Introduction
Antisecretory factor (AF) is a 41 kDa endogenous protein which was originally purified in the pig pituitary gland by Lönnroth et al. (Reference Lönnroth, Lange and Skadhauge1) and since then its gene has been cloned and sequenced(Reference Johansson, Lönnroth and Lange2). It has been established that AF is synthesised in most tissues of mammals and can be found linked to the 26S proteasome or free in the cytoplasm and/or nucleus(Reference Lange, Jennische and Johansson3). AF has been shown to inhibit enterotoxin-induced intestinal hypersecretion in both human and animal subjects(Reference Johansson, Jennische and Lange4, Reference Lange and Lönnroth5). In a rat intestinal loop model, 1 pmol of purified AF caused a significant reduction of cholera toxin (CT) and Escherichia coli toxin-induced secretion(Reference Lange6). Rapallino et al. (Reference Rapallino, Cupello and Lange7) hypothesised that AF may act at the level of intestinal epithelial cells, by regulating chloride homeostasis, thereby possibly counteracting excessive secretion of fluids into the intestinal lumen. The immunohistological distribution of AF suggests that this protein may play a role in the immune system as an anti-inflammatory agent(Reference Davidson and Hickey8). In rat jejunal loops, AF inhibited the inflammatory response caused by toxin A from Clostridium difficile (Reference Johansson, Jennische and Lange4). Intestinal exposure to bacterial toxins(Reference Johansson, Jennische and Lange4) or intake of specific dietary compounds has proved to enhance the antisecretory and anti-inflammatory activity of endogenous AF(Reference Björck, Bosaeus and Ek9).
Nowadays, diarrhoeal diseases remain a major global health concern, especially in developing countries where the transmission of infectious gastrointestinal pathogens is favoured by poor hygienic conditions. The 4th Millennium Goal of the UN stated that between the years 1990 and 2015 childhood diarrhoea should be reduced by two-thirds(10). Recent reports state that childhood diarrhoea is responsible for 18 % of all child deaths; it is estimated that two million children die each year due to excessive fluid loss, despite the introduction of oral rehydration therapy(10, Reference Bryce, Boschi-Pinto and Shibuya11).
Not only is diarrhoeal disease a problem in developing countries, but also it remains an issue of wealthy countries, due to a high incidence of chronic gastrointestinal diseases.
Induction of endogenous AF through nutritional means, in addition to rehydration therapy and/or pharmacological therapy, has been proved to noticeably reduce fluid loss and bowel movements, resulting in an improvement of patients' well-being(Reference Björck, Bosaeus and Ek9, Reference Zaman, Mannana and Lange12, Reference Laurenius, Wängberg and Lange13). A patented hydrothermal process allows cereal kernels to reach an optimal concentration of specific amino acids and oligosaccharides, able to induce endogenous AF secretion(Reference Lange and Lönnroth5). These specially processed cereals (SPC) are purified and available under registered trademarks. Included in the diet of patients affected by intestinal chronic inflammatory diseases, SPC improve illness symptoms, reducing daily bowel movements and tissue damage(Reference Lange and Lönnroth5).
In livestock production, the efficacy of AF-inducing diets could be of great interest, especially as a tool to counteract post-weaning diarrhoea syndrome in piglets. Indeed, weaning is the most stressful event in piglets' lives, reared in intensive piggeries. Weaning is frequently associated with a reduced nutrient intake, severe growth check, impairment of health status and a higher susceptibility towards infections and gut disorders(Reference Lallès, Bosi and Janczyk14, Reference Berkeveld, Langendijk and Soede15).
For more than 50 years, the use of antimicrobial growth promoters (AGP) in animal diets improved growth performance and prevented the side effects of early weaning practice. Increasing concerns about the risk of antibiotic resistance transmission, associated with the widespread agricultural use of antimicrobials, led the European Union to a complete ban of AGP since 1 January 2006. As experienced by the Nordic countries, which banned AGP during the 1980s and 1990s, the main consequences of the elimination of AGP were increased morbidity and mortality of animals, due to an increased incidence of infectious gastrointestinal and respiratory diseases. This has led to an intensification of research to find alternatives to in-feed antibiotics.
AF-inducing diets seem to be interesting alternatives to AGP and a useful tool to improve livestock efficiency, animal health and welfare.
