Epithelial cell dynamics in rabbit cecum and proximal colon P1.

The large intestine of mammals has long been viewed as an osmoregulatory organ, and evidence suggests that fluid and solute transport mechanisms within the intestine are heterogeneous, varying depending on the particular segment involved. Variations in function are often matched by morphological correlates, but despite the widespread use of rabbit large intestine as an experimental model, there is a lack of knowledge about the cellular makeup and dynamics in the colonic mucosal epithelium. The presence of mitotic figures and immunohistochemical localization of proliferating cell nuclear antigen (PCNA) were used to identify the proliferative zone(s). Cellular migration patterns were determined through the use of the thymidine analog 5-bromo-2-deoxyuridine (BrdU) over a 24-, 48-, and 72-hr period. Apoptotic nuclei were identified utilizing terminal deoxynucleotidyl transferase d-UTP nick-end labeling (TUNEL). Both cecum and the initial portion of the proximal colon (P1) exhibited a proliferative zone at or near the crypt base, and migration proceeded upwards toward the surface epithelium lining the intestinal lumen, where apoptosis occurred Turnover time of crypt columnar cells was determined to be about 3 days; that of mucous cells was estimated to be about 5 weeks. Rabbit cecum and proximal colon P1 are similar in their cellular morphology and epithelial cell kinetics. In both, the major proliferative zone is located at or near the crypt base, from which crypt columnar cells migrate toward the lumenal surface epithelium over a period of 3 days. Goblet cell turnover rate is much slower than that of columnar cells.

Endothelial expression of vascular cell adhesion molecule-1 correlates with metastatic pattern in spontaneous melanoma.

OBJECTIVE: Adhesive interactions between tumor cell surface receptors and endothelial cell adhesion molecules are thought to contribute to tumor cell arrest and extravasation during hematogenous metastasis. Recent reports suggest that melanoma cell integrin alpha4beta1 (very late antigen-4, VLA-4) interaction with the inducible cell adhesion molecule, vascular cell adhesion molecule-1 (VCAM-1), is critical for tumor cell arrest. However, no information is available regarding microvascular VCAM-1 expression during spontaneous melanoma metastasis. The objectives of this study were to evaluate VCAM-1 expression in pulmonary and extrapulmonary vascular beds during melanoma progression, and to determine whether there is an organ-specific profile for VCAM-1 expression which corresponds with the clinical pattern of melanoma metastasis. METHODS: The dual-radiolabeled monoclonal antibody (mAb) technique for quantification of VCAM-1 in different vascular beds was applied to a physiological model of melanoma (B16-BL6) metastasis. Measurements of VCAM-1 were obtained when primary tumors reached 5 mm in size, and every 7 days following removal of the primary lesion. Histological examinations were performed, and mice were placed into 2 groups, based on the presence (+colonies) or absence (-colonies) of pulmonary metastases. VCAM-1 measurements obtained from several organ systems were then compared between these 2 groups of mice. Localization of VCAM-1 was achieved through immunohistochemical staining of tissues. Plasma collected from each experimental animal, as well as melanoma-conditioned media, was assayed to determine levels of the cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin-1alpha (IL-1alpha). RESULTS: Data collected from the dual-radiolabeled mAb technique indicate that 3 weeks following removal of the primary lesion, there is a tendency for VCAM-1 expression to increase in cardiac, hepatic, and cerebral vascular beds. Four weeks following primary resection, when pulmonary metastatic burden was maximal, VCAM-1 was significantly upregulated in each of these vascular beds. Results obtained from the lung indicate that VCAM-1 remains unchanged in pulmonary vessels at all time points examined. Immunohistochemical staining of heart and brain support the radiolabeled mAb measurements, and reveals that these organs exhibit an inflammatory phenotype in mice with heavy pulmonary tumor burden. Furthermore, 25% of these mice had histological evidence of melanoma metastases in heart and brain. Transplantation of liver fragments from mice with advanced pulmonary metastases into subcutaneous tissue of donor mice resulted in the formation of melanotic outgrowths. Plasma levels of the cytokines TNF-alpha and IL-1alpha were negligible in both groups of mice. CONCLUSIONS: Our results indicate that upregulation of VCAM-1 is not a prerequisite for the formation of pulmonary metastases during spontaneous melanoma metastases. However, once lung metastases become well established, organ-specific increases in VCAM-1 expression become apparent. Furthermore, these organ-specific increments in VCAM-1 expression correspond with documented clinical patterns of melanoma metastasis. The enhanced expression of VCAM-1 is independent of systemic levels of TNF-alpha and IL-1alpha, but may be the result of melanoma-induced alterations at the local level, as we found evidence of melanoma cell occupation in heart, brain, and liver in pulmonary metastases-bearing mice.

