Anti-Inflammatory Substances in Wheat Malt Inducing Antisecretory Factor.

Extensively malted cereals counteract enterotoxic diarrhea and inflammatory bowel diseases. This effect depends on a protein called antisecretory factor (AF), which is secreted into the blood as a larger complex known as the compleasome. In this study, we identified anti-inflammatory substances in malt and assayed their capacity to induce AF. Guaiacol and quercetin inhibited inflammation in a mouse footpad model, while catechin, sinapic acid, ferulic acid, and quercetin inhibited nitric oxide formation in RAW 264.7 cells. The proteasome activity in these cells was inhibited by vanillic acid and quercetin but not by the other tested phenols. As the transient receptor potential vanilloid 1 (TRPV1) might be involved in AF induction, the TRPV1 antagonist capsazepine was tested and shown to inhibit inflammation in mouse paw and nitric oxide formation. Catechin, ferulic acid, and sinapic acid induced AF in rat blood, and these substances were all increased in malt compared to control wheat. These phenols might therefore be of particular importance for the beneficial effect of malted cereals on inflammatory diseases. Our results further suggest that TRPV1 might play a role in the anti-inflammatory activity of phenols via the induction of AF.

Induction of anti-secretory factor in human milk may prevent mastitis.

AIM: The aim of the study was to try to induce anti-secretory factor (AF) in human milk and possibly prevent mastitis. METHODS: Forty mothers who had normal deliveries and healthy full-term infants were randomly divided into two groups, 3-7 days postpartum. The experimental group received a food inducing AF. The control group received the same type of food, without AF-inducing properties. Milk was tested for AF after the mothers had eaten the cereals for 4-5 wk. AF was determined by intravenous injection of milk samples into rats measuring their capacity to prevent secretion into a gut loop of the rat injected with cholera toxin. RESULTS: The median levels of AF differed between the experimental (n = 12) and control groups (n = 16): 1.1 (0.7-1.25) units vs 0.1 (0.0-0.25) units, Z = -4.492, p < 0.0001 (11 mothers dropped out and one milk sample is missing from one of the control mothers). The frequency of mastitis in the experimental compared with the control group was reduced (p = 0.0086, permutation test). The median AF levels in mothers with or without mastitis differed; 0.0 (0.0-0.1) vs 0.5 (0.2-1.1), Z = -2.399, p = 0.017. CONCLUSION: We suggest a specially treated cereal induces AF in human milk and protects against clinically manifested mastitis.

Increased antisecretory factor reduces vertigo in patients with Ménière's disease: a pilot study.

It has been hypothesized that the symptoms of vertigo in patients with Ménière's disease somehow are related to impaired production and/or transport of endolymph. Antisecretory factor (AF) is a protein known to affect transport processes in the intestine and it has been shown that intake of specially processed cereals (SPC) can increase endogenous AF synthesis. In a prospective open pilot study, 24 patients with severe Ménière's disease (functional level scale 5-6 according to the criteria of AAO-HNS) received SPC for 14-30 days. AF levels in plasma increased by 83% in 20 of the 24 patients studied. The attacks of rotatory vertigo were reduced, to final AAO-HNS functional level scale 1-3, in 12 patients and in three of these hearing was normalized. Twelve patients had no or minor effects of the treatment. The correlation between AF activity after treatment and the final AAO-HNS functional level scale was -0.65, P<0.001. Studies in rats using immunohistochemistry methods showed that AF was localized to the cochlea and the vestibule of the inner ear. The present results suggest that AF might be a new regulator of the endolymph.

Antisecretory factor peptide derivatives specifically inhibit [3H]-gamma-amino-butyric acid/36Cl- out-->in permeation across the isolated rabbit Deiters' neuronal membrane.