The aim of the present review was to summarise the information about the AF protein and its derivatives, evidencing the beneficial effects of AF-inducing diets on both human and animal health and welfare.
The antisecretory factor molecule
Genetics
The AF protein was first purified in rodent extracts of the pituitary gland and intestinal mucosa and later in pig blood(Reference Lönnroth, Lange and Skadhauge1–Reference Lange, Jennische and Johansson3). AF protein is evolutionarily highly conserved and it appears to be a unique protein since no family of AF-like proteins has been identified(Reference Lange and Lönnroth5).
Genes coding for AF protein are present in all mammals and homologues of AF genes have been found only in eukaryotic organisms. In the human genome, potential AF genes are expressed in chromosomes 1, 19 and 23, with pseudogenes located on chromosomes 10 and 15(Reference Lange and Lönnroth5).
The sequence responsible for the antisecretory activity is present only in mammalian proteins while in other eukaryotic organisms the proteins codified by the AF genes act as regulators of proteolytic degradation in the proteasome(Reference Johansson, Lönnroth and Lange2, Reference Lange and Lönnroth5). AF protein from plasma is structurally different from the one obtained from the pituitary gland; Lange & Lönnroth(Reference Lange and Lönnroth5) showed that the first is polymeric, while the second is monomeric.
Active site
Post-translational modifications of AF, such as proteolysis, glycosylation, phosphorylation, or conformational changes, could result in the exposure or hiding of active sites, essential for the exercise of the biological activity(Reference Lange and Lönnroth5, Reference Jennische, Hanner and Johansson16).
Trypsin digestion of the AF protein enhanced two-fold the capability to counteract CT-induced intestinal secretion, indicating that in vivo AF might be activated by hydrolysis(Reference Johansson, Lange and Lönnroth17). The amino acid sequences derived from recombinant human AF trypsin digestion were tested in a rat intestinal loop model in order to identify the active domain of the protein. The antisecretory activity of the truncated forms was related to the presence of a specific amino acid sequence between residues 35′ and 50′ in the N-terminal part; the antisecretory potency was found to be dose dependent. According to Johansson et al. (Reference Johansson, Lange and Lönnroth17), the active region of mammalian AF is probably exerted by a small peptide of eight amino acids (35-IVCHSKTR-42). Compared with other peptides with antisecretory activity, AF appears to be the most potent, since somatostatin is active at nanomolar and enkephalines at micromolar concentrations, while AF is active at 1 pmol dosages(Reference Johansson, Lange and Lönnroth17), since only 10− 12 mol of AF are needed to block CT-induced hypersecretion in a rat intestinal loop model.
The smallest molecule with antisecretory and anti-inflammatory properties is AF-16, a sixteen amino acid-long peptide comprising the sequence VCHSK TRSNP ENNVG L, synthesised using the solid-phase technique(Reference Johansson, Lange and Lönnroth17).
Receptors
AF is present in most body tissues; it can be found in free form in the cytoplasm and/or nucleus or linked to the 26S proteasome(Reference Lange, Jennische and Johansson3).
Bioinformatic models suggest that the AF protein has a von Willenbrand-like motif in the N-terminal part, between residues 5 and 188, which contains the antisecretory sequence(Reference Johansson, Jonson and Bosaeus18). The von Willenbrand-like motif proteins are protein binders; Johansson et al. (Reference Johansson, Jonson and Bosaeus18) hypothesised that AF might exert its biological activities by binding to the receptor with its N-terminal part.
With the C-terminal part, AF binds to polyubiquitin, an intracellular protein, via two sites localised between residues 211–230 and 282–301, without involving the von Willenbrand-like motif. This interaction is probably involved in the regulation of the proteolytic processes in the proteasome(Reference Johansson, Jonson and Bosaeus18).
Sections from rat brain, double-labelled with antibodies against AF and flotillin-1, showed that there is a high degree of co-localisation between AF and flotillin-1 in neurons and the plexus choroideus (Reference Johansson, Jonson and Bosaeus18). Dot-blot analysis has shown that AF interacts with flotillin-1 by the means of its N-terminus, carrying the anti-inflammatory and anti-secretory active site.
