Juan Francisco Madrid

Comparative immunohistochemical study of the gastroenteropancreatic endocrine system of three reptiles

The gastroenteropancreatic (GEP) endocrine system of three reptiles, Testudo graeca, Mauremys caspica, and Lacerta lepida, was investigated by means of immunocytochemistry. Single and double immunostaining methods have demonstrated immunoreactivity for insulin, glucagon, pancreatic polypeptide (PP), somatostatin, serotonin, and peptide tyrosine tyrosine (PYY) in endocrine cells of the pancreas of the reptiles studied. Islet-like structures with insulin-immunoreactive (IR) cells surrounded by glucagon-IR cells were observed only in the splenic portion of the pancreas of M. caspica. Occasionally, somatostatin- and PP-IR cells were associated with glucagon-containing cells. Endocrine cells were also observed in the excretory ducts of the exocrine glands. Serotonin, bombesin, neurotensin, gastrin, glucagon, somatostatin, PYY, and insulin were demonstrated immunocytochemically in open-type GEP cells of the digestive tract of the animals studied. Serotonin, somatostatin, and glucagon-immunoreactive cells were the most abundant endocrine cell types. In L. lepida, PP- and peptide tyrosine tyrosine-immunoreactive cells were also frequently observed. Cells containing cholecystokinin, gastric inhibitory peptide, met- and leu-enkephalin, motilin, secretin, and vasoactive intestinal peptide could not be detected. The present work demonstrates that the reptilian GEP endocrine system is a complex structure containing most of the regulatory peptides similar in structure to those found in higher vertebrates.

Telocytes in neuromuscular spindles.

A new cell type named telocyte (TC) has recently been identified in various stromal tissues, including skeletal muscle interstitium. The aim of this study was to investigate by means of light (conventional and immunohistochemical procedures) and electron microscopy the presence of TCs in adult human neuromuscular spindles (NMSs) and lay the foundations for future research on their behaviour during human foetal development and in skeletal muscle pathology. A large number of TCs were observed in NMSs and were characterized ultrastructurally by very long, initially thin, moniliform prolongations (telopodes – Tps), in which thin segments (podomeres) alternated with dilations (podoms). TCs formed the innermost and (partially) the outermost layers of the external NMS capsule and the entire NMS internal capsule. In the latter, the Tps were organized in a dense network, which surrounded intrafusal striated muscle cells, nerve fibres and vessels, suggesting a passive and active role in controlling NMS activity, including their participation in cell‐to‐cell signalling. Immunohistochemically, TCs expressed vimentin, CD34 and occasionally c‐kit/CD117. In human foetus (22–23 weeks of gestational age), TCs and perineural cells formed a sheath, serving as an interconnection guide for the intrafusal structures. In pathological conditions, the number of CD34‐positive TCs increased in residual NMSs between infiltrative musculoaponeurotic fibromatosis and varied in NMSs surrounded by lymphocytic infiltrate in inflammatory myopathy. We conclude that TCs are numerous in NMSs (where striated muscle cells, nerves and vessels converge), which provide an ideal microanatomic structure for TC study.

Glycoconjugate distribution in the human fundic mucosa revealed by lectin- and glycoprotein-gold cytochemistry

SummaryThe glycoconjugates of the human fundic mucosa were characterized at the ultrastructural level by means of direct (Helix pomatia agglutinin-gold complex) and indirect lectin techniques (Concanavalin A and horseradish peroxidase-gold complex; wheat germ agglutinin and ovomucoid-gold complex). Surface mucous cells and mucous neck cells secreted O-glycoproteins with N-acetylgalactosamine and N-acetylglucosamine residues at the non reducing terminus of the saccharidic chain. The secretory granules of the mucous neck cells showed condensed areas slightly reactive to ConA. The results obtained in the chief cells suggest that these cells secrete N-glycoproteins rich in mannose and/or glucose residues. “Transitional cells”, presenting both morphological characteristics and lectin binding pattern intermediate to the mucous neck and chief cells have been observed. The surface of the intracellular canaliculi of the parietal cell was labelled by HPA, WGA and ConA. In the neck region of the gastric glands, immature parietal cells containing abundant mucous granules reactive to HPA, WGA and ConA were observed. The present results further corroborate the existence of a common cell precursor for surface mucous, mucous neck and parietal cells. In a further step, mucous neck cells gradually differentiate into chief cells the transitional cells being an intermediate stage.

