On the alleged presence of non-argyrophile argentaffin cells in the human gastro-intestinal tract

Summary The relationship between the argentaffin and argyrophile cells of the human gastrointestinal tract has been studied, in foetal and adult material, by a technique involving the staining of sections first by an argentaffin method (Gomori-hexamine silver, Schmorl, Diazonium) and subsequently by an argyrophile method (Bodian). A comparison of the cells staining by the two methods shows that all argentaffin cells of the human gastrointestinal tract are also argyrophile and that there is no evidence to support the claim of Hellweg (1952) and of HamPerl (1952) regarding the presence of non-argyrophile argentaffin cells.W. H. O. fellow from the Department of Anatomy, Medical College, Rohtak, India. — I am very grateful to Professor J. D. Boyd and to the World Health Organisation for having made it possible for me to carry out this research at the Anatomy School, Cambridge. I am indebted to Dr. G. A. Gresham and his staff for their very willing cooperation in providing material from surgical resections. My thanks are also due to Mr. J. F. Crane for the photographs and to Mr. J. W. Cash and Mr. R. Smith for helpful discussions on staining techniques.     

Argyrophile and argentaffin reactions in individual granules of enterochromaffin cells of the guinea pig

Summary Successive staining of cells of the enterochromaffin system of the guinea pig by an argentaffin and an argyrophile method shows that, as in man and rabbit, they can be divided into (a) those in which all granules are apparently argentaffin, (b) those in which all granules are argyrophile but none are argentaffin, and (c) those in which some granules are argentaffin (as well as argyrophile) while others are purely argyrophile. The presence of the third type makes it evident that no hard and fast line of distinction can be drawn between the so called argentaffin and argyrophile cells.I am grateful to Mr. Mohan Lal Sharma for help with the photographs and to Mr. Anand Parkash for technical assistance.     

Argyrophile and argentaffin reactions in individual granules of enterochromaffin cells of reserpine treated guinea pigs

Summary The individual granules of enterochromaffin cells of normal and reserpine treated guinea pigs have been studied by staining slides of the duodenum first by an argentaffin method and subsequently by an argyrophile method. Some argentaffin cells can be shown to contain not only argentaffin granules, but also granules that are purely argyrophile. The relative number of such argentaffin cells is greatly increased following administration of reserpine, as depletion of their 5-hydroxytryptamine content converts argentaffin granules into purely argyrophile ones. On the basis of this finding it is confirmed that the argyrophile granule is merely an argentaffin granule depleted of its 5-HT content, and that the argyrophile (nonargentaffin) and the argentaffin cells represent different phases of a secretory cycle.This investigation was supported by a grant from the Indian Council of Medical Research. I am grateful to Ms. Ciba of India Ltd. for making available reserpine (Serpasil) and solvent for reserpine. It is a pleasure to thank Mr. Anand Parkash for technical assistance and Mr. M. L. Sharma for the photographs.     

The relative proportion of pre-enterochromaffin (argyrophile) and enterochromaffin (argentaffin) cells in the gastro-intestinal tract of human foetuses

Summary The proportion of argyrophile cells that are argentaffin varies considerably over different parts of the gastro-intestinal tract of the human foetus. In the small intestine plots of the number of argentaffin cells against the number of argyrophile cells, at various cranio-caudal levels, reveal no correlation. The percentage of argyrophile cells that are argentaffin decreases in a caudal direction in the proximal part of the small intestine, but markedly increases in the distal part. In the large intestine there is a positive correlation between the numbers of argyrophile and argentaffin cells, but the percentage of argyrophile cells that are argentaffin also varies with cranio-caudal level. The pattern of this variation is not consistent in the various foetuses studied.     

The distribution of enterochromaffin cells in the human small intestine

Summary The distribution of argyrophile and argentaffin cells has been studied in the small intestine of five human adults. In proceeding cranio-caudally the characteristic feature of their distribution is the presence of eight to ten waves of rising and falling density. A progressive decrease in density of cells from duodenum to terminal ileum (described by previous workers) is not present.Re-examination of findings reported earlier in the small intestines of human foetuses shows that a predominant U shaped pattern of distribution is present in younger foetuses. This changes to the adult pattern by full term. The appearance of the adult pattern occurs earlier for argyrophile cells than for argentaffin cells.     