The flotillin protein family is phylogenetically well preserved and consists of two proteins, flotillin-1 and flotillin-2(Reference Vassileva, Ivanov and Nusrat19). Flotillins are ubiquitarious: they have a role in endocytosis, participate in signalling, regulate actin cytoskeleton reorganisation and actin-dependent cell adhesion as well as motility(Reference Vassileva, Ivanov and Nusrat19–Reference Babuke and Tikkanen21).
Flotillin-1 is an integral membrane protein component of the lipid rafts(Reference Johansson, Jonson and Bosaeus18), specialised cell membrane micro-domains rich in cholesterol, sphingolipids and gangliosides(Reference Allen, Halverson-Tamboli and Rasenick22, Reference Rossy, Schlicht and Engelhardt23). In the lipid rafts there are proteins of key importance for cohesion and signal transduction such as receptors, cytoskeletal contacts and ion channel complexes(Reference Katoh, Kamimoto and Yamakawa24).
The lipid rafts appear to play a key role in the assembly of signalling processes and to mediate many crucial cellular processes as endocytosis, oxidative stress, apoptosis, ion homeostasis and protein membrane trafficking and turnover(Reference Allen, Halverson-Tamboli and Rasenick22–Reference Morgan, Kim and Liu25). Though lipid rafts make up a small percentage of the cell membrane, their high concentration of molecules involved in cellular signalling makes them a natural target for bacteria and viruses(Reference Van der Goot and Harder26). CT attaches to the intestinal epithelium by binding to ganglioside GM1, which is localised in the lipid rafts as flotillin-1(Reference Li, Mak and Richards27). Johansson et al. (Reference Johansson, Jonson and Bosaeus18) suggested that the interaction between AF and flotillin-1 could affect secretory processes by regulating the localisation of signal proteins, such as receptors, in the lipid rafts.
AF and flotillin-1 are also co-localised in the central nervous system (CNS) and their interaction might affect the AF modulation of glutamatergic and γ-aminobutyric acid (GABA)ergic synaptic transmissions(Reference Johansson, Jonson and Bosaeus18).
Effect of antisecretory factor on the immune system
In situ hybridisation and immunohistochemical analysis has demonstrated that AF is expressed in various mammalian organs(Reference Lange, Jennische and Johansson3, Reference Davidson and Hickey8).
Monoclonal TLD antibodies, obtained from rats immunised with cultured microglial cells, were screened by Davidson & Hickey(Reference Davidson and Hickey8) with the aim to investigate the roles of perivascular and microglia cells in the inflammatory processes in the CNS.
TLD-1A8A is a monoclonal antibody that appears to specifically recognise the AF molecule cloned from rat microglia; administration of TLD-1A8A in T cell proliferation or mixed-leucocyte response assays increased T cells proliferation(Reference Davidson and Hickey8). T cell proliferation assays performed with rat serum containing AF showed that AF blocked the TLD-1A8A effect on T cells, suggesting a role for AF in the regulation of T cell responses(Reference Davidson and Hickey8).
However, the effects on the immunological response observed in vitro did not clarify the nature of the interaction between AF and its antibody. Davidson & Hickey(Reference Davidson and Hickey28) hypothesised the existence of a receptor for AF on the surface of T cells which might be disrupted by the antibody, blocking the normal suppressing signal. Alternatively, the binding of T cell membrane-associated AF to the antibody might activate the antigen-presenting cells determining T cell proliferation(Reference Davidson and Hickey8). Immunohistochemical analysis performed on rat tissue sections treated with TLD-1A8A showed that in lymphoid organs, such as the spleen, thymus and lymph nodes, AF was highly expressed by macrophages and cells with dendritic morphology. In the gut, AF was expressed by scattered macrophages in the connective tissue of the villi and particularly in the Peyer's patch, important in the immune surveillance of the intestinal tract. In the CNS, AF was found in blood vessels and perivascular cells, a bone marrow-derived cell population with immunoregulatory activity(Reference Davidson and Hickey8, Reference Hickey and Kimura29). Davidson & Hickey(Reference Davidson and Hickey28) observed that macrophages expressed AF even in absence of a direct immunological stimulus, even if the activation of T cells induced an up-regulation of AF expression in macrophages.