MAdCAM mediates lymphocyte-endothelial cell adhesion in a murine model of chronic colitis.

Previous studies have revealed that the expression of several endothelial cell adhesion molecules [e.g., intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and mucosal addressin cell adhesion molecule 1 (MAdCAM-1)] is dramatically elevated in the chronically inflamed colonic vasculature of severe combined immunodeficient (SCID) mice reconstituted with congenic CD4+, CD45RB(high) T lymphocytes. The objective of this study was to define the contribution of different endothelial cell adhesion molecules to the lymphocyte-endothelial cell (L/E) adhesion observed in the colonic microvasculature in this experimental model of inflammatory bowel disease. Fluorescently labeled T lymphocytes, isolated from spleens of normal BALB/C mice, were injected intravenously into SCID mice that had been reconstituted with CD4+, CD45RB(high) T lymphocytes either before (3 wk after reconstitution) or after (7 wk postreconstitution) the onset of clinical signs of colitis (i.e., diarrhea, loss of body wt). Intravital fluorescence microscopy was used to quantify L/E adhesion in different-sized venules of the colonic submucosa during the development of colitis. L/E adhesion was noted in some segments of the vasculature in precolitic SCID mice (3 wk after reconstitution) but not in similar-sized vessels of control (wild type and SCID) mice. L/E adhesion was observed in a greater proportion of venules and occurred with greater intensity in the mucosa of colitic mice (7 wk postreconstitution). Pretreatment with a blocking monoclonal antibody against MAdCAM-1, but not ICAM-1 or VCAM-1, significantly and profoundly reduced L/E adhesion in colitic mice. Immunohistochemical staining also revealed the localization of T cells on colonic endothelial cells expressing MAdCAM-1. These findings indicate that MAdCAM-1 is largely responsible for recruiting T lymphocytes into inflamed colonic tissue.

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.

Surgically induced cryptorchidism-related degenerative changes in spermatogonia are associated with loss of cyclic adenosine monophosphate-dependent phosphodiesterases type 4 in abdominal testes of rats.

The present study was undertaken to investigate the role of phosphodiesterase type 4 (PDE4) enzymes in cryptorchidism-induced apoptosis of the germ cells. Regulation of expression of PDE4 enzymes was studied in the abdominal and scrotal testes of surgically induced cryptorchid rats for 10, 20, and 30 days. In some cases orchidopexy was performed after 30 days of cryptorchidism, and rats were allowed to recover for an additional 50 days. Upon histological examination, marked degenerative changes in the epithelial lining of the seminiferous tubules within abdominal testes were observed compared with contralateral control or age-matched sham-operated rats. These changes included degeneration of some spermatogonia, apoptosis of the secondary spermatocytes, incomplete spermatogenesis, and lack of spermatozoa in the lumen. In contrast, contralateral scrotal testes exhibited normal histology. Significant improvement in the regeneration of spermatogonia was observed in rats after 50 days of recovery following orchidopexy. Immunocytochemical examination suggested the presence of PDE4A in germ cells while PDE4B was predominantly expressed on somatic cells. Western blotting using PDE4 subtype-selective antibodies showed the presence of two PDE4A variants (a 109-kDa PDE4A8 and a previously uncharacterized 88-kDa PDE4A variant) and two PDE4B (78-kDa PDE4B2 and 66-kDa PDE4B variant) bands. In unilaterally cryptorchid animals, the abdominal testis showed a time-dependent decrease in both PDE4A8 and 88-kDa PDE4A variants. In contrast, the expression of 66-kDa PDE4B was markedly increased in a time-dependent fashion in abdominal testes of cryptorchid rats. Animals surgically corrected for cryptorchidism and allowed to recover for 50 days exhibited normal expression of both PDE4A and PDE4B variants compared with aged-matched, sham-operated controls. In conclusion, this study suggests that down-regulation of PDE4A variants in cryptorchid testes may play an important role in the degeneration of spermatogonia and increased apoptotic activity in the germ cells.