The depletion of the granules of argyrophile cells of dog duodenum by reserpine

Summary The influence of reserpine on the argentaffin and non-argentaffin argyrophile cells of the duodenum has been studied in 8 dogs. In two dogs that were given 5 mg/kg of reserpine there was a complete disappearance of both types of cells. In another dog given 5 mg/kg and in dogs given 2.5 mg/kg and 1 mg/kg reserpine, the number of argentaffin cells was reduced to almost nil while the number of non-argentaffin argyrophile cells showed a statistically significant reduction. The degree of depletion of non-argentaffin argyrophile cells was proportional to the dose of reserpine given. The findings show that the non-argentaffin argyrophile cells are closely related to 5-hydroxytryptamine and their distinction from argentaffin cells is only a quantitative one.     

Electron microscopic classification of amine-producing endocrine cells by selective staining of ultra-thin sections

Summary The argyrophil, argentaffin and chromaffin reactions were performed directly on ultra-thin sections for examination in the electron microscope. Glutaraldehyde fixation was appropriate for the argentaffin and chromaffin reactions; additional fixation with osmium tetroxide, however, caused impairment of these reactions. Fixation with formaldehyde, but not with glutaraldehyde, was adequate for the argyrophil reaction; post-fixation with osmium tetroxide did not affect this staining. At the light microscopic level the staining reactions were correlated with fluorescence histochemistry according to the method of Falck and Hillarp. The techniques described were used to study certain amine-producing endocrine cell systems: adrenal medullary cells and thyroid parafollicular cells of the mouse, gastric endocrine cells from the oxyntic gland area of the mouse, rat and rabbit. All these cells stained argyrophil. The adrenal medullary cells and one cell type in the oxyntic gland area of the rabbit were strongly argentaffin and chromaffin. The remainder of the cells were non-argentaffin and non-chromaffin but could be induced to give an argentaffin (and chromaffin) reaction after injection of the animals with l-3,4-dihydroxyphenylalanine or l-5-hydroxytryptophan, a treatment which is known to result in the accumulation of the highly reducing dopamine and 5-hydroxytryptamine, respectively, in these endocrine cells. Without exception the precipitates formed in all the staining reactions accumulated selectively over the secretory granules of the cells.The techniques described permit differential staining of consecutive ultra-thin sections for electron microscopic characterization of one and the same cell. They will provide information necessary for correlative studies of the stainable cells at the light and electron microscopic levels.     

A fluorescent histochemical method for the detection of tissue disulphide groups

Summary Bennett and Yphantis (1948) introduced into histochemistry the use of organic mercurials for the detection of sulphydryl groups. The new fluorescent method which is now reported is similarly based upon the reaction of mercaptans with an organic mercurial, fluorescein mercuric acetate (FMA). This method is sensitive; moreover it is not as elaborate as the dihydroxy-dinaphthyl-disulphide (DDD) technique of Barrnett and Seligman (1952).     

Peptide and amine producing endocrine-like cells in the chicken thymus

Summary The thymus of the chicken contains at least two types of endocrine-like cells predominating in the juxtacortical medulla. One type stores 5-hydroxytryptamine and is stained by the argentaffin, chromaffin and Schmorl methods. Treatment with reserpine markedly reduces its 5-hydroxytryptamine content. The other cell type is devoid of 5-hydroxytryptamine; if supplied withl-dopa it can produce and store dopamine. Both cell types stain with the argyrophil method of Grimelius and with HCl-basic dye methods believed to reflect the presence of peptides with masked carboxyl groups. In the electron microscope both cell types were found to contain numerous cytoplasmic 2000–3000 ? granules similar to those seen in polypeptide hormone-producing cells elsewhere. The cytoplasmic granules in one of the two endocrine-like cell types are argentaffin and chromaffin, indicating that they are the storage site for 5-hydroxytryptamine. It is suggested that the main secretory products of the two endocrine-like cell types are peptides, possibly regulating lymphatic tissue function.     

