pattern is likely to be due to the living conditions of these primates. Sarcoglycans which play a key role in muscle activity by controlling contractile forces may influence the phenotype of muscle fibres, thus determining an adaptation to functional conditions. We hypothesize that this intraspecies variation reflects an epigenetic modification of the muscular protein network that allows baboons to adapt progressively to a different social status.

Effect of hypothyroidism on postnatal conjunctival development in rats.

AIM: To study the conjunctival development in hypothyroid genetically epilepsy-prone rats (GEPRs) with serum T3 and T4 significantly lower than in normal rats. METHODS: A structural, ultrastructural and histochemical study on the conjunctival epithelium of GEPRs and of control Sprague-Dawley (SD) rats before and after eyelid opening, with particular regard to goblet cell differentiation. RESULTS: From birth to day 12, no goblet cells were demonstrated on the conjunctival surface of both strains, so that the epithelium was formed only by a cuboidal basal layer and by a superficial layer of roundish or flattened cells. On day 16, after the eyelid opening, Alcian blue (AB)-positive goblet cells filled with homogeneous granules were demonstrated isolated, in GEPRs, or clustered, in SD rats, in both the fornices and palpebral conjunctiva. The epithelium showed a basal layer and many layers of flattened cells and was taller in SD rats (8-10 layers) than in GEPRs (6-7 layers). At 3 months, the epithelium in SD rats was higher with generally clustered goblet cells, whilst in GEPRs goblet cells were both isolated or clustered. In both strains, the goblet cells showed a marked AB/periodic acid-Schiff positivity all over the conjunctival surface and were filled with granules of different density. In both strains, goblet cells were absent at birth and their appearance, as AB-positive cells, was concomitant with eyelid opening. CONCLUSIONS: Hypothyroid rats showed a conjunctival development different than that of normothyroid rats for both epithelial and goblet cells. It appears that thyroid hormone imbalance may influence conjunctival development.

Actin-associated proteins in ameloblast differentiation.

Focal contacts are systems of adherens junctions of the cell-extracellular matrix type, which allow the transfer of fundamental signals from the extracellular matrix to nuclear compartments, capable of regulating adhesion, proliferation, migration and differentiation of cells. Recently, many authors have concentrated their attention on epitheliomesenchymal interactions which guide organogenesis of dental germ, identifying numerous growth and differentiation factors and having the inner enamel epithelial cells of the enamel organ as a target. Given that the two cellular compartments in their tooth germ are separated by a basal membrane and by an extracellular matrix, which touches it, we wanted to evaluate the presence of focal contacts through the identification of talin and vinculin, proteins of the actin-associated protein complex. In this study we utilized the hemimandibles of young Wistar rats and we extracted the related odontogenic tooth organs present at their apical end. Specimens are processed with antibody against vinculin and talin. Results show that these junctional system proteins are present at the apical poles of both cellular compartments suggesting that putative epithelial-mesenchymal interactions, other than marker molecules, may use focal contacts as a system for transmission of signals.

Immunofluorescence distribution of actin-associated proteins in human seminiferous tubules of adolescent testes, normal and pathologic.

The aim of our study on human seminiferous tubules of adolescent testes was to study the localization of two actin-associated proteins of the adherens junctions, such as vinculin and talin, and to verify if there were modifications in their pattern in varicocele, a frequent disease of the testis in adolescent age. The study group consisted of 8 biopsies from normal testes (i.e., adolescents operated on for hydrocele or inguinal hernia) and 20 biopsies from pathological testes (i.e., adolescents operated on for idiophatic left varicocele). Biopsies were evaluated by indirect immunofluorescence using anti-human vinculin and anti-human talin antibodies. Observation was recorded with a Leica TCS 4D upright confocal microscope. In the normal testes, there was a strong positive immunoreactivity for vinculin, which was localized in the interstitial cells of Leydig, and both basal pole and lateral cell surface of Sertoli cells; the pattern of talin immunoreactivity was the same except that the lateral cell surface of Sertoli cells was not stained. In the varicocele group the pattern was different. Vinculin immunoreactivity showed small patches of fluorescence only in the cytoplasm of Sertoli cells while talin immunoreactivity showed a scanty distribution at the basal surface of Sertoli cells. These results confirm that, similarly to other tissues, vinculin is expressed at cell-cell and cell-matrix adherens junctions, while talin is present at cell-matrix adherens junctions in human seminiferous tubules of normal adolescents. Varicocele alters the patterns of these two proteins both quantitatively and qualitatively.

