Antibodies in the intestinal secretions of rats. Primary and secondary responses to polymeric flagellin.

The antibody responses in serum and secretions obtained from the mucosal surfaces of the small intestine of rats immunized by a parenteral and intestinal route have been compared. Though no significant differences in the mean serum titres were found, the responses of animals immunized via the latter route to large doses of antigen were far less uniform. Apart from the first few days of the primary response, antibody activity was found in three major immunoglobulin classes (IgG2, IgA and IgM), irrespective of the route of immunization. Significant antibody activity appeared in the intestinal surface secretions only after two injections of antigen. In rats immunized parenterally the activity was found only in the IgG2 component. Whilst activity was found in both IgG2 and IgA fractions of the secretions obtained from intestinally immunized rats, it was predominantly of the IgG2 class. The possible significance of this observation is discussed.     

The x-ray microanalysis of the mucosa of the rat small intestine

A rat small intestine mucosa is shown to accumulate significant amount of potassium and chloride. There was found a correlation between the content of these chemical elements and glycoprotein compartmentalization in goblet cell secret, brush border of enterocytes and a mucus layer. In this connection a role of mucus glycoproteins in membrane digestion is discussed. For preparation of samples the cryotechniques of electron microscopy are used.     

Purification and characterization of goblet-cell mucin of high Mr from the small intestine of sheep.

Crude soluble mucus from sheep small intestine was freed of nearly all the nucleic acid contaminants by precipitation with protamine sulphate and treatment with nucleases. After removal of non-covalently bound proteins by equilibrium density-gradient centrifugation in CsCl, a high-Mr glycoprotein was isolated by repeated h.p.l.c. from the partially purified mucin. The high degree of purity of the high-Mr mucin was borne out by (a) the observation of a single boundary on analytical ultracentrifugation in the presence of 5M-guanidinium chloride and (b) the observation of apparent monodispersity on sedimentation-equilibrium analysis. The Mr of the highly purified mucin, determined by sedimentation equilibrium, was 5.0 (+/- 0.1) X 10(6) and was concentration-independent. Finally, only goblet cells and the mucus blanket lining the intestinal epithelial cells were immunofluorescent when guinea-pig anti-(highly purified mucin) serum was used in an indirect immunofluorescence assay. The above antiserum reacted with apparently equal strength with goblet cells and with free mucin in abomasum, caecum and colon. The chemical composition of the glycoprotein was 66% carbohydrate and 34% protein, 45% of the latter being composed of valine and threonine. The glycoprotein migrated anodally on immunoelectrophoresis and contained 7.1% (w/w) sulphate. Neutral hexoses accounted for nearly half of the total carbohydrate content, followed by galactosamine and glucosamine. Whereas fucose and sialic acid were present in only small amounts, uronic acid was not detectable in the highly purified mucus glycoprotein.     

SYNTHESIS OF THE CARBOHYDRATE OF MUCUS IN THE GOLGI COMPLEX AS SHOWN BY ELECTRON MICROSCOPE RADIOAUTOGRAPHY OF GOBLET CELLS FROM RATS INJECTED WITH GLUCOSE-H3

It is known that colonic goblet cells utilize glucose to synthesize the carbohydrate portion of mucus glycoprotein. To determine the intracellular site of this synthesis, glucose-H³ was injected into 10-g rats. At 5, 20, 40 min, 1, 1½, and 4 hr after injection, segments of colon were fixed and prepared for electron microscope radioautography. By 5 min after injection, label had been incorporated into substances present in the flattened saccules of the Golgi complex. At 20 min, both Golgi saccules and nearby mucigen granules were labeled. By 40 min, mucigen granules carried almost all detectable radioactivity. Between 1 and 4 hr, these labeled granules migrated from the supranuclear region to the apical membrane; here, they were extruded singly, retaining their limiting membrane. The evidence indicates that the Golgi saccule is the site where complex carbohydrate is synthesized and is added to immigrant protein to form the complete glycoprotein of mucus. The Golgi saccule, distended by this material, becomes mucigen granules. It is roughly estimated that one saccule is released by each Golgi stack every 2 to 4 min: a conclusion implying continuous renewal of Golgi stacks. It appears that the Golgi synthesis, intracellular migration, and release of mucus glycoprotein occur continually throughout the life of the goblet cell.     