AIMS: Antisecretory factor (AF) is a 41-kDa protein, its main function being the regulation of intestinal ion/water transport, but it also inhibits chloride and gamma-amino-butyric acid transport across nerve cell membranes. The present experiments were designed to evaluate whether the same AF peptide sequence mediates the permeability effects seen at the nerve cell membrane and in the rat small intestine. METHODS: Four peptides were prepared by the solid phase technique with sequences derived from positions 1-51 of the full-length antisecretory factor AF and tested on nerve cell membranes isolated from rabbit Dieter cells. RESULTS: AF peptides containing the active 36-51 peptide exerted a blocking effect of the out-->in permeation of 36Cl- as well as of [3H]-gamma-amino-butyric acid. The minimal dose causing inhibition, however, varied between 10(-11) m (AF10) and 10(-7) m (AF13). The most potent peptides have been shown previously to be active in inhibiting experimental diarrhoea in vivo in small intestinal ligated loops in rats. The non-active sequence AF23-32 did not inhibit any of the two permeation markers in vitro, a result which supports the lack of activity found also in vivo. CONCLUSION: The results suggest that AF, or AF derivatives, counteract intestinal hypersecretion by blocking anion permeation across large anionic pores. Such a blocking effect could also influence the generation of action potentials in enteric nerve cells controlling the intestinal water and ion transport system.

The antisecretory factor: synthesis, anatomical and cellular distribution, and biological action in experimental and clinical studies.

The antisecretory factor (AF) is a 41-kDa protein that provides protection against diarrheal diseases and intestinal inflammation. Its cDNA has been cloned and sequenced. AF is highly potent, with 10(-12) mol of recombinant AF being sufficient to counteract experimentally induced diarrhea in rat. The antisecretory activity is exerted by a peptide located between positions 35 and 50 of the AF sequence. Synthetic peptides based on this sequence are promising candidates for drugs to counteract intestinal hypersecretion, as well as imbalances of fluid transport in other body compartments. AF probably exerts its effects via nerves; AF immediately and potently inhibits ion transport across isolated nerve membranes from Deiters' cells. Immunocytochemistry has shown that AF is present in most tissues in the body, and in situ nucleic acid hybridization has shown that cells that store AF are also capable of AF synthesis. The endogenous plasma level of AF is increased by enterotoxins and by certain food constituents such as hydrothermally processed cereals. These cereals significantly improve clinical performance in patients suffering from inflammatory bowel diseases. AF-enhancing food also protects domestic animals against diarrheal diseases, and such feed has been used successfully in Swedish swine farming for the past 10 years. Increased understanding of AF action might result in expanded clinical applications and confirm that AF is an important regulator of homeostasis.

Food induced stimulation of the antisecretory factor can improve symptoms in human inflammatory bowel disease: a study of a concept.

BACKGROUND: Antisecretory factor (AF), a 41 kDa cloned and sequenced protein, suppresses intestinal inflammation and hypersecretion in animals. Endogenous AF production can be induced by dietary modifications in several animal species, and this feed has been shown to reduce the incidence of diarrhoeal disease in weaning piglets. The role of AF in intestinal disease in humans is not known. AIMS: To study the effects of hydrothermally processed cereals, optimised for AF induction in animals, added to the diet of patients with longstanding symptoms of inflammatory bowel disease (IBD). PATIENTS: Fifty three patients with IBD (ulcerative colitis and Crohn's disease) were entered into the study, and 50 completed follow up. The experimental group consisted of 16 females (mean age 50 (SEM 5) years) and 10 males (41 (4) years) and the placebo group of 12 women (41 (4) years old) and 12 men (51 (5) years). METHODS: Patients were randomised to receive either hydrothermally processed cereals (active treatment) or the same amount of ordinary cereals (placebo treatment) for four weeks in a double blind study design. Baseline diet and medications remained unchanged. Bowel symptoms, plasma levels of AF, and colonic biopsies were evaluated before and after treatment. RESULTS: The active treatment significantly improved subjective ratings of clinical symptoms and increased plasma AF levels compared with placebo. Plasma lipid levels were unaffected. CONCLUSION: Hydrothermally processed cereals can induce AF production in human IBD. This increase in endogenous AF activity is associated with clinical improvement. Further studies are warranted to clarify the exact role of AF in human intestinal disease.