On the glycocalyx,the external coat of the plasma membrane,of some secretory cells

Summary The free surface of epithelial cells of secretory organs (human placenta, lactating mammary gland of the rat, choroid plexus of man and rat) and of the accessory organs of the genital tract of the male rat is characterized by a plasmalemmal differentiation named glycocalyx or surface mucous coat. This structure is built up by filamentous or globular substructures.Two main ultrastructural types of the glyeocalyx were observed: 1) The filamentous type such as in the rat epididymis, which resembles the cat intestinal glyeocalyx (Ito, 1965) and that one of human transitional epithelium (Monis and Zambrano, 1968), and 2) The globular type, as observed in the lumen of the lactating mammary gland of the rat.Sialic acid was demonstrated histochemically in the luminal glyeocalyx of all organs studied. In addition, the glyeocalyx of acinar cells of the lactating mammary gland contains sulfate and phosphate groups which were identified by histochemical technics, using enzymatic digestion procedures, suggesting the chemical heterogeneity of this glyeocalyx.Present investigations follow the working hypothesis that the complex carbohydrates of glycocalyces become part of the product of activity of secreting cells.We thank Mr. Luis Iwakawa, Miss Silvia Falcón, Miss Elsa M. Orgnero for technical help, Miss Graciela Aliaga for secretarial assistance. Photography by Mr. H. Magnani. Dr. Hugo F. Carrer cooperated in the initial stages of this investigation.The authors acknowledge the use of the electron microscope of the Department of Pathology, Córdoba University Medical School, for which they thank Prof. E. Mosquera and Dr. E. Hliba. Dr. Hliba photographed picture number 4.     

Endocrine cells in the large intestine epithelium in various vertebrate species

Amount of argyrophile and argentoffin endocrine cells per 1 mm2 of the large intestine mucous membrane has been counted in the turtle, pigeon, white rat and frog at various periods of their life (winter hibernation, awakening, active life). As the animal's organization becomes more complicated, the amount of the endocrine cells in the epithelium increases both in the initial and in the terminal parts of the large intestine, their amount in the terminal (rectum) part being less than in the initial one. The greatest amount of the endocrine cells is found in simmer frogs at the expense of increasing content of the elements that do not belong to the argentaffin group. The latter do not significantly change during various seasons.     

Endocrine cells of the human gastric mucosa

Summary By light and electron microscopy investigation of the human gastric mucosa five types of ultrastructurally different endocrine cells have been detected: 5-hydroxytryptamine storing enterochromaffin (EC) cells, gastrin storing G cells, and functionally undefined ECL, D and D₁ cells. By direct application of Masson's argentaffin reaction as well as of Sevier-Munger's and Grimelius' argyrophil method to electron microscopy specimens, selective deposition of silver grains upon the endocrine granules of such cells was obtained. In particular, only EC cell granules reacted to the argentaffin method, granules of both EC and ECL cells heavily reacted to Sevier-Munger's technique, granules of EC, ECL, G and D₁ cells reacted to Grimelius' technique, while D cell granules failed to react either to argentaffin or argyrophil methods. By the application of the same silver methods to paraffin sections as well as by other selective staining methods for endocrine granules (5-hydroxytryptamine techniques, lead-haematoxylin, HCl-basic dye method), at least four of the above cell types were also identified under light microscope. This opens the way for extensive studies of such cells in conventional histologie specimens.This investigation was supported in part by grant N.70.01022.04 from the Italian Consiglio Nazionale delle Ricerche.     

Ultrastructure and fluorescence histochemistry of endocrine (APUD-type) cells in tracheal mucosa of human and various animal species

This study describes distrinctive cells with ultrastructural and histochemical features of APUD-type endocrine cells within the tracheal epithelium of human fetuses, newborns and children as well as different animal species. These cells referred to as Kultschitzky cells (K cells) were found to be argyrophilic, but not argentaffin, and are considered analogous to the same type of cells in lung and gastro-intestinal tract. Fluorescence histochemistry demonstrated the presence of intracellular amine within tracheal K cells, but only after in-vitro or in-vivo administration of amine precursor (L-DOPA). Ultrastructurally, these cells are characterized by the presence of numerous cytoplasmic granules (dense core vesicles) which show species related morphologic variations. Two different types of K cells were found in trachea of lamb and armadillo, each type possessing morphologically different dense core vesicles. In human and rabbit tracheas, only one type of K cell was identified. K cells in the trachea are distributed as single cells between other epithelial cells; neuroepithelial bodies such as those found in bronchial mucosa were not identified. Well differentiated K cells were found in tracheas of early human fetuses and throughout gestation, infancy, and childhood. Preservation of K cells in human autopsy material and widespread occurence of these cells in various laboratory animals will permit further studies into the nature and function of tracheobronchial endocrine cells.     

