13 ASTROVIRUS ALTERS THE GUT MUCUS BARRIER AND REDUCES COLONIZATION TO ENTEROPATHOGENIC E. COLI

2020 ◽  
Vol 26 (Supplement_1) ◽  
pp. S28-S28
Author(s):  
Valerie Cortez ◽  
Bridgett Sharp ◽  
Brandi Livingston ◽  
Hannah Rowe ◽  
Ramzi Alsallaq ◽  
...  
Keyword(s):  
Periodic Acid ◽  
Enteric Virus ◽  
Goblet Cells ◽  
Gut Health ◽  
Cell Secretion ◽  
Periodic Acid Schiff ◽  
E Coli ◽  
Degrading Bacteria ◽  
Human Astroviruses ◽  
Mucus Barrier

Abstract Goblet cells, specialized epithelial cells that produce mucus, are essential for gut health. Dysfunctional goblet cell secretion can weaken the mucus barrier, bringing commensal microbes and other lumenal contents in contact with the epithelial barrier, triggering inflammation. We recently discovered that murine astroviruses infect goblet cells in the small intestine, making it the first enteric virus in which this tropism has been recognized in vivo. To first determine whether astrovirus infection alters mucus production in goblet cells, we used periodic acid-Schiff staining of small intestinal tissues and found a thicker mucus layer in infected compared to mock-infected animals (1.85 to 2.51-fold, p<0.001). Because changes to the mucus barrier can alter the microbial environment in the gut, we used 16S metagenomic to characterize the microbiome after infection. We found that the relative frequency of well-characterized mucus-degrading bacteria significantly increased after infection (p<0.001). Using phylogenetic edge principal components analysis, we also identified distinct microbiome shifts between 0 and 14 days post-infection, corresponding to peak infection. Finally, to determine the extent to which these changes in the mucus barrier could have a functional consequence to host disease susceptibility, we developed a neonatal model for astrovirus and tested whether animals inoculated with enteropathogenic E. coli (EPEC), an adherent bacterial pathogen, would be protected from colonization. In comparison to animals inoculated with EPEC alone, co-infected animals had significantly reduced EPEC colonization (p<0.01). Together, these studies reveal a new avenue of enteric virus regulation of gut homeostasis and immunity via goblet cells and highlight astroviruses as a novel model to study the mucus barrier. Future studies are needed to determine whether astrovirus-induced increases in the mucus barrier protect from other gastrointestinal diseases and how these data translate to human astroviruses, which predominately infect children <1-year-old and coincides with the development of gut immunity.

Epithelial response to intestinal anaphylaxis in rats: goblet cell secretion and enterocyte damage

1984 ◽  
Vol 247 (6) ◽  
pp. G632-G637 ◽  
Author(s):  
M. H. Perdue ◽  
J. F. Forstner ◽  
N. W. Roomi ◽  
D. G. Gall
Keyword(s):  
Goblet Cell ◽  
Immunoglobulin E ◽  
Periodic Acid ◽  
Goblet Cells ◽  
High Serum ◽  
Cell Secretion ◽  
Periodic Acid Schiff ◽  
Cellular Debris ◽  
Ige Mediated

The effects of immunoglobulin E (IgE)-mediated reactions on the intestinal epithelium were examined during intestinal anaphylaxis in the rat. Rats sensitized by intraperitoneal injection of egg albumin (EA) plus alum developed high serum titers of IgE anti-EA antibodies after 14 days; sham-treated littermate controls had no anti-EA antibodies. Two isolated loops of jejunum were prepared in vivo in anesthetized rats. The loops were injected with EA in saline or saline alone, and intraluminal contents of each loop were examined after 4 h. Mucosal histamine decreased in sensitized rat intestine exposed to EA. Luminal mucin, measured by radioimmunoassay, was not increased by antigen challenge. In contrast, DNA, protein, and sucrase activities were elevated in contents from the isolated segments exposed to EA in sensitized rats. Histology revealed that periodic acid-Schiff-stained material was contained in goblet cells in sections prepared from these segments after antigen exposure. Cellular debris was present over the tips of the villi. These findings suggest that IgE-mediated reactions in the intestine cause epithelial damage and loss of material from cells other than goblet cells. The results indicate that release of goblet cell mucus is not a feature of intestinal anaphylaxis.