Beneficial effects of peroxynitrite decomposition catalyst in a rat model of splanchnic artery occlusion and reperfusion.

The aim of the present study was to investigate the protective effect of the peroxynitrite decomposition catalyst 5,10,15, 20-tetrakis(2,4,6-trimethyl-3,5-disulfonatophenyl)-porphyrinato iron (III) (FeTMPS) in a model of splanchnic artery occlusion shock (SAO). SAO shock was induced in rats by clamping both the superior mesenteric artery and the celiac trunk for 45 min, followed by release of the clamp (reperfusion). At 60 min after reperfusion, animals were killed for histological examination and biochemical studies. There was a marked increase in the oxidation of dihydrorhodamine 123 to rhodamine (a marker of peroxynitrite-induced oxidative processes) in the plasma of the SAO-shocked rats after reperfusion, but not during ischemia alone. Immunohistochemical examination demonstrated a marked increase in the immunoreactivity to nitrotyrosine, an index of nitrogen species such as peroxynitrite, in the necrotic ileum in shocked rats. SAO-shocked rats developed a significant increase of tissue myeloperoxidase and malonaldehyde activity, and marked histological injury to the distal ileum. SAO shock was also associated with a significant mortality (0% survival at 2 h after reperfusion). Reperfused ileum tissue sections from SAO-shocked rats showed positive staining for P-selectin localized mainly in the vascular endothelial cells. Ileum tissue sections obtained from SAO-shocked rats and stained with antibody to ICAM-1 showed a diffuse staining. Administration of FeTMPS significantly reduced ischemia/reperfusion injury in the bowel, and reduced lipid and the production of peroxynitrite during reperfusion. Treatment with PN catalyst also markedly reduced the intensity and degree of P-selectin and ICAM-1 staining in tissue sections from SAO-shocked rats and improved survival. Our results clearly demonstrate that peroxynitrite decomposition catalysts exert a protective effect in SAO and that this effect may be due to inhibition of the expression of adhesion molecules and the tissue damage associated with peroxynitrite-related pathways.

Beneficial effects of melatonin in a rat model of splanchnic artery occlusion and reperfusion.

The aim of the present study was to investigate the protective effect of the pineal secretary product melatonin in a model of splanchnic artery occlusion shock (SAO). SAO shock was induced in rats by clamping both the superior mesenteric artery and the celiac trunk for 45 min, followed thereafter by release of the clamp (reperfusion). At 60 min after reperfusion, animals were sacrificed for tissue histological examination and biochemical studies. There was a marked increase in the oxidation of dihydrorhodamine 123 to rhodamine (a marker of peroxynitrite-induced oxidative processes) in the plasma of the SAO-shocked rats after reperfusion, but not during ischemia alone. Immunohistochemical examination demonstrated a marked increase in the immunoreactivity to nitrotyrosine, an index of nitrogen species such as peroxynitrite, in the necrotic ileum in shocked rats. SAO-shocked rats developed a significant increase of tissue myeloperoxidase and malondialdehyde activity, and marked histological injury to the distal ileum. SAO shock was also associated with a significant mortality (0% survival at 2 hr after reperfusion). Reperfused ileum tissue sections from SAO-shocked rats showed positive staining for P-selectin, which was mainly localized in the vascular endothelial cells. Ileum tissue sections obtained from SAO-shocked rats with anti-intercellular adhesion molecule (ICAM-1) antibody showed a diffuse staining. Melatonin (applied at 3 mg/kg, 5 min prior to reperfusion, followed by an infusion of 3 mg/kg per hr), significantly reduced ischemia reperfusion injury in the bowel as evaluated by histological examination. This prevented the infiltration of neutrophils into the reperfused intestine, is evidenced by reduced myeloperoxidase activity and reduced lipid peroxidation. This was evaluated by malondialdehyde activity which reduced the production of peroxynitrite during reperfusion, markedly reduced the intensity and degree of P-selectin and ICAM-1 in tissue section from SAO-shocked rats and improved their survival. Taken together, our results clearly demonstrate that melatonin treatment exerts a protective effect and part of this effect may be due to inhibition of the expression of adhesion molecule and peroxynitrite-related pathways and subsequent reduction of neutrophil-mediated cellular injury.