Silver stains for identification of neuroendocrine cells. A study of the chemical background

Summary The chemical background of silver stains used for visualization and characterization of peripheral neuroendocrine cells in the gastrointestinal tract and pancreas, and of their corresponding tumours, was studied in tissue sections and by a dot-blot technique. Sequential staining of pancreatic islets with an immunohistochemical procedure and silver staining of the same tissue section revealed that chromogranin A immunostained cells also displayed an argyrophil reaction with the Grimelius method, but no argentaffin reaction with the Masson technique. Accordingly, purified chromogranin A (15 g or less) treated in formalin and applied to nitrocellulose did not show any argentaffin reaction but displayed a dose-related argyrophil reaction. Equal quantities of other polypeptide components did not give rise to any silver reaction. Further dot-blot studies showed that the tryptophan and tyrosine metabolites, dopamine, norepinephrine, 5-hydroxytryptamine and 5-hydroxindole caused strongly argentaffin and argyrophil reactions while epinephrine, 5-hydroxyindole-3-acetic acid and 5-hydroxytryptophan gave only the former reaction. Among other chemical components studied, only guanine displayed weak silver staining. The results indicate that the reaction products between aldehydes and the granular content of biogenic amines synthesized from tryptophan and tryosine display an argentaffin reaction and that the granular chromogranin A caused an argyrophil but no argentaffin reaction.     

Ultrastructure of transitional epithelium of man

Summary Blocks of human normal renal pelvis and ureter obtained at the time of surgery were fixed in glutaraldehyde and osmium with or without ruthenium red, for electron microscopic observations. The transitional epithelium is arranged in three cell layers: basal, intermediate and superficial. All epithelial cells show numerous microvilli and contain the characteristic vesicles of transitional epithelium, bundles of cytoplasmic filaments, microtubules and numerous free ribosomes. The epithelial extracellular compartment is notably large and appears as an intricate, tridimensional network of canaliculi and cisternae which are wider in the intermediate and superficial layers and in which microvilli and cytoplasmic folds of vicinal cells are often attached or interdigitated. At these sites there are desmosomes.The surface of all transitional epithelial cells is covered by a fibrillar mucous coat which is more developed at the plasmalemma of the free border of luminal cells in which microvilli are also seen. Ruthenium red stains selectively the plasmalemma and the mucous coat of the free surface of the epithelium, indicating the presence of an acid polysaccharide. With this technic (Luft, 1965), it is observed, radiating from the plasmalemma, branching filaments which measure 100 Å in diameter forming a zone of varying density which is about 400 m wide and which corresponds, at the light microscopic level, to the luminal border of the transitional epithelial cells in which a sialomucin has been identified. The slender filaments have a beaded appearance. At the free border, superficial cells are attached by functional complexes in which tight junctions seal the epithelial intercellular space, which is opened at the level of the basement membrane where only desmosomes are observed.The ultrastructure of human transitional epithelium of urinary tract resembles the duct cells of the salt gland of certain marine birds (Fawcett, 1962) and the amphibian epidermis (Farquhar and Palade, 1965) in which there are active processes of transport. The mucous surface coat, selectively stained by the ruthenium red, contains a sialomucin (Monis and Dorfman, 1965, 1967).The ultrastructure and histochemistry of the mucous fluffy coat of man transitional epithelium and the observations of Porter and Tamm (1955), on the ultrastructure of preparations of the Tamm and Horsfall mucoprotein (1952) are bases for suggesting that transitional epithelium of urinary tract of man is the site of biosynthesis of certain urinary mucoids. Present investigations are directed to obtain evidence to substantiate this hypothesis.General Abbreviations B basal cell - E exfoliating cell - I intermediate cell - L lumen - S superficial cell - SC surface coat - bm basement membrane - ci cell infolding - d desmosome (macula adhaerens) - f fibroblast - fi cytoplasmic filaments - is intercellular space - jc junctional complex - ly lysosome - lym lymphocyte - mt microtubules - m mitochondria - mv microvilli - n nucleus - r ribosomes - rv round vesicle - zo zonula occludens - za zonula adherens Dr. Monis wishes to thank Dr. E. De Robertis for the use of the electron microscope facilities of the Instituto de Anatomía General y Embriologia, Facultad de Medicina, Universidad de Buenos Aires. — Prof. E. Trabucco and Dr. R. J. Borzone (Cátedra de Clinica Genitourinaria de la Facultad de Medicina, Universidad de Buenos Aires) generously supplied the specimens which were the bases of this study. — Thanks are due to Mrs. A. M. Novara and Mrs. Defilippi-Novoa for efficient technical help and to Miss Rosa Gentile for secretarial assistance. Photomicrography by Mr. M. A. Saenz.Dr. Zambrano is investigator (CNICT).     