Interferon-gamma and interleukin-10 reciprocally regulate endothelial junction integrity and barrier function.

Inflammatory bowel disease (IBD) is associated with Th1/Th2 cytokine dysregulation, leukocyte extravasation, and tissue edema, but the mechanisms for cytokine-mediated vascular dysfunction are not understood. To investigate how cytokines might control edema in IBD, we determined vascular permeability and IFN-gamma expression in two models of murine colitis: SCID mice reconstituted with CD45RB(high T-lymphocytes (CD45RB(high)/SCID mice), and interleukin-10 gene deficient (IL-10(-/-)) mice. We also investigated the in vitro effects of IFN-gamma and IL-10 on human endothelial solute barrier and junction protein expression. Vascular permeability in CD45RB(high)/SCID and IL-10(-/-) mice was quantified using tissue (131)I-IgG accumulation. The IFN-gamma message was quantified using the ribonuclease protection assay. Endothelial barrier integrity in vitro was measured by transmonolayer electrical resistance, and junctional proteins were examined by immunoblotting and fluorescence microscopy. Both CD45RB(high)/SCID and IL-10(-/-) mice exhibit enhanced colonic microvascular leakage and IFN-gamma message levels compared to their respective controls. In vitro, IFN-gamma also reduced endothelial barrier (monolayer electrical resistance, increased albumin permeability) and reduced tight junction (occludin) expression and staining. These effects were reversed by pretreatment of monolayers with IL-10. Therefore, in vivo IFN-gamma and IL-10 may modulate microvascular leakage in IBD partly by controlling the expression of intestinal endothelial tight junctional proteins.

Quantification of murine endothelial cell adhesion molecules in solid tumors.

Coordinated adhesive interactions between lymphocyte receptors and endothelial cell adhesion molecules (CAMs) are a prerequisite for effector cell entry into tumor stroma. Whereas the diminished leukocyte-endothelial cell interactions observed in tumor microvessels have been attributed to a reduced expression of endothelial CAMs, there is no quantitative data bearing on this issue. The dual-radiolabeled monoclonal antibody technique was used to quantify constitutive and tumor necrosis factor (TNF)-alpha-induced expression of intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), ICAM-2, P-selectin, E-selectin, and platelet-endothelial cell adhesion molecule 1 (PECAM-1) in different vascular beds of normal (C57Bl/6) and RM-1 tumor-bearing mice. When corrected for endothelial surface area, the constitutive expression of selectins in tumor vessels was higher than that observed in other vascular beds. Both constitutive and induced expression of endothelial CAMs in peripheral vascular beds did not differ between normal and tumor-bearing mice. Within the tumor, the magnitude of the upregulation of P-selectin, ICAM-1, and VCAM-1 after TNF-alpha was similar to that within other vascular beds. E-selectin expression in tumors was refractory to TNF-alpha, whereas PECAM-1 and ICAM-2 expression were significantly reduced. Our findings suggest that the presence of a solid tumor does not influence endothelial CAM expression in other vascular beds and that the higher density of selectins in nonstimulated tumor vessels may promote the recruitment of rolling leukocytes in this tissue.

Influence of arteriovenular pairing on PAF-induced capillary filtration.