The expulsion of Echinostoma trivolvis from C3H Mice: differences in glycoconjugates in mouse versus hamster small intestinal mucosa during infection

1996 ◽  
Vol 70 (2) ◽  
pp. 115-121 ◽  
Author(s):  
T. Fujino ◽  
B. Fried
Keyword(s):  
Lectin Binding ◽  
Periodic Acid ◽  
Helix Pomatia ◽  
Goblet Cells ◽  
Small Intestinal Mucosa ◽  
Periodic Acid Schiff ◽  
C3h Mice ◽  
Small Intestines ◽  
Echinostoma Trivolvis ◽  
Lectin Binding Sites

AbstractMucosal glycoconjugates were examined in C3H mice and in hamster small intestines infected with Echinostoma trivolvis and in uninfected rodents, using periodic-acid Schiff (PAS) and high-iron diamine-alcian blue (HID-AB) staining and three different fluorescein-conjugated lectins: Triticum vulgaris agglutinin (WGA), Helix pomatia agglutinin (HPA) and Griffonia simplicifolia agglutinin (GSA-II). Lectin-labelling by electron microscopy was also undertaken with WGA and HPA lectin-gold probes. HID-AB stain demonstrated that the most mature goblet cells of the mouse villi contain sulfomucins, whereas those of hamsters contain sialomucins. The expression of lectin-binding sites and the intensity of the lectin binding in the small intestines were changed by echinostome infection. Specific differences in the reaction to mucin glycoproteins were clearly observed between the mouse and hamster intestines infected with E. trivolvis; lectin-binding to hyperplastic goblet cells and crypts in the infected mice increased, while no marked increase in the number of goblet cells and reaction to the glycoconjugates were observed in the infected hamsters. These findings indicate that the expression of terminal N-acetyl-D-galactosamine, sialic acid and N-acetyl-D-glucosamine increased in mucins secreted from hyperplastic goblet cells associated with E. trivolvis infection in mice. No marked increase in these glycoconjugates occurred in hamster infections. These findings reflect clear differences in infectivity of E. trivolvis in C3H mice versus hamsters.

A physiological, biochemical and histological study of goose tracheal mucin and its secretion

1977 ◽  
Vol 279 (969) ◽  
pp. 513-540 ◽  
Keyword(s):  
Sialic Acid ◽  
Nerve Stimulation ◽  
Histological Study ◽  
Periodic Acid ◽  
Gel Filtration ◽  
Goblet Cells ◽  
Mucin Secretion ◽  
Electron Microscopic ◽  
Periodic Acid Schiff ◽  
Tracheal Mucus

Tracheal mucin secretion has been measured from a segment of trachea, isolated in situ , in anaesthetized geese by a method that involves radioactive labelling of tracheal mucus glycoproteins (Gallagher et al. 1975). Goose tracheal mucus comes entirely from goblet cells, since the goose trachea does not contain submucosal mucous or serous glands, and this method has been used to investigate the nervous and pharmacological control of the mucin secretion from these epithelial goblet cells. The mucins secreted have been collected, fractionated, and chemically analysed. Intracellular mucin has been examined histochemically, and the results of electron microscopic observations of epithelial cells and nerves are presented. Acetylcholine increased tracheal mucin secretion, and this effect was completely blocked by atropine. Neither α- nor β-stimulant sympathomimetic amines affected tracheal mucin secretion. Stimulation of the peripheral cut ends of the descending oesophageal nerves increased tracheal mucin secretion and the majority of this response, approximately three-quarters, appeared to be cholinergic since this proportion was blocked by atropine. The mediator for the atropine-resistant part of the response is not known, but it appears not to be a β-adrenoreceptor stimulant since the response to nerve stimulation was unaffected by propranolol given at 34 μm intrasegmentally. Other possibilities are discussed. Atropine itself decreased the resting level of tracheal mucin secretion. The local anaesthetic, lignocaine, increased tracheal mucin secretion, while at the same time blocking the responses to acetylcholine and descending oesophageal nerve stimulation. The implications of this are discussed. The electrophoretic, gel filtration and ion-exchange properties of goose tracheal mucins showed that they represented high molecular mass, negatively charged glycoproteins which could be labelled biosynthetically with [ 35 S]sulphate, [ 3 H]- and [ 14 C]glucose. These mucins could be stained with Alcian blue or periodic acid Schiff reagent. The carbohydrate composition was unusual for an epithelial glycoprotein in that fucose was absent and mannose was present in small quantities. The monosaccharides present in larger quantity were galactose, N -acetylglucosamine, N -acetylgalactosamine and sialic acid. Histochemical analysis of tissue sections of gosling tracheas demonstrated that nearly all of the glycoprotein in epithelial goblet cells contained both sialic acid and sulphate residues. Sialated mucin was present also, but to a lesser extent, and many cells contained a mixture of sialated and sulphated mucins. The adult goose trachea had a high proportion of sialated glycoprotein. Electron microscopy showed a range of epithelial cell types and intra-epithelial nerves also. Many of the nerves had neurosecretory vesicles suggestive of motor function and some were near to goblet cells.