Morphological changes induced by prolonged darkness in the retinal pigment epithelium of the turtle (Pseudemys scripta elegans).

The retinal pigment epithelium of Vertebrates was shown to be sensitive to cyclic oscillations of light and darkness. The morphological changes induced by prolonged darkness on the retinal epithelial cells of the freshwater turtle were studied, with particular regard to their localization and to their reversibility if animals are recovered under cyclic light. The eyes were processed for light and electron microscopy and a morphological and morphometric analysis was performed on the specimens. After 7 days of prolonged darkness, the vitreal extremity of some epithelial cells was partially detached; on the basal zone the infoldings were missing and vesicles and tubules, often arranged in rows, were observed. After 30 days of prolonged darkness, partial or complete double layers of epithelial cells were present: the superficial layer was connected, by means of the apical fringes, to the photoreceptors, whilst the deepest layer showed vesicles and tubules on its basal zone. After 7 days of recovery to L:D = 12:12, no cyclic activity was demonstrated and only occasional double layers of cells were present; on the basal surface isolated basal infoldings were present where two adjacent cells were joined together. It could be concluded that the detachment of the apical part of some cells, rapidly covered by the lateral sliding of the adjacent cells, and the substitution of the basal infoldings with vesicles and tubules could represent the morphological response of the retinal epithelium to the functional changes induced by prolonged darkness.

The protective role of endogenous glutathione in carrageenan-induced pleurisy in the rat.

In the present study we investigated the protective role of endogenous glutathione, a known free radical scavenger, in rats subjected to carrageenan-induced pleurisy. In vivo depletion of endogenous glutathione pools with L-buthionine-(S,R)-sulfoximine (BSO, 1 g/kg for 24 h, intraperitoneally) enhances the carrageenan-induced degree of pleural exudation and polymorphonuclear leukocyte migration in rats subjected to carrageenan-induced pleurisy. Lung myeloperoxidase activity and lipid peroxidation were significantly increased in BSO pretreated rats. However, the inducible nitric oxide (NO) synthase in lung samples was unaffected by BSO pretreatment. Immunohistochemical analysis for nitrotyrosine revealed a positive staining in lungs from carrageenan-treated rats, which was massively enhanced by BSO pretreatment. Furthermore, in vivo BSO pretreatment significantly increased peroxynitrite formation as measured by the oxidation of the fluorescent dye dihydrorhodamine 123, enhanced the appearance of DNA damage, the decrease in mitochondrial respiration and partially decreased the cellular level of NAD+ in ex vivo macrophages harvested from the pleural cavity of rats subjected to carrageenan-induced pleurisy. In vivo treatment with exogenous glutathione (50 mg/kg i.p.) significantly reverts the effects of BSO and exerts anti-inflammatory effects. Thus, endogenous glutathione plays an important protective role against carrageenan-induced local inflammation.

Experimental colitis increases small intestine permeability in the rat.