Cytochemical characterization of glycoproteins in the developing acrosome of rats

SummaryThe composition and distribution of rat acrosomal glycoproteins during spermiogenesis have been investigated at light and electron microscopic level by means of a variety of morphological techniques including the application of lectins conjugated to peroxidase, digoxigenin and colloidal gold, enzyme and chemical deglycosylation procedures and conventional histochemistry. Results obtained with lectin histochemistry in combination with β-elimination reaction and endoglucosaminidase F/peptide N-glycosidase F digestion suggest that glycoproteins of mature acrosomes contain both N- and O-linked oligosaccharides. N-linked chains of acrosomal glycoproteins contain mannose and external residues of N-acetylglucosamine and galactose. They also have fucose residues linked to the core region of the oligosaccharide side chains. O-linked oligosaccharide chains contain external residues of both galactose and N-acetylgalactosamine. Mannose, fucose, galactose and N-acetylglucosamine residues were detected in acrosomes at all steps of spermiogenesis. N-acetylgalactosamine residues were only observed in the late steps of the spermiogenesis. N-acetylneuraminic acid residues were not detected throughout the acrosomal development. At initial stages of acrosome formation, glycoproteins were preferentially distributed over the acrosomic granules. In cap phase spermatids, lectin binding sites were homogeneously distributed throughout the acrosomes; however, in mature spermatozoa, glycoproteins were predominantly located over the outer acrosomal membrane.

Characterization of glycoconjugates in developing rat respiratory system by means of conventional and lectin histochemistry

SummaryThe glycoconjugates of the respiratory system of rats from 15 days of gestation through the adult period have been characterized by means of both conventional and lectin histochemistry. The main changes occurred at 20–21 days of gestation immediately before birth. An increase of acidic groups in the glycoproteins of the lung and airway epithelium was observed by conventional mucin histochemistry. The combined use of neuraminidase digestion and lectin histochemistry demonstrated an increase of sialic acid residues at the terminal position of the glucidic moieties of the glycoproteins. The sialic acid residues were linked α (2–3, 6) to d-galactose (β1–3)-N-acetylgalactosamine, thus masking the PNA-reactivity detected on the luminal surface of Clara cells and pneumonocytes before birth. In the adult period, α-l-fucose residues, detected by UEA-I, were localized in the glycoproteins contained in goblet cells and periciliary layer of the rat airway epithelium. The modifications observed in the lung of developing rats are similar to those previously described in human fetal and neonatal lungs. This suggests that the rat represents a useful model to study the glycoprotein synthesis during lung development.

Histochemical characterization of glycoconjugates in the epithelium of the extrapulmonary airways of several vertebrates.

SummaryThe glycoconjugates of the extrapulmonary airways of 11 tetrapode vertebrates have been characterized by means of both conventional and lectin histochemistry. Abundant sialosulphomucins were detected in the secretory cells and periciliary layer of turtles, snakes, birds and mammals while only sialomucins were observed in amphibians. Neutral and traces of acidic mucins were detected in the secretory cells of lizards. The secretory cells of the amphibian airways were reactive to Con-A, DBA and WGA. No α-l-fucose residues reactive with UEA-I or LTA were detected in amphibians. The goblet cells of the turtles were stained by DBA, SBA and WGA. Secretory cells of snakes and lizards reacted with Con-A and WGA. The mucous goblet cells of the birds were reactive to Con-A, LTA and WGA. In the chicken, they also showed affinity for PNA and SBA. The ciliated cells ofthe avian species studied were stained by Con-A and WGA. Mammalian goblet cells were reactive to Con-A, UEA-I and WGA. In the rat, affinity for DBA and SBA was also observed. The present results reveal the existence of marked differences in the sugar residues of the glycoconjugates of the extrapulmonary airways of tetrapode vertebrates. Only sialic acid residues appear to be constant constituents of the glycoconjugates of the airways of all species studied.

Influence of sulphate groups in the binding of peanut agglutinin. Histochemical demonstration with ligh- and electron-microscopy

SummaryThe influence of sulphation of mucus glycoproteins in the binding of peanut agglutinin (PNA) to tissue sections has been investigated by means of histochemical techniques at the light- and electron-microscopic level. A sequential methylation-saponification procedure was applied for the desulphation of tissue samples. Labelling by peroxidase- and colloidal gold-conjugated PNA was compared in control and desulphated samples of rat intestinal mucosa. The high-iron-diamine (HID) technique was used as a control for the effectiveness of the desulphation technique, and the Alcian Blue, pH 2.5 (AB 2.5), PAS and phosphotungstic acid-HCl (acid-PTA) techniques served as controls for the integrity of the oligosaccharide chains, respectively. In general, a marked increase of PNA reactivity was observed in desulphated samples when compared with control sections. These findings indicate that sulphation of galactose inhibits the binding of PNA to carbohydrate moieties in tissue sections. Staining patterns obtained with HID, PNA and the desulphation-PNA sequence in the goblet cells of the large intestine suggest a modification of the secretory product stored in these cells as the cell matures and moves from the lower crypt region toward the luminal surface. These modifications were not detected in the small intestine. Ultrastructural detection of PNA-binding sites suggests that galactose residues are incorporated into the oligosaccharide chains of O-liked glycoproteins at the medial cisternae of the Golgi apparatus. However, sulphation occurs at the trans side of the Golgi complex and the trans Golgi network. In conclusion, desulphation procedures are useful for revealing PNA-binding sites.