Tritiated thymidine autoradiographic study on the origin and renewal of argentaffin cells in the pyloric gland of hamsters

Summary The origin and renewal of the argentaffin cells in the pyloric glands of hamsters were studied by flash, cumulative and pulse labelling autoradiography with ³H-thymidine. The argentaffin cells were identified by the Diazo Method using Fast Red B Salt.By flash labelling autoradiography, it was shown that the argentaffin cells located from the middle to the lower level of the pyloric mucosa were not labelled with ³H-thymidine, indicating that this cell type has no proliferative activity. On the 10th and the 20th day of cumulative labelling, 31% and 63% of the argentaffin cells in the gland were found to be labelled, respectively. The labelled argentaffin cells were concentrated in the upper part of the gland (around the region of the isthmus), and no label was found over nuclei of the cells at the lowermost level of the gland. These labelled cells were shown to undergo a downward migration in the days following pulse labelling. They were replaced by unlabelled (and weakly or very weakly labelled) cells which arose at the region of the isthmus. The argentaffin cells in the pyloric gland are thought to arise from epithelial precursor cells at the region of the isthmus.The labelled argentaffin cells in the gland were found to decrease in number almost exponentially after pulse labelling. This indicates that the life span of argentaffin cells is not fixed, but their renewal conforms to the random loss system. The half time of turnover of this cell population was 15 days on average.Supported by a Grant-in-Aid for Cancer Research from the Ministry of Education, Science and Culture, Japan     

Immunohistochemical evidence for the presence, localization and partial coexistence of insulin, insulin-like growth factor I and relaxin in the protochordate Ciona intestinalis

The occurrence and coexistence of peptides of the insulin-like growth factor (IGF)/insulin superfamily were investigated in the ovary and gastro-intestinal tract of the protochordate Ciona intestinalis. Antisera specific for mammalian IGF-I, insulin and relaxin were used in a double-immunofluorescence method on paraffin sections and with an immunogold technique on consecutive semi-thin sections. IGF-I and relaxin immunoreactions but no insulin immunoreactions occurred in the ovary and were confined to medium-sized and mature follicle cells. Two subpopulations of reacting follicular cells were present: those containing only IGF-I immunoreactivity (5%) and those containing IGF-I and relaxin immunoreactivities (95%). In the gastro-intestinal tract, IGF-I and insulin immunoreactions coexisted, whereas no relaxin immunoreactions were obtained. Gel chromatography and radioimmunoassay in Ciona ovary revealed IGF-I immunoreactivity in two peaks with apparent molecular masses of approximately 16 kDa and 3 kDa. The present results indicate that (1) the same IGF-I-related peptide probably occurs in gastro-intestinal tract and ovary, (2) three different members of the insulin/IGF family of peptides are probably present in protochordates, (3) different types of coexistence of these peptides seem to exist in protochordates, i.e. an IGF-I-related peptide and an insulin-related peptide in the digestive tract and, as shown previously, in central nervous system, and the IGF-I-related peptide and relaxin in the ovary, (4) an IGF-I-related peptide and relaxin may be involved in oocyte maturation in the protochordate ovary.     