Small intestine permeability is frequently altered in patients with Crohn's disease and is thought to play a pathogenic role. The aim of this study was to investigate the permeability and structure of small intestine tight junctions (TJ) in an animal model of chronic distal colitis. Seven days after colitis induction with trinitrobenzenesulfonic acid/ethanol, the duodenal and ileal TJ were studied by means of transmission electron microscopy using lanthanum nitrate, freeze fractures, and immunohistochemistry of occludin, ZO-1, and cingulin. Animals treated with intrarectal ethanol alone served as controls. In controls, 7.5% of duodenal and 9.6% of ileal TJ were permeable to lanthanum, whereas in colitis, permeability increased significantly (79.5% and 72.9%, respectively; p < 0.001, both segments compared with controls). In colitis, the percentage of "leaky" junctions in duodena as well as in terminal ilea correlated positively with the macroscopic colon damage score (p < 0.02 and p < 0.005, respectively). Freeze-fracture analysis and immunohistochemistry of cingulin and ZO-1 did not reveal any difference between control and treated animals, whereas the signal of the transmembrane protein occludin was found to be disrupted and irregular in both small intestine segments. Distal colitis induces an increase of TJ permeability throughout the entire small intestine, and the extent of alterations correlates with colonic damage. Alterations in the transmembrane protein occludin seem to be responsible for the observed changes. Further investigation is needed to elucidate the mechanism of TJ alterations by a remote focus of inflammation.

Morphological changes of the conjunctival epithelium in contact lens wearers evaluated by impression cytology.

PURPOSE: Conjunctival changes induced by rigid, gas-permeable and soft contact lenses (CL) were investigated using impression cytology. A scoring system based on seven parameters (specimen cellularity, cell-to-cell contacts, nucleus/cytoplasm ratio, chromatin, goblet cell distribution, keratinisation, inflammatory cells) was used to evaluate the morphological results. METHODS: One hundred and seventy-two CL-wearing eyes and 40 control eyes were examined. The population was divided into three groups: group 1, control eyes; group 2, asymptomatic CL-wearing eyes; group 3, CL-wearing eyes with intolerance problems. Impression cytology was carried out and all specimens were evaluated according to a scoring system. RESULTS: Group 1 specimens always showed normal features in the conjunctival epithelium. In group 2, rigid and gaspermeable lenses produced greater changes in conjunctival morphology than soft lenses, especially as regards cellularity, nucleus/cytoplasm ratio, goblet cell distribution and total score. On the contrary, in group 3 wearers of soft lenses showed higher partial and total scores than wearers of gas-permeable and rigid lenses. CONCLUSIONS: In asymptomatic CL wearers conjunctival morphology was better preserved in wearers of soft lenses. In patients with intolerance problems, wearers of soft lenses showed the worst cytological features. Impression cytology, evaluated with this scoring system, can be used to demonstrate epithelial damage occurring in CL-wearing patients.

Ultrastructural study of the conjunctival epithelium in the Mongolian gerbil (Meriones unguiculatus).

The conjunctival epithelium of the Mongolian gerbil (Meriones unguiculatus) was studied using the transmission and scanning electron microscopy. The superficial cells of the conjunctival epithelium were isoprismatic in shape and were covered, on their luminal surface, with microplicae. They were connected with the adjacent cells by junctional complexes and desmosomes. Some of the superficial cells were partially or completely detached from the underlying intermediate cells: this morphological pattern was interpreted as an expression of cellular renewal of the conjunctival surface. Goblet cells were either isolated or gathered in clusters: in any case, they were firmly connected with the adjacent epithelial cells. The apical part of the goblet cells was covered with a great number of long microvilli: they showed a variety of morphological aspects, which were interpreted as occurring in a sequential pattern during the secretory process. The Mongolian gerbil can be considered among the laboratory animals used for ophthalmic research.

Morphological and morphometric analysis on the rabbit conjunctival goblet cells in different hormonal conditions.