The antisecretory factor: synthesis and intracellular localisation in porcine tissues.

The antisecretory factor, AF, is a 41-kDa protein, cloned and sequenced from a human pituitary library. AF is a potent inhibitor of experimental intestinal hypersecretion in rats and pigs. An antiserum against the C-terminal of the truncated, recombinantly produced AF protein was raised in rabbits. The affinity-purified antiserum was used to study the expression of AF in mucosal membranes and in the pituitary gland of the pig; distinctly stained cells were found in lymphoid cells in the connective tissue of all parts of the gastrointestinal, respiratory and urinary tracts. Cytoplasmic AF was demonstrated in endocrine and epithelial cells in the pituitary gland. In situ hybridisation with a digoxigenin-labelled mRNA probe also demonstrated specific cytoplasmic staining in epithelial and lymphoid cells in all of these tissues. The cells stained by either method were similarly distributed topographically within the tissues. The results suggest that a specific defined cell population in these various tissues possesses the capability of both synthesising and storing the AF protein within the cellular cytoplasmic compartment.

Recombinant or plasma-derived antisecretory factor inhibits cholera toxin-induced increase in Evans blue permeation of rat intestinal capillaries.

The effect of cholera toxin on small intestinal capillary function, utilizing the Evans blue dye method, was analyzed. The modulatory influence of plasma-derived or recombinant human antisecretory factor on this variable was also investigated. Male Sprague-Dawley rats were briefly anesthetized with ether, and a jejunal loop was constructed that was challenged for 90 min with phosphate-buffered saline or cholera toxin. Five minutes prior to death, the rats received an intravenous injection of Evans blue. The tissue content of dye in the loop was quantitated spectrophotometrically or demonstrated histochemically. Cholera toxin increased the recovery of Evans blue; extravasation of the dye was prominent in the top of the villi, while the crypts were spared. It is suggested that the toxin caused increased transcapillary permeation of albumin in a heterogenous fashion in the gut wall. This effect of the toxin was prevented by pretreatment with the antisecretory factor.

Purification and identification of Haemophilus ducreyi cytotoxin by use of a neutralizing monoclonal antibody.

Haemophilus ducreyi produces a cytotoxin responsible for the killing of cultured human epithelial cells. Cytotoxin-neutralizing antibodies were detected in the majority of sera from patients with culture-proven chancroid, and a significantly higher level of such antibodies in patients than in blood donors was noted both in areas where the disease is endemic and those where it is not. We produced neutralizing monoclonal antibodies (MAbs) in mice with a crude osmotic preparation of the cytotoxin. These antibodies, with high capacity to neutralize cytotoxicity, were used for purification and identification of the cytotoxin. Purification was performed by a two-step procedure which included Sephacryl S-200 filtration followed by immunoaffinity chromatography. The purification resulted in poor cytotoxin protein recovery and contamination with MAbs from the affinity column. The results of the gel filtration experiments and immunoblotting indicate that the active cytotoxin consists of a single, small protein with an approximate molecular mass of 20 kDa. Cytotoxins from different strains seem to have the same or similar epitopes. The cytotoxin protein was not detected in preparations from nontoxic strains. The N-terminal amino acid sequence of the 20-kDa band was E-S-N-P-D-P-T-T-Y-P-D-V-E-L-S-P-P-P. This sequence does not resemble that of any currently known bacterial protein.

Identification of an active site in the antisecretory factor protein.

