Ivar Lönnroth

The antisecretory factors: inducible proteins which modulate secretion in the small intestine

1. Cholera toxin and glucose induce the synthesis of antisecretory factors (ASF) of isoelectric points 5.0 and 4.3, respectively, and of a molecular mass of ca 60,000. 2. ASF, in nanogram amounts, inhibit intestinal secretion induced by cholera toxin, Campylobacter toxin, E. coli heat-stable toxin, C. difficile toxin A, and Dinophysis toxin. 3. Intraspinal injection of cholera toxin and glucose induces the synthesis of pituitary ASF much more effectively than does either peroral or intranasal administration. 4. Cholera toxin and glucose seem to act synergistically while inducing ASF. 5. Vagotomy abolishes both the intestinal effects of ASF and the peroral, but not the intraspinal induction of pituitary ASF. 6. ASF has no effect on ion transport across isolated intestinal mucosa from either pig or hen. 7. The results suggest that both the induction and the intestinal effects of ASF are mediated via the central and intestinal nervous system.

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 Crohns 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.

Broad Up-Regulation of Innate Defense Factors during Acute Cholera

ABSTRACT We used a whole-genome microarray screening system (Affymetrix human GeneChips covering 47,000 different transcripts) to examine the gene expression in duodenal mucosa during acute cholera. Biopsies were taken from the duodenal mucosa of seven cholera patients 2 and 30 days after the onset of diarrhea, and the gene expression patterns in the acute- and convalescent-phase samples were compared pairwise. Of about 21,000 transcripts expressed in the intestinal epithelium, 29 were defined as transcripts that were up-regulated and 33 were defined as transcripts that were down-regulated during acute cholera. The majority of the up-regulated genes characterized were found to have an established or possible role in the innate defense against infections; these genes included the LPLUNC1, LF, VCC1, TCN1, CD55, SERPINA3, MMP1, MMP3, IL1B, LCN2, SOCS3, GDF15, SLPI, CXCL13, and MUC1 genes. The results of confirmative PCR correlated well with the microarray data. An immunohistochemical analysis revealed increased expression of lactoferrin in lamina propria cells and increased expression of CD55 in epithelial cells, whereas increased expression of the SERPINA3 protein (α1-antichymotrypsin) was detected in both lamina propria and epithelial cells during acute cholera. The expression pattern of CD55 and SERPINA3 in cholera toxin (CT)-stimulated Caco-2 cells was the same as the pattern found in the intestinal mucosa during acute cholera, indicating that the activation of the CD55 and SERPINA3 genes in intestinal epithelium was induced by CT. In conclusion, during acute cholera infection, innate defense mechanisms are switched on to an extent not described previously. Both direct effects of CT on the epithelial cells and changes in the lamina propria cells contribute to this up-regulation.

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 fromClostridium 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 μg of toxin A or 3  μg of okadaic acid administered before or after injection of 0.1  μg 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.

Purification and characterization of the antisecretory factor: a protein in the central nervous system and in the gut which inhibits intestinal hypersecretion induced by cholera toxin

The antisecretory factors (ASF) are hormone-like proteins which inhibit cholera toxin-induced intestinal hypersecretion. Although ASF concentrations in young control rats were low, those in old control rats and toxin-treated rats were high. Toxin-treated rats had 200 ED50 units/g wet weight of ASF in the pituitary gland, while their intestinal mucosa, bile and milk contained 3, 0.5 and 0.5 units/g. In adult man and in 8-9-month-old pig the pituitary level was about 20 units/g. The isoelectric points of ASF from pig and rat were 4.8 and 5.0, respectively, while the molecular size as determined by gel filtration on Bio-Gel P-150 was the same in both cases (Kav 0.43). The molecular weight as determined by SDS-polyacrylamide gel electrophoresis was 60,000 for ASF from porcine pituitary gland. One ED50 unit of the purified porcine ASF corresponded to about 10(-13) mol (1-5 ng) of protein. There were two different ASF from human pituitary gland: pI 5.2, Kav 0.43; and pI 4.5, Kav 0.6. Since antibodies against porcine ASF failed to neutralize the latter protein, it may be unrelated to porcine ASF; the human pI 5.2-protein and rat ASF were both neutralized, but less effectively than was porcine ASF. All the ASF molecules attached to agarose gel, from which they dissociated again in methyl alpha-D-glucose: porcine and rat ASF were eluted at 0.3-0.9 M methyl alpha-D-glucose, human pI 5.2-ASF at 0.1-0.9 M, and human pI 4.5-ASF at 0.1-1.5 M methyl alpha-D-glucose.

Effects of the antisecretory factor in pigs

The effect of the antisecretory factor (ASF) on experimental porcine enterotoxin-induced jejunal secretion was tested. The heat-labile enterotoxin (LT) fromEscherichia coli and cholera toxin (CT) was used for challenge in ligated intestinal loops. Less than 10 units of ASF inhibited the LT-induced secretion, while that due to CT required more than 10 units of ASF. ASF was effective only when administered prior to toxin challenge, and could be given either intravenously or intra-intestinally. Mixing of ASF with specific anti-ASF antibodies prior to injection abolished its antisecretory effect. LT- and CT-induced secreted fluid contained equal concentrations of Na+, K+ and Cl−, and the ionic concentration was not affected by ASF. Less than 0.1 units of ASF per pituitary gland was present in 3- and 5-week old pigs, while it increased to 4.5 units in 28-week old animals, and to 12.2 units in pigs older than two years. However, after intra-intestinal vaccination with 2.0 mg CT, the pituitary ASF content in the 5-week old animals increased to 2.0 units within 24h.

Enterotoxins from Clostridium difficile; diarrhoeogenic potency and morphological effects in the rat intestine.

The action of toxins A, B, and C from Clostridium difficile was studied in the small intestine and colon of rats. All three caused fluid accumulation in the small intestine, maximal secretion being induced by 1 micrograms of toxin A, 20 micrograms of B, and 15 micrograms of C. Both toxins A and C caused shedding of epithelial cells from the villi without visible damage to crypt cells; toxin A caused further extensive necrosis and bleeding. Toxin B caused secretion without visible damage to the epithelial cells, though this activity was unstable and decreased significantly after one week of storage. In the colon, toxin A caused secretion and shedding of surface epithelial cells without damage to crypt cells, toxin C caused only a weak secretion, and toxin B had no effect at all. In terms of immunohistochemistry, it was found that toxin A bound to the enterocytes at the tips of the villi but not to goblet and crypt cells. The complex expression or interaction of the toxins produced by Cl difficile may explain the broad spectrum of disease (diarrhoea, colitis, and pseudomembranous colitis) associated with this micro-organism.

The antisecretory factor: synthesis and intracellular localisation in porcine tissues

Abstract 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.

Cholera toxin induces expression of ion channels and carriers in rat small intestinal mucosa

Cholera toxin causes cyclic adenosine monophosphate (cAMP)‐induced electrolyte and water secretion in the small intestine. The toxin‐induced change in gene expression in rat small intestine was evaluated with microarray technique and the results were confirmed by semiquantitative polymerase chain reaction (PCR). The transporter CNT2 for nucleosides was upregulated between 6 and 18 h after challenge, whereas the level of GLUT1 transporter for glucose became elevated at 6 h. Both changes probably facilitate uptake of these nutrients in the gut. At 18 h, the major chloride channel in the villus, ClC2, was upregulated. Aquaporin 8 was downregulated at 6 h and two mucin‐producing genes were upregulated 18 h after toxin challenge. The expression was back to normal after 72 h, which is the turnover time for intestinal epithelial cells.