scholarly journals Histochemical reactivity of peanut lectin-horseradish peroxidase conjugate.

1980 ◽  
Vol 28 (9) ◽  
pp. 979-990 ◽  
Author(s):  
P J Stoward ◽  
S S Spicer ◽  
R L Miller
Keyword(s):  
Sialic Acid ◽  
Horseradish Peroxidase ◽  
Periodic Acid ◽  
Apical Cell ◽  
Goblet Cells ◽  
Surface Epithelium ◽  
Renal Tubules ◽  
Peanut Lectin ◽  
Periodic Acid Schiff ◽  
Terminal Galactose

A peanut lectin-horseradish peroxidase (PL-HRP) conjugate has been applied to histochemical staining of paraffin sections of various mouse organs. The PL-HRP conjugate has selectively reacted with secretory bodies, the Golgi zone, and the apical cell surface in various cell types. Some positive sites, including lingual and tracheal serous glands, Brunner's glands, and the brush border of the proximal straight nephron, contained periodic acid-Schiff (PAS)-positive glycoconjugate with no affinity for basic reagents. The stored secretion in these sites was interpreted as containing neutral glycoprotein with terminal galactose residues which could, in part at least, account for the PAS reactivity. Duodenal goblet cells, which exhibited basophilia attributable to sulfate esters, also bound PL-HRP. As the binding was affected by prior sialidase digestion, the secretory glycoprotein in the duodenal goblet cells was judged to contain oligosaccharides with sulfate esters and terminal galactose uncapped by sialic acid. All sites known from their basophilia to form sialomucin failed to stain with the PL-HRP conjugate, but consistently gained reactivity following sialidase digestion and were inferred, therefore, to possess glycoproteins with oligosaccharide side chains containing subterminal galactose and terminal sialic acid. Lingual mucous glands, known to secrete a mucosubstance with basophilic properties indicative of the presence of sulfate esters but not sialic acid, stained with PL-HRP only after sialidase digestion and, accordingly, were reinterpreted as containing both sulfate esters and terminal galactose-sialic acid dimers. Staining of gastric surface epithelium demonstrated a srongly PAS-reactive neutral glycoprotein, and that of goblet cells in the cecum disclosed PAS-positive sulfated glycoprotein. The latter two sites lacked PL-HRP affinity without or with prior sialidase treatment and apparently possessed neither terminal galactose residues nor galactose-sialic acid dimers. PL-HRP affinity was observed exclusively in the Golgi cisternae of some epithelial cells, thus indicating that galactose occurs transiently as a terminal residue in this site. A few histologic sites, such as pancreatic and gastric zymogen cells and renal tubules, were devoid of both PAS reactivity and basophilia indicative of the presence of complex carbohydrate but stained strongly with the PL-HRP conjugate by means which are not understood. Galactose in the PL-HRP solution blocked or reversed the PL-HRP binding in most of the structures with an affinity for the conjugate, supporting the conclusions that the reagent is specific for galactosyl residues.