The purpose of this study was to determine the basic mechanism by which leukocyte-endothelial cell adhesion mediates platelet-activating factor (PAF)-induced increases in capillary fluid filtration rate. A modified Landis technique was used to observe fluid filtration from capillaries in the rat mesentery. Hypothetical mechanisms of increased filtration that were tested included 1) a direct action of leukocytes on endothelial cells during transit through the capillaries; 2) an upstream propagated response (via gap junction communication by adjacent endothelial cells) originating at sites of venular leukocyte adhesion; and 3) venule-to-arteriole communication, where mediators produced at the site of venular leukocyte adhesion reach a nearby paired arteriole that delivers the mediators to branching capillaries. Evidence was obtained in opposition to the first two hypotheses. However, in support of the third hypothesis, a significant correlation was found between the extent of arteriolar pairing to venules and the PAF-induced increase in capillary fluid filtration rate. These findings suggest that venule-to-arteriole communication might modify capillary filtration rate during acute inflammation.

Expression of zonula occludens and adherens junctional proteins in human venous and arterial endothelial cells: role of occludin in endothelial solute barriers.

OBJECTIVE: The purpose of this study was to correlate the expression of occludin and VE-cadherin with the solute barrier properties of arterial and venous endothelial monolayers. METHODS: Immunofluorescent confocal and traditional microscopy were used to determine junctional protein localization in endothelium in vivo and in vitro respectively, and western and northern analysis used to determine protein and gene expression levels. Permeability of endothelial monolayers was examined under normal, low calcium, and cytochalasin-D treatment conditions. Antisense oligonucleotide experiments for occludin were performed to determine the contribution of occludin to solute barrier. RESULTS: Occludin protein in endothelial monolayers is more concentrated in arterial junctions than in venous junctions both in vivo and in vitro. Arterial endothelial cells express 18-fold more occludin protein and nine times more occludin mRNA compared to venous endothelial cells. In vivo, both endothelial cells demonstrate VE-cadherin staining; and in vitro, only venous endothelial cells express VE-cadherin protein and mRNA. Occludin antisense experiments suggest that both arterial and venous barrier properties are due to these different amounts of occludin expression. Venous barrier was remarkably sensitive to low extracellular calcium, while arterial barrier was more sensitive to cytochalasin-D. CONCLUSIONS: These findings suggest strongly that arterial and venous endothelial barrier reflects the level of expression of different adhesion molecules and that modulation of these proteins, especially occludin, may regulate the level of endothelial solute barrier.

Proliferation of goblet cells and vacuolated cells in the rabbit distal colon.

Previous studies of colonic epithelial cell kinetics in mice and rats revealed a pattern similar to small intestine, where basally located stem cells proliferate, differentiating as they migrate towards the surface epithelium. Vacuolated and goblet cells are assumed to co-migrate at the same rate. The present study indicates that rabbit distal colon has more complicated epithelial cell kinetics. The zone of proliferation was detected immunohistochemically using proliferating cell nuclear antigen (PCNA) and confirmed with the use of colchicine to arrest dividing cells in metaphase. Migrating cells were tracked from the zero-hour position (PCNA labeling, mitosis) to positions 24, 48, 72 hrs by monitoring cell migration with the thymidine analog 5-Bromo-2-Deoxyuridine (BrdU). PCNA revealed a major proliferative zone in the upper third of the crypt column and the presence of mitotic figures after colchicine corroborated these results. Differentiated vacuolated cell proliferation was detected at three crypt sites: base, middle, and top of the crypt, while columnar cells arose from a population of dividing cells at the top of the crypt. Turnover of columnar and vacuolated cells occurred within 72 hrs. Goblet cells exhibited maximal proliferation at the crypt base and migrated at a much slower rate than the other cell types. In rabbit distal colon, populations of proliferating cells exist at multiple levels of the crypt column. Vacuolated and goblet cells differ in their labeling indices and migration rates, suggesting that the two cell types arise and migrate independently.

Unique crystalline inclusions in neonatal pig small intestine epithelial cells.