The antisecretory factor (AF) is a new regulatory protein, produced in the human pituitary gland, which reverses intestinal fluid secretion induced by cholera toxin. We have previously described the cDNA-cloning and characterization of the expressed gene. The aim of this study was to identify the region responsible for the antisecretory activity in the AF-molecule. The recombinant full-length AF has an increased ability to inhibit hypersecretion after treatment with trypsin, indicating that the activity of AF is achieved by smaller peptide fragments. To localize the active region of AF, we expressed truncated forms of the recombinant protein and examined their antisecretory activity against cholera toxin-induced fluid secretion in rat. Nine recombinant AF peptides and four smaller peptides made by solid phase synthesis were tested. Five of the peptides lacked all activity, whereas seven of them were highly active, a dose between 4 and 15 pmol causing a half-maximal inhibition. All the active peptides contained amino acid 36-42 of the AF sequence, whereas none of the inactive peptides contained this sequence. Our results suggest that the site of the antisecretory activity resides in a small region (I)VCHSKTR between position 35 and 42 of the AF molecule.

Antisecretory factor suppresses intestinal inflammation and hypersecretion.

BACKGROUND: Antisecretory factor (AF) is a recently identified regulatory protein which inhibits the intestinal fluid secretion induced by cholera toxin. AIMS: To test the effect of AF on: (a) inflammation and hypersecretion induced by toxin A from Clostridium difficile; and (b) morphological changes and hypersecretion induced by okadaic acid (the blue mussel toxin) in rat intestinal mucosa. METHODS: Morphological changes and fluid accumulation were observed in intestinal loops challenged with 1 microgram of toxin A or 3 micrograms of okadaic acid administered before or after injection of 0.1 microgram of recombinant AF (rAF). RESULTS: The cytotoxic and inflammatory reaction caused by toxin A was abolished after treatment with rAF given either intraveneously or intraluminally prior to the toxin or one hour after the toxin. The intestinal fluid response induced by toxin A and okadaic acid was reduced 55-80% by rAF. However, the characteristic increase in goblet cells at the tips of villi in the okadaic acid treated mucosa was not inhibited by rAF. CONCLUSION: Results suggest that AF might be involved in protection against inflammation and in counteracting dehydration caused by enterotoxins. Both effects are probably mediated via the enteric nervous system.

Molecular mimicry in the recognition of glycosphingolipids by Gal alpha 3 Gal beta 4 GlcNAc beta-binding Clostridium difficile toxin A, human natural anti alpha-galactosyl IgG and the monoclonal antibody Gal-13: characterization of a binding-active human glycosphingolipid, non-identical with the animal receptor.

Glycoconjugates with terminal Gal alpha 3Gal beta 4GlcNAc beta sequences have been shown to be recognized by three carbohydrate-binding proteins; toxin A of Clostridium difficile, human natural anti alpha-galactosyl IgG and the monoclonal antibody Gal-13. However, the biological significance of this binding specificity in humans is unclear, since unsubstituted Gal alpha 3Gal beta 4GlcNAc beta sequences are not found in human tissues, due to suppression of the gene coding for the enzyme Gal beta 3-transferase. To explore this inconsistency, the binding of toxin A, human natural anti alpha-galactosyl IgG, and the Gal-13 monoclonal antibody to various glycosphingolipids was examined using the thin-layer chromatogram binding assay. The binding to Gal alpha 3Gal beta 4GlcNAc beta-terminated glycosphingolipids of rabbit erythrocytes was confirmed. A minor binding-active compound was also detected in the non-acid glycosphingolipid fraction of human erythrocytes. This glycosphingolipid was isolated and characterized by EI mass spectrometry, gas chromatography-EI mass spectrometry after degradation, and proton NMR spectroscopy, as GalNAc beta 3Gal beta 4GlcNAc beta 3Gal beta 4Glc beta 1Cer, corresponding to the x2 glycosphingolipid isolated before form this source. Two additional binding-active glycosphingolipids were found. One was GalNAc alpha 3Gal beta 4GlcNAc beta 3Gal beta 4 Glc beta 1 Cer, produced from blood group A-active GalNAc alpha 3 (Fuc alpha 2) Gal beta 4GlcNAc beta 3Gal beta 4Glc beta 1 Cer by acid-induced defucosylation. The other was GlcNAc beta 3 Gal beta 4 GlcNAc beta 3 Gal beta 4 Glc beta 1 Cer, generated from NeuGc alpha 3Gal beta 4GlcNAc beta 3Gal beta 4GlcNAc beta 3Gal beta 4Glc beta 1Cer by enzymatic hydrolysis. A number of other glycosphingolipid sequences, including the human Le(x), Le(y), and I blood group determinants, suggested to act as receptors for toxin A, were not recognized by the three ligands. Despite the different terminal substituents and anomerity of the binding-active glycosphingolipids, calculated minimum energy conformations demonstrated topographical similarities in the spatial orientation of the terminal trisaccharides, possibly accounting for the cross-reactivity.