scholarly journals Loss of NHE8 expression impairs intestinal mucosal integrity

2015 ◽  
Vol 309 (11) ◽  
pp. G855-G864 ◽  
Author(s):  
Aiping Wang ◽  
Jing Li ◽  
Yang Zhao ◽  
Malin E. V. Johansson ◽  
Hua Xu ◽  
...  
Keyword(s):  
Periodic Acid ◽  
Paneth Cell ◽  
Distal Colon ◽  
Goblet Cells ◽  
Paneth Cells ◽  
Wild Type ◽  
Periodic Acid Schiff ◽  
Mucin Production ◽  
Mucosal Integrity ◽  
Mucin 2

The newest member of the Na+/H+ exchanger (NHE) family, NHE8, is abundantly expressed at the apical membrane of the intestinal epithelia. We previously reported that mucin 2 expression was significantly decreased in the colon in NHE8−/− mice, suggesting that NHE8 is involved in intestinal mucosal protection. In this study, we further evaluated the role of NHE8 in intestinal epithelial protection after dextran sodium sulfate (DSS) challenge. Compared with wild-type mice, NHE8−/− mice have increased bacterial adhesion and inflammation, especially in the distal colon. NHE8−/− mice are also susceptible to DSS treatment. Real-time PCR detected a remarkable increase in the expression of IL-1β, IL-6, TNF-α, and IL-4 in DSS-treated NHE8−/− mice compared with DSS-treated wild-type littermates. Immunohistochemistry showed a disorganized epithelial layer in the colon of NHE8−/− mice. Periodic acid-Schiff staining showed a reduction in the number of mature goblet cells and the area of the goblet cell theca in NHE8−/− mice. Phyloxine/tartrazine staining revealed a decrease in functional Paneth cell population in the NHE8−/− small intestinal crypt. The expression of enteric defensins was also decreased in NHE8−/− mice. The reduced mucin production in goblet cells and antimicrobial peptides production in Paneth cells lead to disruption of the intestinal mucosa protection. Therefore, NHE8 may be involved in the establishment of intestinal mucosal integrity by regulating the functions of goblet and Paneth cells.

scholarly journals Identification of Brachyspira spp. in the cecum of broiler chickens using histology and in situ diagnostic assays

2021 ◽  
Vol 42 (5) ◽  
pp. 2813-2824
Author(s):  
Monica Regina de Matos ◽  
◽  
Aline Patrícia Grzegozevski ◽  
Alessandra da Cruz ◽  
Arthur Colombari Cheng ◽  
...  
Keyword(s):  
Broiler Chickens ◽  
Periodic Acid ◽  
Goblet Cells ◽  
Ffpe Tissue ◽  
Economic Losses ◽  
Periodic Acid Schiff ◽  
Tissue Samples ◽  
Rabbit Polyclonal Antibody ◽  
Formalin Fixed Paraffin Embedded

The genus Brachyspira corresponds to the group of bacteria formerly classified into the genus Serpulina and includes several commensal and pathogenic intestinal spirochetes that affect pigs, poultry, and other animal species, including humans. In birds, some pathogenic species of this genus causes a condition known as avian intestinal spirochetosis, which remains underdiagnosed, thereby causing serious economic losses. Brachyspira is a fastidious organism that necessitates the employment of fast and efficient identification techniques. The aim of this study was to identify Brachyspira spp. using histology, immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) in formalin-fixed paraffin embedded (FFPE) tissue samples from the cecum of commercial poultry. Samples were collected from 129 birds aged between 35 and 45 days from commercial broiler farms. For evaluation, routine histology processing (H&E) and the histochemical technique, periodic acid–Schiff (PAS) were done. Additionally, FFPE tissue samples were evaluated for FISH and IHC. The histological lesions were analyzed and graded after H&E staining, and the goblet cells were counted and compared using PAS staining with the positive and negative samples obtained through FISH and IHC. For FISH, probes labeled with Brachyspira spp., B. pilosicoli, B. hyodysenteriae, and B. intermedia were used, whereas rabbit polyclonal antibody specific for Brachyspira spp. was used for IHC. Of 129 samples, 82 were positive with IHC and 86 were positive with FISH. The samples positive for the genus Brachyspira in the FISH technique were tested for B. pilosicoli, B. hyodysenteriae, and B. intermedia in which 56 were positive for B. pilosicoli, 75 for B. hyodysenteriae and 80 for B. intermedia. There was an increase in goblet cells in the samples positive for FISH and IHC. The techniques used were effective and gave corresponding results, thus serving as a fast and efficient tool for diagnosis.