In this study we describe the presence of novel crystalline inclusions in the jejunal and ileal epithelium of newborn pigs. Hampshire/Yorkshire piglets (0-3 days) were anesthetized with pentobarbital and tissues were recovered following laparotomy. Sections of jejunum and ileum were prepared with a variety of fixatives for paraffin and methacrylate embedment for the light and electron microscopy. The characteristics of these crystalline structures have been demonstrated by cytochemical staining, immunohistochemical staining, and ultrastructural studies. Using light microscopy, it was determined that 10.8% of villus epithelial cells in the jejunum contained inclusions, whereas inclusions were present in 26.6% of the villus epithelial cells in the ileum. Inclusions were absent in crypt epithelial cells of both areas of the intestine, and the percentage of cells with inclusions increased towards the villus tip. Preliminary histochemical analysis has been unrevealing. The inclusions are periodic acid/Schiff's (PAS) reagent-negative, and do not stain for the presence of concentrated proteins. Using electron microscopy, the inclusions are found to be clustered in the supra-nuclear region of the cells and are generally oriented with the long axis of the cell. The crystals vary in size and have a dense core region surrounded by a regular less dense periphery.

Role of bacterial adherence and the mucus barrier on bacterial translocation: effects of protein malnutrition and endotoxin in rats.

OBJECTIVE: The purpose of the study was to investigate the potential relations between mucosal bacterial adherence, intestinal mucus and mucin content, and bacterial translocation. SUMMARY BACKGROUND DATA: The attachment of bacteria to mucosal surfaces is the initial event in the pathogenesis of most bacterial infections that originate at mucosal surfaces, such as the gut. The intestinal mucus layer appears to function as a defensive barrier limiting micro-organisms present in the intestinal lumen from colonizing enterocytes. Consequently, studies focusing on the biology of bacterial adherence to the intestinal mucosa likely are to be important in clarifying the pathogenesis of gut origin sepsis. METHODS: To explore the relations between intestinal bacterial adherence, mucus bacterial binding, and bacterial translocation, two models were used. One (protein malnutrition) in which profound alterations in intestinal morphology occurs in the absence of significant translocation and one (endotoxin challenge) in which bacterial translocation occurs and intestinal morphology is relatively normal. RESULTS: Protein malnutrition was not associated with bacterial translocation and measurement of enteroadherent, mucosally associated bacterial population levels documented that the total number of gram-negative enteric bacilli adherent to the ileum and cecum was less in the protein-malnourished rats than in the normally nourished animals (p < 0.01). Furthermore, there was an inverse relation between the duration of protein malnutrition and bacterial adherence to the intestinal mucosa (r = 0.62, p < 0.002). In contrast, after endotoxin challenge, the level of enteroadherent bacteria was increased and bacterial translocation was observed. The binding of Escherichia coli to immobilized ileal mucus in vitro was decreased significantly in protein-malnourished rats, whereas E. coli binding to insoluble ileal mucus was increased in the rats receiving endotoxin. CONCLUSIONS: This study indicates that the adherence of bacteria to the intestinal mucosal surface is an important factor in bacterial translocation, that intestinal mucus modulates bacterial adherence, and that increased levels of mucosally associated bacteria are associated with a loss intestinal barrier function to bacteria.

Sulfasalazine or enteral diets containing fish oil or oligosaccharides attenuate chronic colitis in rats.