The immunological stimulus, due to experimentally induced autoimmune encephalomyelitis in rats, modified the AF expression level and distribution in the inflamed nervous system, where the expression was enhanced in parenchymal microglia and infiltrating macrophages(Reference Davidson and Hickey28). In inflamed spinal cords, the mRNA expression showed a particular kinetics, with a drop at the onset of the clinical symptoms, a peak in coincidence with the highest disease severity and a decrease to baseline levels in correspondence with the complete recovery of the rats(Reference Davidson and Hickey28). Despite the accumulation of AF-positive leucocytes in the CNS during the course of the disease, no overt changes were apparent in either expression levels or distribution of AF in peripheral organs, such as the spleen and lymph nodes. Since AF levels are at almost undetectable levels on circulating monocytes, Davidson & Hickey(Reference Davidson and Hickey28) suggested a mechanism whereby AF is up-regulated on these cells at the area of inflammation upon macrophage activation and/or as a result of interaction with activated T lymphocytes. Pro-inflammatory factors, such as lipopolysaccharide (LPS) and interferon-γ, up-regulated AF expression in rat alveolar macrophages only 72–96 h after the treatment. The delayed kinetics was attributed to the AF anti-inflammatory effect; the AF up-regulation was associated with a redistribution of the protein within the cell, mainly in the cytoplasm and at the cell surface(Reference Davidson and Hickey28).
Administration of the anti-AF antibody TLD-1A8A to rats with induced experimental autoimmune encephalomyelitis increased the severity of clinical symptoms and the duration of the disease, with up-regulation of pro-inflammatory cytokine expression (IL-18 and IL-6) and decreased anti-inflammatory IL-10 levels.
Davidson & Hickey(Reference Davidson and Hickey8, Reference Davidson and Hickey28) hypothesised that the increased AF expression during the course of experimental autoimmune encephalomyelitis might have counteracted the pro-inflammatory environment leading to a reduced tissue damage.
In a recent experiment, performed at the Katholieke Universiteit of Leuven in collaboration with Professor T. A. Niewold, we investigated the anti-inflammatory activity of the AF-16 peptide in LPS-stimulated RAW 264.7 macrophages (C ulgheri and TA Niewold, unpublished results). Indeed, macrophages play a critical role in the initiation, maintenance and resolution of inflammation and, upon activation, they release a set of primary inflammatory mediators, such as interleukins, reactive oxygen and nitric oxide species, which are involved in both beneficial and detrimental outcomes in inflammation(Reference Fujiwara and Kobayashi30). LPS, as an outer membrane component of bacteria, triggers the secretion of NO, which is extremely reactive and has a wide range of physiological activities involved in the immune response. It was found that treatment with AF-16 reduced NO production by LPS-stimulated macrophages in a dose-dependent fashion, with major effects observed at the highest dosages (10− 4 m), showing a mild anti-inflammatory effect only at the highest dosage (Fig. 1).
Fig. 1 NO production (μm) by RAW 264.7 macrophages grown with or without (control) 10− 4 m of antisecretory factor (AF). Values are means. * Mean value was significantly different from that for the control macrophages (P < 0·01).
Effects of antisecretory factor on the nervous system
The amino acids GABA and glutamate are, respectively, the most common inhibitory and excitatory neurotransmitters in the CNS(Reference Galofré, Babot and García31). Once in the synapse, they act on ionotropic receptors of GABA (GABAA) and glutamate. In mammals, overstimulation of GABAA activity results in central depression, whereas inhibition of GABAA receptor activity leads to general excitatory symptoms and convulsions(Reference Galofré, Babot and García31–Reference Medina and Chudotvorova33). Excitotoxicity occurs when ionotropic glutamate receptors are excessively activated; it is characterised by excitatory symptoms and degeneration of neurons(Reference Leist and Nicotera34, Reference Olney35). GABA exerts its effect on both the CNS and enteric nervous system by increasing the Cl− conductance of the cell membrane.
