Ewa Johansson

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.

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.

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.

Development of monoclonal antibodies for detection of Antisecretory Factor activity in human plasma

Antisecretory Factor (AF) is expressed in most tissues and can be demonstrated in plasma and other body fluids. Most of the AF in plasma is in an inactive form and activation of AF occurs after exposure to bacterial toxins or after intake of various dietary components. Patients with chronic diseases involving disturbances in inflammatory and secretory processes may benefit from an AF-inducing diet. The aim of the present study was to develop an in vitro assay for the analysis of AF-activity in human plasma. Monoclonal antibodies were raised against a native form of AF prepared from human placenta. Nine clones of the monoclonal antibodies recognizing AF and AF peptides were identified. With the aid of these antibodies, we developed a sensitive ELISA method for direct detection of AF-activity in human plasma. The AF activity in plasma from five healthy volunteers was low, 0.112+/-0.022 (absorbance at 405 nm), before intake of the AF-inducing diet with the SPC-Flakes, and increased significantly (p<0.05) to 0.444+/-0.068 after >or=6 weeks on the diet. A comparison of the plasma-AF values, obtained by the bioassay and the immunogenic assay (indirect ELISA), shows that there is a significant correlation (r=0.85) between the values from the two methods. The results indicate that the ELISA measures AF-activity and has the potential to be an important tool for the analysis of AF-activity in further clinical studies on AF-therapy.

Diet-induced antisecretory factor prevents intracranial hypertension in a dosage-dependent manner

Intake of specially processed cereal (SPC) stimulates endogenous antisecretory factor (AF) activity, and SPC intake has proven to be beneficial for a number of clinical conditions. The aim of the present study was to investigate the dosage relationship between SPC intake and plasma AF activity and to further correlate achieved AF levels to a biological effect. SPC was fed to rats in concentrations of 5, 10 or 15% for 2 weeks. A further group was fed 5% SPC for 4 weeks. AF activity and the complement factors C3c and factor H were analysed in plasma after the feeding period. Groups of rats fed the various SPC concentrations were subjected to a standardised freezing brain injury, known to induce increases in intracranial pressure (ICP). The AF activity in plasma increased after intake of SPC, in a dosage- and time-dependent manner. The complement factors C3c and factor H increased in a time-dependent manner. Measurements of ICP in animals fed with SPC prior to the brain injury showed that the ICP was significantly lower, compared with that of injured rats fed with a standard feed, and that the change was dose and time dependent. AF activity increases, in a dosage- and time-dependent manner, after intake of SPC. The inverse relationship between ICP after a head injury and the percentage of SPC in the feed indicate that the protective effect is, to a large extent, due to AF.

Cholera toxin protects against action by Clostridium difficile toxin A The role of antisecretory factor in intestinal secretion and inflammation in rat

The protein antisecretory factor (AF) inhibits intestinal fluid secretion induced by the cholera toxin (CT) and Clostridium difficile toxin A (CDA). The present work investigated whether CT‐induced AF protects against the enterotoxin action by CDA. Rats were pretreated perorally with CT or buffer as control, whereafter CDA‐induced fluid secretion and cytotoxicity was tested in vivo in ligated intestinal loops; the mucosal level of AF was estimated using the Western blot technique. Rats given repeated peroral doses of CT became tolerant to CDA, the inhibition of fluid secretion and of cytotoxicity being 79% in eight out of nine animals. The repeated CT‐treatment also induced long‐lasting rise of AF in the mucosal epithelium. Recombinant AF given either perorally or intravenously inhibited both fluid secretion and cytotoxicity by CDA; similar results were obtained with a truncated 16‐mer AF peptide. In conclusion: peroral CT‐treatment induced tolerance to CDA in rat small intestine. The tolerance was probably mediated by AF induced via action of cholera toxin on the enteric nervous and immune system.

Immunohistochemical staining patterns using epitope-specific antibodies indicate conformation variants of antisecretory factor/S5a in the CNS†

Antisecretory factor (AF/S5a/Rpn10) was originally identified through its ability to counteract pathological secretion. AF is also a potent anti‐inflammatory agent, a neuromodulator, and an important component of the proteasome. Human AF has a calculated molecular mass of 41 kDa and a pI of 4.7. No family of AF‐like proteins has been identified. AF has multiple functions in the cell, and different functional forms could exist as a result of post‐translational modifications. Epitope‐specific antibodies covering the entire length of AF were used to investigate whether modified forms of AF could be detected in the porcine spinal cord by Western blots, 2D gels, and immunohistochemistry (IHC). Western blot and 2D gels showed that all antisera detected a single protein with very similar molecular mass and pI. However, IHC resulted in an epitope‐specific subcellular staining pattern. Antisera recognizing epitopes in the N‐terminal part of AF, containing the antisecretory activity, showed a more restricted localisation than antisera directed at the C‐terminal part, containing the ubiquitin‐binding sites. We suggest that AF can exist in several conformational variants, perhaps due to differences in redox state and/or pH in the various cellular compartments. Such conformational changes could be of functional importance.

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 intestinalcapillary function, utilizing the Evans blue dye method,was analyzed. The modulatory influence of plasma-derivedor recombinant human antisecretory factor on this variable was also investigated. MaleSprague-Dawley rats were briefly anesthetized withether, and a jejunal loop was constructed that waschallenged for 90 min with phosphate-buffered saline or cholera toxin. Five minutes prior to death, therats received an intravenous injection of Evans blue.The tissue content of dye in the loop was quantitatedspectrophotometrically or demonstrated histochemically. Cholera toxin increased the recovery of Evansblue; extravasation of the dye was prominent in the topof the villi, while the crypts were spared. It issuggested that the toxin caused increased transcapillary permeation of albumin in a heterogenous fashionin the gut wall. This effect of the toxin was preventedby pretreatment with the antisecretory factor.

Genotyping of Pseudomonas aeruginosa isolates from lung transplant recipients and aquatic environment- detected in-hospital transmission

Lung infection with Pseudomonas aeruginosa is common in lung transplant recipients and may lead to severe complications. Bacteriological surveillance aims to detect transmission of microbes between hospital environment and patients. We sought to determine whether genotyping of P. aeruginosa isolates could improve identifications of pathways of infection. From 2004 to 2009, we performed genotyping with multiple‐locus variable number of tandem repeats analysis (MLVA) and pulsed‐field gel electrophoresis (PFGE) of P. aeruginosa isolates cultured from lung transplant recipients at Sahlgrenska University Hospital, Gothenburg. During a small outbreak in 2008, cultivation and genotyping of isolates from sink and drains samples from the hospital ward were performed. Pseudomona aeruginosa from 11/18 patients were genotyped to unique strains. The remaining seven patients were carriers of a P. aeruginosa strain of cluster A genotype. Pseudomona aeruginosa was isolated in 4/8 water samples, typed by MLVA also as cluster A genotype and confirmed by PFGE to be similar or identical to the isolates from four transplanted patients. In conclusion, genotyping of isolates revealed a clonal relationship between patient and water isolates, indicating in‐hospital transmission of P. aeruginosa. We suggest genotyping with MLVA for rapid routine surveillance, with the PFGE method used for extended, confirmatory analyses.