: Recent studies have suggested that n-3 fatty acids from fish oil (FO) as well as short-chain fatty acids may attenuate some of the gut injury and inflammationassociated ulcerative colitic (UC). The objectives of this study were to (a) assess the antiinflammatory activity of sulfasalazine (SAZ), a drug known to be effective in the treatment of human UC in a model of chronic granulomatous colitis in rats and (b) determine whether enteral diets supplemented with either FO or two indigestible oligosaccharides (fructooligosaccharide, FOS; xylooligosaccharide, XOS) could attenuate the inflammation observed in a model of chronic granulomatous colitis. In one series of experiments, female Lewis rats were randomized into three groups consisting of a sham-operated control group, a colitic group, and a colitic group in which rats were given oral sulfasalazine (SAZ) immediately after induction of colitis and continued for 3 weeks. Chronic granulomatous colitis with liver and spleen inflammation was induced by subserosal (intramural) injection of purified peptidoglycan-polysaccharide (PG/PS) into the distal colon. Sham-operated rats were injected with human serum albumin. All rats received standard lab chow. In a second series of experiments, female Lewis rats were randomized into six groups consisting of four colitic groups fed enteral diets, a colitic group fed chow, and a sham-operated group fed a control enteral diet. Enteral diets (300 kcal/kg/day) contained either FO, FOS/ gum arabic, XOS/gum arabic, or no bioactive ingredient (control diet). All rats were fed for 1 week before induction of colitis. Rats consumed the diets for 3 additional weeks before being killed. SAZ significantly attenuated the PG/PS-induced increases in myeloperoxidase (MPO) activity as well as significantly reduced the PG/PS-induced increases in liver and spleen weights. Control (enteral diet) as well as the FO and XOS diets significantly attenuated the increase in colon weight when compared with chow-fed rats. We also found that the FO and XOS diets significantly attenuated the PG/PS-induced increases in colonic MPO activity and colon weight. The FOS and XOS diets significantly attenuated the PG/PS-induced increases in liver weights when compared with PG/PS + chow-fed animals. The antiinflammatory activity of these diets was confirmed by means of histological inspection showing an inhibition of inflammation and maintenance of crypt cell integrity. These results demonstrate that a complete enteral diet supplemented with either FO, FOS, or XOS exhibited antiinflammatory activity that was similar in efficacy to the known antiinflammatory drug SAZ in this model of colitis.

Isolation and characterization of cDNA clones encoding pig gastric mucin.

Polyclonal antibodies raised to deglycosylated pig gastric mucin were used to screen a cDNA library constructed with pig stomach mucosal mRNA. Immunocytochemistry indicated that the antibody recognizes intracellular and secreted mucin in surface mucous cells of pig gastric epithelium. A total of 70 clones producing proteins immunoreactive to this antibody were identified, two of which (PGM-2A,9B) were fully sequenced from both ends. Clone PGM-9B hybridized to a polydisperse mRNA (3-9 kb) from pig stomach, but not liver, intestine or spleen, nor to mRNA from human, mouse, rabbit or rat stomach. Sequence analysis indicated that PGM-9B encodes 33 tandem repeats of a 16-amino-acid consensus sequence rich in serine (46%) and threonine (17%). Using the restriction enzyme MwoI, which has a single target site in the repeat, it was demonstrated that PGM-9B consists entirely of this tandem repeat. Southern-blot analysis indicated that the repeat region is contained in a 20 kb HindIII-EcoRI fragment, and BamHI digestion suggested that most of the repeats are contained in a 10 kb fragment. In situ hybridization with an antisense probe to PGM-9B showed an intense signal in the entire gastric gland. Clone PGM-2A also contains the same repeat sequence as 9B, but, in addition, has a 64-amino-acid-long non-repeat region at its 5' end. Interestingly the non-repeat region of PGM-2A has five cysteine residues, the arrangement of which is identical with that reported for human intestinal mucin gene MUC2.

Microvascular responses to inhibition of nitric oxide production. Role of active oxidants.

The objective of this study was to assess the potential contribution of hydrogen peroxide (H2O2) to the leukocyte-endothelial cell adhesion and increased microvascular permeability observed in rat mesenteric venules after inhibition of nitric oxide synthesis with NG-nitro-L-arginine methyl ester (L-NAME). Leukocyte adherence and emigration and leakage of fluorescein isothiocyanate-labeled albumin were monitored in postcapillary venules before and after exposure of the tissue to L-NAME. H2O2 production in mesenteric tissue was monitored by using dihydrorhodamine 123 (DHR), the H2O2-sensitive fluorochrome. L-NAME elicited a rapid increase in both the rate of albumin extravasation and oxidation of DHR, which was followed by an increased adherence and emigration of leukocytes in postcapillary venules. Treatment with either catalase or dimethylthiourea attenuated the L-NAME-induced oxidative stress, albumin leakage, and leukocyte-endothelial cell adhesion. Oxidation of DHR was enhanced in animals treated with either 3-amino-1,2,4-triazole (ATZ), an inhibitor of endogenous catalase, or a combination of ATZ and maleic acid diethyl ester, which depletes intracellular glutathione. Animals receiving a CD11/CD18-specific antibody to prevent leukocyte adhesion/emigration exhibited a reduced oxidation of DHR in response to L-NAME. These findings indicate that most of the H2O2 (and secondarily derived oxidants) generated in mesenteric tissue exposed to an inhibitor of nitric oxide production is due to accumulation of activated leukocytes.