Distribution patterns of neurotensin-like immunoreactive cells in the gastro-intestinal tract of higher vertebrates

Summary The endocrine system of the gastro-intestinal tract of selected species representing the five higher vertebrate classes was investigated with reference to occurrence and distribution of neurotensin-like immunoreactive cells. Using antibodies against C-terminal and N-terminal fragments of neurotensin and against the C-terminal sequence of xenopsin it was demonstrated that the intestine of all species studied contains endocrine, neurotensin-like immunoreactive cells. However, large differences in localization and frequency of these neurotensin-like immunoreactive cells were found. Except for a teleostean fish, neurotensin-like immunoreactive cells in the gastro-intestinal tract were more frequent in non-mammalian vertebrates than in mammals. In contrast to mammals, where the highest density of neurotensin-like immunoreactive cells was present in the ileal mucosa, in the non-mammalian vertebrates studied the corresponding cells were most abundant in the pyloric-duodenal junction. The exact mapping of neurotensin-like immunoreactive cells is presented throughout the entire gastro-intestinal tract of six species (Rattus, Coturnix, Lacerta, Rana, Xenopus, Carassius) including a quantitative evaluation of sequential serial sections.     

Complete Suppression of the Gut Microbiome Prevents Acute Graft-Versus-Host Disease following Allogeneic Bone Marrow Transplantation

The hypothesis that elimination of facultative and strict anaerobic microorganisms from the gastro-intestinal tract by antimicrobial drugs in the period of time around allogeneic bone marrow transplantation (BMT) prevents acute graft-versus-host disease (GVHD), was examined in a cohort of 112 children grafted between 1989 and 2002 for hematological malignancies. All patients received T-cell replete marrow from human leukocyte antigens (HLA) matched sibling donors under identical transplantation conditions. To eliminate microorganisms from the gastro-intestinal tract, total gastro-intestinal decontamination (GID) was applied by high doses of non-absorbable antimicrobial drugs while the graft recipient was maintained in strict protective isolation. About half of the children (51%) proved to be successfully decontaminated, and about half (49%) unsuccessfully. One recipient got acute GVHD in the first group and 8 in the second group (p = 0.013). The degree of success of total GID was decisive for the occurrence of acute GVHD, irrespective of the presence of other risk factors such as higher age of recipient and/or donor, female donor for male recipient and carriership or reactivation of herpesviruses. Our results demonstrate that successful total GID of the graft recipient prevents moderate to severe acute GVHD. We suppose that substantial translocation of gastro-intestinal microorganisms or parts of these, functioning as microbial-associated molecular patterns (MAMP''s), triggering macrophages/dendritic cells via pattern recognizing receptors (PRR''s) is prohibited. As a consequence the initiation and progression of an inflammatory process leading to acute GVHD is inhibited.     

Occurrence of members of the insulin superfamily in central nervous system and digestive tract of protochordates

Antisera specific for mammalian insulin-like growth factor 1 (IGF-1) and mammalian insulin and the double immunofluorescence technique were used for this study. IGF-1-like-immunoreactivity was localized in entero-endocrine cells in the gastro-intestinal tract of the protochordates Ciona intestinalis and Branchiostoma lanceolatum. Some of the specimens also showed IGF-1-like-immunoreactive (-IR) perikarya and fibers in the central nervous system. Whilst in rat endocrine pancreas, IGF-1-IR and insulin-IR occurred in different cell populations, in Ciona and Branchiostoma the vast majority of entero-endocrine cells and central neurons were IGF-1-like-+insulin-IR. A minor portion exhibited IGF-1-like-IR alone. For further characterization of the IGF-1-like-IR material, in Ciona intestinalis, peptides related to IGF-1 were identified by radioimmunoassay and gel chromatography. In accordance with the immunohistochemical results, IGF-I-like-IR was detected both in cerebral ganglion and in gastro-intestinal tract. Using acid gel chromatography, in Ciona gastro-intestinal tract the IGF-1-like-IR was found to occur in two peaks, with apparent molecular weights of approximately 16 kDa and 3 kDa. Absorption studies with insulin- and IGF-related peptides, with crude extracts and the peak material obtained after gel chromatography, indicated that the IGF-1-like peptides in Ciona are different from mammalian insulin and IGF-1. The findings are in accordance with the presence of a common insulin/IGF precursor molecule in protochordates.