scholarly journals Mucosubstances in the porcine gastrointestinal tract: Fixation, staining and quantification

2019 ◽  
Vol 63 (2) ◽  
Author(s):  
Juliane Rieger ◽  
Barbara Drewes ◽  
Hana Hünigen ◽  
Johanna Plendl
Keyword(s):  
Tissue Section ◽  
Periodic Acid ◽  
Goblet Cells ◽  
Staining Procedure ◽  
Periodic Acid Schiff ◽  
Tissue Samples ◽  
Feeding Experiments ◽  
Neutral Mucin ◽  
Electron Microscopical ◽  
Triple Staining

Mucins are of great interest in intestinal research and histochemical methods are often employed to identify them. Since it is in the nature of mucins that they are “hard to hold onto” once they come into contact with water, a frequently used medium in histochemistry, there are a number of challenges that may decrease diagnostic accuracy. As the outcome of methods published for microscopic detection of mucosubstances proved to be unsatisfactory in our hands, the aim was the establishment of a reliable and reproducible protocol. Tissue samples were available from pig feeding experiments. In the present study, we focus on a fixation / staining procedure without making comparisons between differently fed pigs. Several fixation and staining procedures were evaluated for their use in semiautomatic quantification and quality assessment of different mucus fractions simultaneous on one tissue section. Cryostat sectioning, subsequent fixation steps with heat, ethanol and modified Bouin’s solution, followed by triple staining with high iron diamine, alcian blue and periodic acid-Schiff turned out to be the best method to identify sulfomucin, sialomucin and neutral mucin simultaneous on one tissue section. This methodology resulted in very good morphology of goblet cells with intact mucin containing vesicles within the cells, which was comparable to ultrastructural electron microscopical observations. Semiautomatic quantification of different mucins was possible. In conclusion, reliable mucus quantification and assessment of mucus quality requires strictly tested procedures. According to our experience, the most important aim after cryosectioning is fast fixation of the mucosubstances, which requires a combination of different fixation steps.

The Cytochemistry of the Amoebocytes and Intestinal Epithelium of Venus Mercenaria (Lamellibranchiata), with Remarks on a Pigment Resembling Ceroid

1955 ◽  
Vol s3-96 (33) ◽  
pp. 57-71
Author(s):  
SUMNER I. ZACKS
Keyword(s):  
Intestinal Epithelium ◽  
Periodic Acid ◽  
Goblet Cells ◽  
Neutral Red ◽  
Serum Cholinesterase ◽  
Periodic Acid Schiff ◽  
Intestinal Mucus ◽  
Specific Granules ◽  
Sulphydryl Groups ◽  
Metachromatic Granules

The properties of the amoebocytes and intestinal epithelium of Venus mercenaria were studied by a variety of cytochemical procedures designed to demonstrate proteins, enzymes, carbohydrates, and lipids. The cytoplasm of the amoebocytes contains specific granules which are constantly present and which are interpreted as being atypical mitochondria. Identification of their mitochondrial nature rests on their staining with Janus green B, their positive reaction for phospholipid by Baker's test, and the presence of dehydrogenase activity. Unlike typical mitochondria, the specific granules are eosinophil. Protein-bound carbonyl groups and disulphide and sulphydryl groups are present in both the specific granules and the cytoplasm. The sulphydryl groups may in part be associated with the presence of dehydrogenase, lipase, and serum cholinesterase. Amoebocytes also ontain glycogen and a material that is resistant to diastase and positive to the periodic acid / Schiff test; this material may be a neutral polysaccharide, unsaturated lipid, or mucoprotein. Cytoplasmic structures which are inconstantly present in amoebocytes include sudanophil droplets, neutral red vacuoles, metachromatic granules, and granules of an excretory pigment resembling ceroid. The sudanophil droplets may be stored neutral fat or lipid associated with the Golgi apparatus. The neutral red vacuoles are not preformed inclusions, but form as the dye accumulates within the cells. Metachromatic granules, which are confined solely to the intestinal amoebocytes, consist of phagocytosed intestinal mucus liberated from goblet cells. The histochemical reactions of the columnar intestinal epithelium suggest that these cells may be active in the digestion and absorption of nutrients, since eosinophil granules, lipid droplets, alkaline phosphatase, lipase, and serum cholinesterase are present in them. Masses of a ceroid-like excretory pigment and goblet cells containing mucus are present between the columnar intestinal epithelial cells. The pigment contains phospholipid and apparently arises as an oxidized end-product of lipid metabolism.