Epidermal growth factor attenuates jejunal mucosal injury induced by oleic acid: role of mucus.

Epidermal growth factor (EGF) is present in biliary, pancreatic, and Brunner's gland secretions. The aim of the present study was to assess the effects of EGF on lipid-induced mucosal injury. The proximal jejunum of anesthetized rats was cannulated for perfusion of the lumen with emulsified oleic acid (40 mM oleic acid in 20 mM sodium taurocholate; pH 6.0). Mucosal epithelial integrity was monitored by measuring the blood-to-lumen clearance of 51Cr-labeled EDTA. Perfusion of the lumen with emulsified lipid increased EDTA clearance. Addition of EGF (0.5 ng/ml) to the lipid emulsion ameliorated the lipid-induced increase in EDTA clearance. Perfusion of the lumen with EGF alone stimulated mucus secretion from goblet cells. This effect of EGF was abolished by atropine. In addition, in atropinized animals there was 1) an exaggeration of the lipid-induced injury and 2) a loss of the protective effect of EGF. Our findings provide evidence supporting the hypothesis that EGF provides protection against lipid-induced mucosal injury, in part, by stimulating mucus production.

Secretory immunoglobulin A, intestinal mucin, and mucosal permeability in nutritionally induced bacterial translocation in rats.

OBJECTIVE: The authors investigated the role of mucin and secretory immunoglobulin A (slgA) in a model of nutritionally induced bacterial translocation. BACKGROUND: Parenteral and certain elemental diets have been shown to impair intestinal barrier function, whereas fiber has been shown to protect against nutritionally induced bacterial translocation. However, the factors responsible for these phenomenon have not been fully determined. METHODS: Intestinal mucin levels, mucosal protein content, slgA, intestinal morphology, and permeability to horseradish peroxidase, bacterial translocation, and intestinal bacterial population levels were measured in rats 7 days after receiving total parenteral nutrition (TPN) solution (28% glucose, 4.25% amino acids; 307 kcal/kg/day) enterally (ORAL-TPN) or parenterally (IV-TPN) with or without enteral bulk fiber supplementation. Chow-fed rats served as control subjects. RESULTS: The incidence of bacterial translocation in the ORAL-TPN and IV-TPN groups was reduced significantly by the provision of fiber (p < 0.05). Mucosal protein, slgA, and insoluble mucin levels were decreased in the jejunum of the ORAL-TPN and IV-TPN groups, with mucosal protein levels being decreased to a greater extent than slgA or mucin. Although similar decreases in these parameters were observed in the fiber-fed groups, fiber appeared to improve intestinal barrier function as measured by horseradish peroxidase permeability. CONCLUSIONS: The provision of bulk-forming fiber improves intestinal barrier function as measured by peroxidase permeability and bacterial translocation, but does not restore mucosal protein content, intestinal mucin, or slgA levels to normal.

Effects of nitric oxide synthase inhibition on the pathophysiology observed in a model of chronic granulomatous colitis.