Isolation and partial characterization of rat duodenal-gland (Brunner''s-gland) mucus glycoprotein.

A mucus glycoprotein was isolated from the duodenal glands of the rat and purified by repeated density-gradient centrifugation. The characterized glycoprotein is unique to the mucous cells of the duodenal glands and is not present in parts of the small intestine devoid of these glands. The chemical composition of the purified glycoprotein is characteristic for glycoproteins of the mucin-type. Its protein content is relatively high and amount to 35% by weight. No neuraminic acid and little sulphate (2%) is present. Evidence is presented that the native glycoprotein is built up from subunits held together via disulphide bridges in a non-glycosylated region of the protein core.     

Dietary supplementation with ovine serum immunoglobulin is associated with an increased gut luminal mucin concentration in the growing rat

The mucus layer covering the gut epithelium is pivotal to host defence and is affected by various dietary components. Part of the reported beneficial effect of dietary immunoglobulins (Igs) on gut health may be due to effects on the gut mucus layer. The aim was to determine whether orally administered ovine serum Ig influence goblet cell count, mucin gene expression and digesta mucin protein content in the gut of the growing rat. Fourteen Sprague-Dawley male growing rats were used in a 21-day study and were fed either a casein-based control diet (CON; no Ig) or a similar diet but containing freeze-dried ovine Ig (FDOI). Daily food intake and growth rate were not affected by the dietary treatments. When compared to the rats consuming CON diet, those consuming the FDOI diet had significantly (P < 0.05) more intact and cavitated goblet cells in the intestinal villi. A similar result was found for crypt goblet cells in the small intestine and colon. Ileal Muc2, Muc3, Muc4 and stomach Muc5Ac mRNA expressions for the FDOI animals were higher (P < 0.05) compared to the the CON animals. Mucin protein content was higher (P < 0.05) in the stomach, ileum and colonic digesta of rats fed the FDOI diet. In conclusion, orally administered FDOI influenced gut mucins in the growing rat as evidenced by increased mucin gene expression and digesta mucin protein concentrations as well as an increased goblet cell count.     

Age-related changes in chemical composition and physical properties of mucus glycoproteins from rat small intestine.

Mucus glycoproteins from newborn and adult rat small intestine were radiolabelled in vivo with Na2 35SO4 and isolated from mucosal homogenates by using Sepharose 4B column chromatography followed by CsCl-density-gradient centrifugation. Non-covalently bound proteins, lipids and nucleic acids were not detected in the purified glycoproteins. Amino acid, carbohydrate and sulphate compositions were similar to chemical compositions reported for other intestinal mucus glycoproteins, as were sedimentation properties. There were, however, important differences in the chemical and physical characteristics of the mucus glycoproteins from newborn and adult animals. The buoyant density in CsCl was higher for the glycoproteins from newborn rats (1.55 g/ml versus 1.47 g/ml). On sodium dodecyl sulphate/polyacrylamide/agarose-gel electrophoresis, the glycoprotein from newborn rats had a greater mobility than the adult-rat sample. Although both preparations had similar general amino acid compositions, variations were observed for individual amino acids. The total protein content was greater in the glycoprotein from newborn animals (27%, w/w, versus 18%, w/w). The molar ratio of carbohydrate to protein was less in the newborn, primarily owing to a decreased fucose and N-acetylgalactosamine content. Comparison of the molar ratio of fucose and sialic acid to galactose for both glycoproteins demonstrated a reciprocal relationship similar to that described by Dische [(1963) Ann. N.Y. Acad. Sci. 106, 259-270]. The sulphate content was greater in the glycoprotein from newborn rats (5.5%, w/w, versus 0.9%, w/w). Both had similar sedimentation coefficients in a dissociative solvent. These results suggest an age-related difference in the types of mucus glycoproteins synthesized by small intestine.     