scholarly journals Granulomatous colitis: more than a canine disease? A case of Escherichia coli-associated granulomatous colitis in an adult cat

2017 ◽  
Vol 3 (2) ◽  
pp. 205511691773116
Author(s):  
Rodolfo Oliveira Leal ◽  
Kenny Simpson ◽  
Mélanie Fine ◽  
Jean-Charles Husson ◽  
Juan Hernandez
Keyword(s):  
Escherichia Coli ◽  
Periodic Acid ◽  
Diagnostic Testing ◽  
In Situ Hybridisation ◽  
Clinical Signs ◽  
Fluorescence In Situ Hybridisation ◽  
Granulomatous Colitis ◽  
Periodic Acid Schiff ◽  
E Coli ◽  
Adult Cat

Case summary This report describes a 4-year-old cat with chronic intermittent haematochezia and faecal incontinence of 7 months’ duration. Investigation revealed severe colonic multifocal mucosal ulcerations and infiltration of the mucosal lamina propria by large numbers of periodic acid–Schiff-positive macrophages. Fluorescence in situ hybridisation analysis of colonic biopsies revealed multifocal clusters of intracellular Escherichia coli. Treatment with fluoroquinolones for 6 weeks led to a complete resolution of clinical signs. Relevance and novel information The findings reveal that mucosally invasive E coli can also be associated with granulomatous colitis in cats and indicate the need for diagnostic testing of mucosal samples for E coli and other infectious agents.

Malakoplakia of the Bladder in a 4-Month-Old Puppy

2019 ◽  
Vol 55 (5) ◽  
pp. 261-265
Author(s):  
Caroline Benzimra ◽  
Chloé Job ◽  
Quentin Pascal ◽  
Stéphane Bureau ◽  
Anaïs Combes ◽  
...  
Keyword(s):  
Histological Examination ◽  
Periodic Acid ◽  
Granulomatous Inflammation ◽  
Abdominal Ultrasound ◽  
Wall Thickening ◽  
Periodic Acid Schiff ◽  
E Coli ◽  
Mass Lesions ◽  
Lost To Follow Up

ABSTRACT A 4 mo old female Staffordshire bull terrier puppy was presented with chronic Escherichia coli cystitis. Ultrasound and cystoscopic examination revealed innumerable, intraluminal, finger-like proliferations arising from the dorsal urinary bladder (UB) wall. Histological examination of mucosal biopsies obtained by cystoscopy was suggestive of granulomatous cystitis. The proliferative lesions were removed surgically and submitted for histological examination. The UB submucosa was heavily infiltrated by macrophages with periodic acid-Schiff–positive cytoplasm exhibiting rare Michaelis-Gutmann bodies, leading to the diagnosis of malakoplakia. The puppy was prescribed with sulfamethoxazole-trimethoprim. The urinary signs disappeared despite the persistent UB wall thickening revealed by abdominal ultrasound. Urine culture performed during the ninth week of treatment showed a persistent infection by E coli resistant to sulfamethoxazole-trimethoprim. The dog was switched to doxycycline but was then lost to follow-up. Malakoplakia is a chronic granulomatous inflammation well documented in humans. Its pathophysiology is not fully understood, but bacterial infection, immunodepression, and a defective lysosomal function may lead to the intracytoplasmic accumulation of partially degraded bacteria that can subsequently mineralize to form the Michaelis-Gutmann bodies. Malakoplakia should be suspected when UB mass lesions are identified in a young dog with bacterial cystitis.

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