Molecular cloning and expression of a pituitary gland protein modulating intestinal fluid secretion.

Antisecretory factor (AF) is a protein known to inhibit intestinal fluid secretion induced by cholera toxin. cDNA clones, expressing immunoreactivity to AF were isolated from a human pituitary gland library and sequenced. The sequence contained 1309 base pairs plus a poly(A) tail; Northern blot analysis of pituitary RNA confirmed this size. One large open reading frame was found to code for 382 amino acids. The protein was expressed in pGEX-lambda 1T/Escherichia coli and purified. The recombinant AF was extremely potent, 9 ng (2.10(-13) mol), giving a significant antisecretory activity against cholera toxin-induced fluid secretion in rat. Antiserum against recombinant AF was used in immunohistochemical and Western blot analysis. Sections from human pituitary glands manifested specific intracellular staining in cells exclusively located in the anterior part. Both recombinant AF and AF extracted from pituitary gland appeared in SDS-polyacrylamide to have a molecular mass of 60 kDa, although the renal value was 41 kDa. The protein sequence manifested homology (29% identity) with one protein, a putative Saccharomyces cerevisiae 30-kDa protein of unknown function.

Concentrations of antisecretory factor in eggs and in chicken blood plasma.

1. In the present study, the concentration of antisecretory factor (ASF), a lectin with hormone-like properties, and with the capacity to regulate water and electrolyte transport in the small intestine, was higher in hen's egg yolk than in egg white (1.20 v. 0.46 units/ml). 2. The blood plasma concentrations of ASF activity were higher in d-old chicks (1.03 units/ml) than in 7-d-old (0.42 units/ml) or 21-d-old birds (0.18 units/ml); the contents were found to be high again in 35-d-old chickens, but to have decreased by 60 to 90% after transport to slaughter 1 day later. 3. The amount of ASF activity was lower in two groups of chickens manifesting patent loose droppings at slaughter (0.15 units/ml in one group, and 0.08 units/ml in the other), than in two groups with normal faecal consistency (0.65 units/ml in one group, and 0.72 units/ml in the other). 4. The results demonstrate the presence of ASF in eggs, and a variation of ASF activity in chickens blood plasma in relation to age, stress and faecal consistency. The interpretation of these data suggests a regulatory influence of ASF on chicken intestinal transport of water.

Antisecretory factor enhances in vivo internalization of cholera toxin and of horseradish peroxidase into rat enterocytes.

The in vivo effect of antisecretory factor (ASF, derived from pig plasma) on the ability of cholera toxin (CT) and of horseradish peroxidase (HRP) to bind to and penetrate into epithelial cells of the rat small intestine was evaluated in the absence of anesthetics. The potency of intravenously administrated ASF was demonstrated by some 70% inhibition of CT-induced secretion in ligated small intestinal loops. Using immunohistochemical methods for visualization, we found ASF to enhance internalization of both CT and HRP after 30 to 60 min of challenge, without interfering with the initial binding to the enterocyte brush border region. The internalization process started in the upper 2/3 of the villus region. After 5 h, no CT or HRP could be seen bound to the enterocytes. The results suggest that ASF might enhance small intestinal absorption.

Feed-induced lectins in piglets. Feed-induced lectins and their effect on post-weaning diarrhoea, daily weight gain and mortality.