An altered conjunctival mucous secretion was reported in pregnancy or oral contraceptive use. Four groups of rabbits (males, dioestrous females, oestrous females, pregnant) were studied to determine whether sex and/or different physiological conditions could influence conjunctival goblet cells structure and ultrastructure. Light microscopy, transmission electron microscopy, and morphometry were performed. In males and in oestrous females the intracytoplasmic secretory granules were filled with granular material, whilst in pregnant and dioestrous females the granules were formed by a more homogenous and dense secretory material. The number of goblet cells was not statistically different in the groups studied, whilst pregnant animals showed the largest mean diameter. As to the secretory granules, their mean area was larger in dioestrous females, whilst their optical density was highest in pregnant animals. These observations indicate that the morphology of conjunctival goblet cell may vary according to sex and to different physiological conditions: this may account for the peculiar mucous secretion demonstrated during pregnancy.

Morphological differentiation of the conjunctival goblet cells in the chick (Gallus domesticus).

BACKGROUND: These is no consensus in the literature regarding the differentiation of conjunctival goblet cells in vertebrates. METHOD: The conjunctival epithelium of the chick was studied before and after hatching in order to demonstrate the morphological evolution of the goblet cells. The entire conjunctiva was processed for light microscopy either on semithin sections stained with toluidine blue-pironine or on traditional sections stained with Alcian blue pH 2.5-PAS. RESULTS: It was possible to demonstrate that goblet cells underwent remarkable changes in their secretory activity. At 12 h after hatching, isolated Alcian blue-positive cells were present in the fornix. At 24 h after hatching, cells positive for both Alcian blue and PAS were scattered among epithelial cells. Two days after hatching, cells which reacted positively only to PAS were also present. CONCLUSION: It is suggested that the differentiation of conjunctival goblet cells occurs first in the fornix, probably due to the particular vascular environment of this region, and then spreads all over the conjunctiva.

Prostaglandin E2 induction of binding activity to CRE and AP-2 elements in human T lymphocytes.

Prostaglandins of the E series are immunomodulatory agents which exert inhibitory as well as stimulatory effects on a variety of immune responses. Since it is known that PGE2 is able to increase cAMP levels, we investigated whether it can affect gene expression through the activation of the transcription factors which bind enhancer elements in the promoter regions of cAMP-regulated genes. Using electrophoretic mobility shift assay, we demonstrated that a short treatment of human T lymphocytes with PGE2 induces specific binding activity to CRE and AP-2, but not AP-1, DNA elements. Since the okadaic acid, a potent protein phosphatase inhibitor, prolongs the induction of the binding activity, phosphorylation events are likely to occur. This activity seems to be due to increased cAMP levels because forskolin and IBMX mimic the effects of PGE2. More interestingly, transfection experiments with CRE-CAT plasmide show that PGE2 activates the transcription of a CRE-containing promoter. These data support the positive role for PGE2 on some immune functions.

Impression cytology of the conjunctival epithelium in patients with vernal conjunctivitis.

The alterations in the conjunctival epithelium during the course of vernal conjunctivitis were examined by conjunctival impression cytology. The study was carried out on 53 patients with vernal conjunctivitis and 20 normal subjects as control. The results of impression cytology demonstrated that all cytological parameters were significantly modified in vernal conjunctivitis patients; the earliest alterations were found in the distribution of goblet cells, in the intercellular junctions, in the chromatin morphology and in the degree of keratinisation. The morphometric comparison showed that in vernal conjunctivitis patients the mean number of goblet cells per field was significantly higher than in controls. Moreover the mean diameter of goblet cells was smaller in patients than in controls. Impression cytology can, therefore, be a simple, non-invasive and cheap method for the study of the ocular surface in vernal conjunctivitis.

Embryological considerations on a multiple vascular anomaly in a child.