Mucus Properties and Goblet Cell Quantification in Mouse,Rat and Human Ileal Peyer's Patches

Peyer''s patches (PPs) are collections of lymphoid follicles in the small intestine, responsible for scanning the intestinal content for foreign antigens such as soluble molecules, particulate matter as well as intact bacteria and viruses. The immune cells of the patch are separated from the intestinal lumen by a single layer of epithelial cells, the follicle-associated epithelium (FAE). This epithelium covers the dome of the follicle and contains enterocyte-like cells and M cells, which are particularly specialized in taking up antigens from the gut. However, the presence and number of goblet cells as well as the presence of mucus on top of the FAE is controversial. When mouse ileal PPs were mounted in a horizontal Ussing-type chamber, we could observe a continuous mucus layer at mounting and new, easily removable mucus was released from the villi on the patch upon stimulation. Confocal imaging using fluorescent beads revealed a penetrable mucus layer covering the domes. Furthermore, immunostaining of FAE from mice, rats and humans with a specific antibody against the main component of intestinal mucus, the MUC2 mucin, clearly identify mucin-containing goblet cells. Transmission electron micrographs further support the identification of mucus releasing goblet cells on the domes of PPs in these species.     

Relationships between cell-mediated immunity and the IgE antibody response: I. Lymphotoxin production to DNP-Ascaris conjugates

The purpose of this paper is to present evidence for the suitability of a lymphotoxin (LT) assay as an in vitro correlate of cell-mediated immunity (CMI) to a hapten-carrier conjugate known to stimulate a high IgE antibody response. This would be used in a study of the factors influencing the relationship(s) between CMI and IgE antibody responses to the same antigen. Antigen-induced LT was assayed on actinomycin-inhibited, chromium-51-labeled L929 fibroblasts, using supernatants obtained from spleen and lymph node cells of animals immunized with 2,4-dinitrophenyl-Ascaris conjugates. Although LT was present at all times tested, beginning at Day 10, its concentration varied with time after immunization, adjuvant used, and cell type assayed. The induction of LT was T-cell dependent and conjugate specific. LT was produced by nonadherent cells. Adherent cells from immunized animals produced L-cell cytotoxin in the absence of antigen stimulation when tested before Day 10.     

Partial characterization of the gastrointestinal weight changes produced in the female rat by 16,16-dimethylprostaglandin E2

Oral and subcutaneous administration of 16,16-dimethylprostaglandin E2 (16,16-dimethyl PGE2) resulted in an increase in the dry weight of the stomach and small intestine of the female rat. This weight response was rapid, controlled rather than continuously progressing, dose dependent and reversible. The dry weight of the colon also increased but this was not studied in detail. Two-day treatment with 16,16-dimethyl PGE2 caused an increase in the incorporation of 3H-thymidine into the duodenum, jejunum and colon suggesting an increase in cell number. Incorporation into the stomach and ileum was not changed. The number of goblet cells per crypt was increased by prostaglandin treatment in all parts of the small intestine. Since these are mucus producing cells, the small intestine may have increased in cell number and mucus production. Both anti-secretory and cytoprotective doses of 16,16-dimethyl PGE2 caused weight increases in the stomach and small intestine. However, the weight gain by itself was not sufficient to protect the stomach or small intestine from necrotic agents after the prostaglandin was discontinued.     