Ultrastructural localization of glycoconjugates in human bronchial glands: the subcellular organization of N- and O-linked oligosaccharide chains.

We investigated the glycoconjugates of the human bronchial glands at light and electron microscopic level by means of lectin histochemistry in combination with neuraminidase digestion and beta-elimination reaction. Both direct and indirect techniques using lectin-peroxidase, lectin-gold, and glycoprotein-gold complexes were applied. The binding pattern of the six lectins (ConA, HPA, DSA, WGA, LEA, and PNA) used in the present study suggests that mucous and serous cells of human bronchial glands contain both N- and O-glycosylated proteins in the secretory granules. Asparagine-linked oligosaccharides containing Gal(beta-1,4) GlcNAc and Man residues were abundant in serous cells. The demonstration of both the terminal Neu 5Ac (alpha-2,3, or 6) Gal (beta-1,4) GlcNAc sequence in the N-linked oligosaccharides of mucous cells and the terminal disaccharide Gal (beta-1,4) GlcNAc in the N-linked oligosaccharide chains of serous cells suggests the existence of complex type sugar chains N-glycosidically linked to the peptide region of the glycoproteins. The binding pattern of the DSA and the neuraminidase-DSA sequence provides evidence for the existence of sialyltransferase activity in the forming mucous granules of mucous bronchial cells.

Histochemistry of glycoconjugates in the gallbladder epithelium of ten animal species

SummaryA battery of seven lectins and several conventional mucin histochemical techniques were used to identify the epithelial mucins of the gallbladder of ten species: man, rabbit (Oryctolagus cuniculus, mammalia), hamster (Mesocricetus auratus, mammalia), chicken (Gallus gallus, bird), sparrow (Passer domesticus, bird), moorish gecko (Tarentola mauritanica, reptilia), ladder snake (Elaphe scalaris, reptilia), lake frog (Rana perezi, amphibia), natterjack toad (Bufo calamita, amphibia) and gilthead sea bream (Sparus auratus, fish). Glycogen was found in the epithelial lining of the reptilian and amphibian gallbladders. Sulphate and carboxyl groups were frequently found in the same species, except in the ladder snake and natterjack toad gallbladders where only sulphate groups were identified. Sialic acid residues were detected in man, rabbit, bird, T. mauritanica, R. perezi and fish gallbladders. ConA binding pattern was similar in the ten species studied. In the human gallbladder only PNA failed to label the luminal surface, while the glands were only unreactive to DBA. The human gallbladder showed a large variety of saccharides. The present results suggest that no relation exists between the composition of the gallbladder mucins and the situation of the species in the phylogenetic scale.

Behaviour of telocytes during physiopathological activation.

We consider CD34+ stromal cells/telocytes (CD34+ SC/TCs) in normal and pathological conditions. These cells are involved in organisation and control of the extracellular matrix, structural support, creation of microenvironments, intercellular communication, neurotransmission, immunomodulation and immunosurveillance, inhibition of apoptosis, and control, regulation and source of other cell types. CD34+ SC/TCs are widely reported in the origin of interstitial cells of Cajal and in regeneration in the heart, skeletal muscle, skin, respiratory tree, liver, urinary system and the eye. In addition, we contribute CD34+ SC/TC hyperplasia associated with several processes, including neurogenous hyperplasia (neuroma of the appendix), hyperplasia of Leydig cells in undescended testes (Cryptorchidism), peripheral areas of inflammatory/repair processes (pericicatricial tissue and transitional zones between diseased segments in Crohns disease and normal bowel), benign tumours (neurofibromas, Antoni-B zones of neurilemmomas, granular cell tumours, and melanocytic nevi) and in some lesions with myxoid, oedematous and degenerative changes (Reinkes oedema, myxomatous mitral valve degeneration, thyroid-associated ophthalmopathy and basophilic degenerative changes of the collagen in the dermis). We pay particular attention to the role of CD34+ SC/TCs during repair through granulation tissue, including morphologic changes, loss of CD34 expression and gain of αSMA expression with myofibroblast transformation, and interactions with pericytes, endothelial and inflammatory cells. Finally, we consider CD34 or αSMA expression in stromal cells of malignant epithelial tumours, and the role of CD34+ SC/TCs in the origin of carcinoma-associated fibroblasts (CAFs) and myofibroblasts. In conclusion, CD34+ SC/TCs play an important role in the maintenance and modulation of tissue homeostasis and in morphogenesis/renewal/repair.