The effect on various biological variables of a special weaner feed (FD) with the capacity to induce endogenous synthesis of feed induced lectins (FIL), and a commonly used commercial diet (CD) was evaluated in 891 piglets in four different experiments. The FD diet was found to induce a high activity of FIL (FIL = 0.94 +/- 0.03 units/ml. blood), as compared to CD (FIL < 0.20 units/ml. blood). The FIL values did not decrease after weaning. The incidence of diarrhoea in piglets fed FD 0-14 days post-weaning was 12 +/- 5 per cent, as compared to 41 +/- 9 per cent in the CD group, the difference being significant. Daily weight gain 0-35 days after weaning was 67 +/- 15 grams greater in the FD group than among those fed CD. The consumption of FD was double that of CD in the period before weaning (at 5 weeks), and some 40 per cent more in the postweaning period up to 9 weeks of age.

Plasma level of antisecretory factor (ASF) and its relation to post-weaning diarrhoea in piglets.

Antisecretory factor (ASF) is a regulatory peptide which counteracts diarrhoea in the pig; ASF is rapidly absorbed from the pig intestine, and significantly reduces the incidence of neonatal diarrhoea in the suckling offspring. ASF is synthesized in the central nervous system, and released to the blood stream via the pituitary gland. In two different experiments (n = 8 and n = 4), the blood concentration of ASF was followed in 5-weeks old piglets from day 7 before weaning up to day 12 days after weaning. In both experiments ASF concentrations were significantly (p < 0.01) lower on day three post-weaning, than either before weaning or on days 7 and 12 post-weaning. In another experiment, where plasma ASF activity was determined in relation to clinical signs of diarrhoea seven days post-weaning, it was found to be 0.87 +/- 0.08 units/ml (mean +/- SEM) in healthy weaners (n = 15), but only 0.22 +/- 0.05 units/ml in piglets suffering from diarrhoea (n = 15), the difference being significant. The faecal flora both of healthy weaners and of their matched controls suffering from diarrhoea were subjected to bacteriological examination before and after weaning, and found to be similar in both groups, namely a mixture of aerobic and anaerobic Gram negative rods, Campylobacter jejuni, Staphylococcus aureus/epidermidis, and Enterococcus faecalis. No particular pathogen was predominant in any of the diseased animals.

Induction of anti-secretory factor in mice by the nonintestinal parasite Schistosoma mansoni.

Anti-secretory factor (ASF) was determined by the rat intestinal loop model in the pituitary gland and in the blood at various intervals after experimental infection of mice with Schistosoma mansoni. ASF could be detected in the pituitary gland by 6 hr after percutaneous infection with cercariae and in the blood at 24 hr after infection. A peak level was reached 3 days after infection and, after a decline, 2 more peaks were noted between 4 and 8 wk after infection. Characterization of ASF induced by S. mansoni by means of isoelectric focusing revealed 2 distinct peaks at approximately pH 4 and pH 5, respectively. Upon affinity chromatography on agarose, the activity dissociated in a gradient of methyl-alpha-D-glucoside at 1.0 M but not at 0.1 or 0.3 M concentrations.

Clostridium difficile toxin A induces a specific antisecretory factor which protects against intestinal mucosal damage.

Peroral challenge with toxin A from Clostridium difficile induced the formation of antisecretory factor in rats. The animals were given 100 micrograms of the toxin, which was followed by a pronounced diarrhoea and by the appearance of antisecretory factor in the pituitary gland. In electrofocusing, the induced antisecretory factor separated in two peaks (pI 5.4 and 5.0); both fractions showed a lectin-like binding to agarose. The pI 5.4 fraction inhibited cholera toxin as well as toxin A induced fluid secretion, while pI 5.0 inhibited toxin A induced secretion only. Immunohistochemistry showed that an antisecretory factor of pI 5.0 protected the mucosa from the cytotoxic effect of toxin A, but did not affect the binding of toxin A to the intestinal epithelium. Sodium dodecyl-sulphate-polyacrylamide gel electrophoresis of the pI 5.0 protein showed two major fractions to be present, one of molecular weight 60 kDa, the other of 30 kDa, the latter probably being a degradation product of the former.