Characteristics of rodent intestinal mucin Muc3 and alterations in a mouse model of human cystic fibrosis

Human mucin MUC3 and rodent Muc3 are widely assumed to represent secretory mucins expressed in columnar and goblet cells of the intestine. Using a 3'-oligonucleotide probe and in situ hybridization, we observed expression of rat Muc3 mostly in columnar cells. Two antibodies specific for COOH-terminal epitopes of Muc3 localized to apical membranes and cytoplasm of columnar cells. An antibody to the tandem repeat (TR) sequence (TTTPDV)3, however, localized to both columnar and goblet cells. On CsCl gradients, Muc3 appeared in both light- and heavy-density fractions. The lighter species was immunoreactive with all three antibodies, whereas the heavier species reacted only with anti-TR antibody. Thus Muc3 is expressed in two forms, a full-length membrane-associated form found in columnar cells (light density) and a carboxyl-truncated soluble form present in goblet cells (heavy density). In a mouse model of human cystic fibrosis, both soluble Muc3 and goblet cell Muc2 were increased in amount and hypersecreted. Thus Muc2 and Muc3 contribute to the excess intestinal luminal mucus of cystic fibrosis mice.     

Heligmosomoides polygrus (equals nematospiroides dubius): humoral and intestinal immunologic responses to infection in mice

Measurements of serum IgG₁, IgG_2a, IgM, and IgA levels and antibody titers in these immunoglobulin classes were made at intervals after initial infection and challenge infection of mice immunized by two or three previous infections. Identical measurements were made on the content of the small intestine in mice which had been exposed to the same infection schedule. Sections of small intestine taken after initial infection and challenge infection were examined by the fluorescent antibody technique for changes in populations of immunoglobulin-containing cells and by routine histologic procedures for histopathologic changes.In serum, only IgG₁ was consistently increased after initial infection, and antibody in IgG₁ was detected within the first 2 wk of infection. In immunized animals, only IgG₁ and antibody of this class always responded to challenge infection, although antibody in other immunoglobulin classes was detected.IgA concentration of the intestinal content did not differ significantly after initial infection or challenge infection of immunized mice. Immunized mice had about twice the IgG₁ concentration in intestinal content as singly infected animals. No intestinal antibody was detected after initial infection; only IgG₁ antibody was detected in the intestinal content of immunized and challenged mice.Cell infiltrates in the intestinal mucosa and submucosa of immunized animals contained numerous IgG₁-containing cells. Mast cells and globular leukocytes were observed in the intestine of immunized animals.     

Purification and immunohistochemistry of Griffonia simplicifolia agglutinin-II-binding mucus glycoprotein in rat stomach

Gastric mucus is thought to protect the gastric wall from mechanicaltrauma, desiccation, pathogenic microorganisms, acid and proteases.We purified Griffonia simp1icifolia agglutinin-II (GSA-II)-bindingmucus glycoprotein (GMG) from rat gastric mucosa by solubilizationin a guanidine- containing buffer, gel permeation chromatography,Ricinus communis agglutinin-I (RCA-I)-affinity chromatographyand GSA-II-affinity chromatography. Rat GMG showed high molecularweight on a Sephacryl S-1000 column, and a single band in 0.5%agarose-2% polyacrylamide composite gels and blots. A proteinof {small tilde}60 kDa was contained in the GMG preparation.GMG was deglycosylated with trifluoromethanesulphonic acid treatment.An antibody was raised against deglycosylated GMG (deGMG). Theantibody recognized deGMG, GMG, periodic acid-treated deGMGand O-glycanase-digested deGMG, but did not react to trypsin-digesteddeGMG. These results suggest that the antibody recognizes proteinase-sensitiveregion or peptide backbone of GMG. In immunohistochemistry,the mucous gel layer of the stomach luminal surface was stainedwith antibody. The antibody recognized not only gastric mucousneck cells and pyloric gland cells, but also gastric surfacemucous cells, mucous cells in the duodenal gland, and gobletcells in the small intestine and colon. These results indicatethat GMG is a component of rat gastric mucus, and that the antibodyrecognizes mucous-secreting cells in rat stomach and intestine. antibody immunohistochemistry lectin-affinity gel chromatography mucus glycoprotein rat stomach     

Rat serum antibodies binding to high-molecular-weight glycoprotein(s) on syngeneic colon carcinomas,demonstrated by competition with rat monoclonal antibody