Coiling and kinking of the internal carotid artery are considered of remarkable importance in the pathogenesis of cerebral ischaemic episodes, especially in adults. The authors describe the case of a seven months old infant who underwent aortic and bilateral cerebral angiography because of severe neurological symptomatology. Besides serious malformations of the aorta and major vessels, both coiling of the left internal carotid artery and kinking of the right internal carotid artery were found. The arterious anomalies of the aortic branches are probably due to the deranged development of the two subclavian arteries and, in particular, of the right one. Owing to the patient's age, the carotid alterations are also consequent to an abnormal development of the arterious district deriving from the primitive aortic archs, especially the third arch.

[The synaptic ribbons in the retina of the turtle (Pseudemys scripta elegans): ultrastructural and chronobiological study]. / I nastri sinaptici nella retina della tartaruga (Pseudemys scripta elegans): indagini ultrastrutturali e cronobiologiche.

The Authors investigate the morphology and the behaviour of the synaptic ribbons (NS) in the outer plexiform layer of the retina of the turtle Pseudemys scripta elegans during the 24 hrs cycle; the animals were subjected to a light-dark cycle = 12:12 hours. New ultrastructural features are demonstrated in the rod arciform density and in the fibers connecting the central axis of the ribbon with the presynaptic membrane (Figs. 1 A-B). As to chronobiological data, the existence of different kinds of ribbons, each typical of a single period of the 24 hrs cycle is excluded: identical NS can likewise be observed at different hours of the day and under various environmental light stimulations (Figs. 2-3-4). Furthermore, it is also demonstrated that during the light period, every rod synapse in the outer plexiform layer has a typical ultrastructural topography of its own (Figs. 5 A-B-C). During the dark period, the ribbon with its neighbouring vesicles moves towards the nucleus, thus leaving the synaptic region (Figs. 6 A-B-C). From the analysis of our results, we are of the opinion that all previously demonstrated changes in number are only in part dependent on cyclic disruption and reconstruction. On the contrary, they can be the result of a different topographical arrangement of the organelles. When NS modify their relationship with the presynaptic membrane of the rods, they may behave as switch on the nervous circuits of the retina and play a facilitatory or inhibitory role on the discharge of synaptic vesicles during the different periods of the 24 hrs cycle.

[Post-natal maturation of the retina in the albino rat. I. The pigment epithelium]. / Maturazione post-natale della retina nel ratto albino: I. L'epitelio pigmentato.

The Authors studied the postnatal development of the retinal pigment epithelium in the albino rat, in order to elucidate its morphological and functional evolution, correlated to the numerous functional roles played in Vertebrates (Scheme 1). At birth, epithelial cells show few cytoplasmic organules and the apical surface provided of small depressions. From the third to the fifth postnatal day the first apical microfolds surround the depressions. From the seventh to the ninth day inner segments develop, whilst the apical surface of the epithelial cells is covered by many finger-like microfolds. During the eleventh postnatal day the buds of the outer segments and many lamellar microfolds can be demonstrated. During the sixteenth day the retina reaches its adult morphology. It is therefore well-evident that birth, similarly to many other Vertebrates, is not the last step, but only a moment, in the development of the retina: this process is completed only during postnatal life, when environmental light is able to stimulate every ocular structure.

Effects of a stable analogue of PGE2 (11-deoxy-13,14-didehydro-16 (S)-methylester methyl PGE2: FCE 20700) on the secretory processes of conjunctival goblet cells of rabbit.

The effects of the instillation of a PGE2-analogue (11-deoxy-13,14-didehydro-16 (S)-methyl PGE2 methylester: FCE 20700) in the conjunctival sac of the rabbit were studied by means of two methods. The former is a clinical study (Dohlman test), the latter is a morphological investigation (semithin sections) on specimens of the conjunctival mucosa. From both methods it was possible to demonstrate that the FCE 20700 instillation enhanced the mucous lacrimal secretion in rabbits.