The objective of this study was to assess the role that nitric oxide (NO) may play in mediating the colonic inflammation observed in a model of chronic granulomatous colitis using two pharmacologically different inhibitors of nitric oxide synthase (NOS). The NOS inhibitors NG-nitro-L-arginine methyl ester (L-NAME; 15 mumol/kg/day) and aminoguanidine (AG; 15 mumol/kg/day) were administered to rats in their drinking water, beginning 3 days before the induction of colitis and continuing for the entire 3-week period. We found that chronic NOS inhibition by L-NAME or AG significantly attenuated the peptidoglycan/polysacchride (PG/PS)-induced increases in macroscopic colonic inflammation scores and colonic MPO activity. Only AG, and not L-NAME, attenuated the PG/PS-induced increases in colon dry weight. Both L-NAME and AG significantly attenuated the PG/PS-induced increases in spleen inflammation, whereas neither drug significantly attenuated the PG/PS-induced liver inflammation. Although both L-NAME and AG inhibited NO production in vivo, as measured by decreases in plasma nitrite and nitrate levels, only AG was found to attenuate these values significantly (38 +/- 3 vs. 83 +/- 8 microM, respectively; P < .05). Finally, administration of L-NAME, but not of AG, significantly increased mean arterial pressure from 83 mm Hg in colitic animals to 105 mm Hg in the PG/PS+L-NAME-treated animals (P < .05). We conclude that NO may play an important role in mediating some of the pathophysiology associated with this model of chronic granulomatous colitis.

Role of mucin, mannose, and beta-1 integrin receptors in Escherichia coli translocation across Caco-2 cell monolayers.

Our previous work suggests that Caco-2 cells play an active role in bacterial translocation (BT). Since bacterial enterocyte interactions may be receptor-mediated, the current study was performed to investigate the role of beta 1 integrin and mannose receptors as well as the general protective effect of the mucous layer in this process. Caco-2 cells grown to confluence on semipermeable membranes contained in the upper compartment of a two compartment system were utilized. BT was assessed by quantitating the number of Escherichia coli crossing the monolayers after challenge with 10(8) E. coli C25. Pretreatment of the Caco-2 cells with the beta 1 integrin receptor competitive inhibitors fibronectin or RGD did not inhibit BT; while pretreatment of Caco-2 cells with the LFA-1 (lectin) receptor competitive inhibitor mannose (12 mg/ml) or purified mucin (8 mg/ml) decreased BT compared to control membranes (p < .001). Transepithelial resistance was similar among all the groups indicating maintenance of tight junction integrity. These studies suggest that E. coli BT in the Caco-2 system can be reduced by mannose and that intestinal mucin contributes to the barrier function of the monolayer.

Mechanisms of acute and chronic intestinal inflammation induced by indomethacin.

The objective of this study was to characterize the mechanisms of acute and chronic intestinal mucosal injury and inflammation induced by subcutaneously injected indomethacin (Indo). One injection of Indo (7.5 mg/kg) produced acute injury and inflammation in the distal jejunum and proximal ileum that were maximal at three days and completely resolved within one week. Two daily subcutaneous injections of Indo produced a more extensive and chronic inflammation that lasted in an active form in more than 75% of the rats for at least two weeks. Epithelial injury, as measured by enhanced mucosal permeability, was significantly elevated only at one day in the acute model (one injection) but was persistently elevated in the chronic model (two injections). Bile duct ligation completely attenuated increased mucosal permeability in the acute model, however, depletion of circulating neutrophils had no effect. Neither Indo (0-0.1 mg/ml) nor normal bile was cytotoxic to cultured rat intestinal epithelial cells; however, they synergistically promoted significant cytotoxicity. Bile collected from rats treated with Indo was cytotoxic towards the epithelial cells in a dose-dependent manner. Sulfasalazine and metronidazole (100 mg/kg/day, both) attenuated enhanced mucosal permeability in the chronic model. Massive bacterial translocation into the mesenteric lymph nodes, liver, and spleen following two injections of Indo was significantly attenuated by metronidazole. We conclude that: (1) a single injection of Indo produces acute intestinal mucosal injury and inflammation that resolve completely within three to seven days, whereas two daily injections of Indo produce both acute and chronic injury and inflammation, (2) enterohepatic circulation of Indo is important in promoting the acute phases of injury and inflammation, (3) circulating neutrophils do not play a role in the pathogenesis of this model, and (4) endogenous bacteria play an important role in exacerbating and/or perpetuating the chronic phases of injury and inflammation.