Summary Sera from rats immunized to syngeneic 1,2-dimethylhydrazine colon carcinomas were analyzed for their ability to inhibit the binding of a syngeneic rat IgM monoclonal antibody (10B12) specific for high-molecular-weight glycoprotein(s) from rat colon carcinoma. Immunization with irradiated tumour cells or with tumour tissue extracts resulted in the appearance of a strong inhibiting activity. Sera of animals with established growing tumours and of females shortly after partus also inhibited binding of the monoclonal antibody, while unimmunized animals or animals immunized with irrelevant antigens had no inhibiting antibodies in their sera. Dimethylhydrazinetreated animals showed an increased titer of antibodies binding to the high-molecular-weight glycoprotein, but showed no inhibition of binding of the 10B12 monoclonal antibody. The syngeneic 10B12 rat antibody obviously does not reflect a rare event captured from a hyperimmune animal by the hybridoma technique but rather represents an antibody specificity frequently appearing in the immune response to tumours expressing the high-molecular-weight glycoprotein.     

Electron probe analysis of calcium transport by small intestine

Calcium transport in small intestine of rat and chick has been studied at the cellular level using the electron probe X-ray microanalyzer. Tissues were examined directly after removal as well as after incubation in a calcium solution. In both preparations, discrete calcium localizations were found associated with intracellular and extracellular goblet cell mucus. The in vitro preparations showed calcium in transit across the absorptive epithelium in discrete localizations. Although the primary path of transport was along lateral cell borders, some localizations were found in the cytoplasm in a supranuclear position. The effect of vitamin D depletion and repletion was to decrease and increase, respectively, the number of calcium localizations in transit across the epithelium. These results suggest that calcium is transported while in a sequestered form and indicate that goblet cell mucus plays a role in this transport process.     

Histochemical characteristics of mucins in the small intestine. A comparative study of normal mucosa, benign epithelial tumours and carcinoma

Synopsis The histochemical properties of the mucins in seven benign epithelial tumours and 15 carcinomas distributed along the duodenum, jejunum and ileum were investigated and compared with normal controls. This study reveals that (a) goblet cells in normal small intestine contain neutral and sialomucins but no sulphated material; (b) the proportion of the different types of mucins in the goblet cells vary along the crypts and villi with an increasing amount of sialomucins towards the villus top; (c) mucin composition also changes from duodenum to ileum particularly in the proportions of sialic acid types and in the presence of traces of sulphomucins in the ileal mucosa close to the ileo-caecal valve, suggesting a gradual transition through the small intestine to the colon; (d) benign tumours show the same mucin pattern as normal mucosa; (e) the mucosa adjacent to carcinoma shows increasing amounts of sialomucins and sulphomucins; (f) carcinomas present a variety of mucin patterns, and thus the study of mucins seems to be of no value in differentiating tumours of the small intestine from those elsewhere in the gastrointestinal tract. A working hypothesis based on the Unitary Theory of the origin of the intestinal epithelial cells is proposed to explain the variations in glycoprotein synthesis with cell differentiation and carcinogenesis.     

Partial characterization of the gastrointestinal weight changes produced in the female rat by 16,16-dimethylprostaglandin E2

Oral and subcutaneous administration of 16,16-dimethylprostaglandin E₂ (16,16-dimethyl PGE₂) resulted in an increase in the dry weight of the stomach and small intestine of the female rat. This weight response was rapid, controlled rather than continuously progressing, dose dependent and reversible. The dry weight of the colon also increased but this was not studied in detail.Two-day treatment with 16,16-dimethyl PGE₂ caused an increase in the incorporation of ³H-thymidine into the duodenum, jejunum and colon suggesting an increase in cell number. Incorporation into the stomach and ileum was not changed.The number of goblet cells per crypt was increased by prostaglandin treatment in all parts of the small intestine. Since these are mucus producing cells, the small intestine may have increased in cell number and mucus production.Both anti-secretory and cytoprotective doses of 16,16-dimethyl PGE₂ caused weight increases in the stomach and small intestine. However, the weight gain by itself was not sufficient to protect the stomach or small intestine from necrotic agents after the prostaglandin was discontinued.     

Acute distal intestinal obstruction in gnotobiotic rats. Intestinal morphology and cell renewal.

1. Complete mechanical obstruction of the distal small intestine was produced in gnotobiotic rats. 72 h after the operation small intestinal morphology and epithelial cell renewal were investigated proximal and distal to the site of obstruction. 2. Proximal to the site of obstruction there were minor changes in villus height, base length and in villus cell number, a large increase in depth and diameter of the crypts and an approximately threefold increase in cell renewal. 3. Distal to the site of obstruction there were no differences between the intestines of rats with obstruction and controls. 4. The apparent lack of secretion by the goblet cells and the reduced number of intraepithelial leucocytes suggest that the barrier function of the small intestine is impaired in obstruction.     

Selective loss of sialic acid from rat small intestinal epithelial cells during postnatal development: demonstration with lectin-gold techniques

Post-embedding lectin-gold cytochemistry was employed to investigate the distribution of sialic acid and fucose residues in rat small intestinal epithelial cells during postnatal development. During the suckling phase (postnatal day 1) the apical and basolateral plasma membranes of epithelial cells, as well as the goblet cell mucus was intensely stained with the sialic acid-specific Sambucus nigra L. lectin I-gold complex (SNL I-g). By the weaning period (postnatal day 23), the entire villus contained both SNL I-g-positive and negative cells. In adult small intestine, the plasma membranes of all epithelial cells were unreactive with SNL I-g; however, abundant staining was detectable in goblet cell mucus, cells of the lamina propria, and smooth muscle cells. The distribution of fucose residues as detected with a Ulex europaeus lectin I-gold complex (UEL I-g) was virtually opposite that of sialic acid. At postnatal day 1, staining was restricted to goblet cell mucus, whereas by postnatal day 23, a portion of epithelial cells displayed UEL I-g binding sites along the apical and basolateral plasma membranes. In the adult, the apical and basolateral plasma membranes of all epithelial cells, as well as goblet cell mucus were stained with UEL I-g. These results support biochemical data demonstrating a shift from sialylation to fucosylation of intestinal microvillar glycoconjugates during the weaning phase of postnatal development. Moreover, the results indicate that rather than a general decrease in cellular sialylation, specific individual cells at all positions along the crypt-to-villus axis become devoid of sialic acid.     

Antioxidant effects of dehydroepiandrosterone and 7alpha-hydroxy-dehydroepiandrosterone in the rat colon, intestine and liver

This study examined in healthy male Wistar rats the in vivo antioxidant effect of dehydroepiandrosterone (DHEA) and 7alpha-hydroxy-DHEA administered by intraperitoneal injections (50 mg/kg body weight) for 2 or 7 days. Markers of oxidative damage to lipids (thiobarbituric acid-reacting substances, TBARS) and to proteins (protein carbonyls) were assessed in colon, small intestine, and liver homogenates. DHEA and 7alpha-hydroxy-DHEA caused a decrease in body weight. DHEA treatment significantly increased liver, colon, and small intestine cell weights. After 7 days, DHEA exerted an antioxidant effect in all organs studied. In the colon, oxidative damage protection was accompanied by a goblet cell proliferation and increase in acidic mucus production. After 2 days, the antioxidant effect of 7alpha-hydroxy-DHEA was mainly observed in the liver. Nonprotein sulfhydryl groups (mostly glutathione levels) were altered by DHEA in the liver whereas they remained unchanged after 7alpha-hydroxy-DHEA treatment. The results indicate that in healthy animals, DHEA exerts a protective effect, particularly in the colon, by reducing the tissue susceptibility to oxidation of both lipids and proteins. This effect was not limited to a specific tissue, whereas the metabolite 7alpha-hydroxy-DHEA exerted its antioxidant effect towards the two markers of oxidative damage earlier than DHEA, and mainly in the liver.