AF is expressed in the CNS and repeated challenges of rat intestine with CT were found to increase the expression of AF(Reference Lange, Lönnroth and Palm36). In vitro studies, with isolated Deiters' cell membrane from rabbit glia, have advanced the knowledge about the effects of AF and its mechanism of action on the nervous system(Reference Rapallino, Cupello and Lange7, Reference Lange, Lönnroth and Palm36, Reference Lange, Lönnroth and Palm37). AF purified from pig pituitary gland and its peptides were shown to be potent blockers of Cl− and GABA permeation across isolated rabbit Deiters' cell membrane in a dose-dependent fashion(Reference Lange and Lönnroth5). The AF concentration causing half-maximal inhibition was found to be 3 × 10− 10 m, more than a thousand times lower than [Met5]enkephalin, an opioid messenger considered to be one of the most potent inhibitors of GABA membrane permeation in vitro (Reference Lange and Lönnroth5).
AF might exerts its effect on the post-synaptic stage since in vitro studies have shown that AF binding to Deiters' cell membrane is reversible while AF had no effect on [3H]GABA transport in integral cells(Reference Lange, Lönnroth and Palm36).
Nipecotic acid and bicuculline, blocker of carrier-mediated transport of GABA and selective blocker of GABAA receptor, respectively, abolished the effects of AF on the neuronal membrane, while picrotoxin improved the AF inhibitory effect on Cl− permeation(Reference Lange, Lönnroth and Palm37). Kim et al. (Reference Kim, Wasling and Xiao38) suggested that AF acts as a regulator of chloride homeostasis, thus possibly counteracting an excessive secretion of fluids in the intestinal lumen. AF protein is expressed in rat hippocampus and can pass the blood–brain barrier(Reference Kim, Wasling and Xiao38). Extracellular application of exogenous AF peptide to hippocampal slices resulted in a 40 % reduction of GABAergic transmission while glutamatergic transmission was not affected(Reference Kim, Wasling and Xiao38). The same effect was also observed in hippocampal slices from rats immunised with CT administration or fed a diet containing hydrothermally processed cereals. To explain the AF-mediated effect on GABA transmission, it was hypothesised that AF might decrease the post-synaptic response by an internalisation of GABAA receptors and subsequent degradation in the proteasome(Reference Kim, Wasling and Xiao38).
The enteric nervous system, enclosed in the wall of the gastrointestinal tract, regulates motility and fluid transport, mainly without conscious control(Reference Gwynne and Bornstein39). Intestinal secretion is controlled by local reflexes that probably involve GABAergic neurons. When bound to GABAA receptors, GABA has depolarising and excitatory effects on the enteric nervous system(Reference Krantis40). In vitro studies showed an inhibitory effect of GABA on motor activity of the guinea-pig distal colon, inducing a relaxation in smooth muscle cells(Reference Minocha and Galligan41, Reference Bayer, Crenner and Aunis42). Two AF peptides, differing in length and molecular weight and containing the same antisecretory domain, were tested to investigate the role of AF on colon contractions in the guinea-pig(Reference Bayer, Crenner and Aunis42). Both peptides increased the frequency of contractions, though at different levels, probably due to conformational differences that might have influenced the interaction with GABAA receptors. It was suggested that the positive effect of AF on the colon contraction frequency during pathological conditions could be a way to protect the mucosa from noxious agents(Reference Harrison, Bartels and Erlwanger43). If the antisecretory factor acts as a negative modulator of GABAergic transmission also in the gut, it may counteract hypersecretion by decreasing the activity in secretory reflexes(Reference Kim, Wasling and Xiao38).
Antisecretory factor-inducing feed in animal nutrition
Since the European ban of in-feed antimicrobials, the reduction of post-weaning diarrhoea incidence is a major goal for the pig industry.
The Swedish government ban of AGP in 1986 prompted research for the development of antibiotic-free post-weaning feeds for piglet nutrition that were economically and ecologically competitive(Reference Lange and Lönnroth5). According to Göransson(Reference Göransson, Garnsworthy and Wiseman44), AF-inducing diets could be an important alternative to AGP and a useful tool to improve livestock efficiency and animal health and welfare.
AF is present in sows' colostrum and milk and there is a correlation between AF content in milk, its level in piglets' blood and the incidence of diarrhoea in the litter(Reference Lönnroth, Lange and Skadhauge1). AF is transferred from the sow to the fetus across the placenta(Reference Sigfridsson, Lange, Lönnroth and van Arendonk45). In piglets, AF activity in plasma has a cyclic variation; it declines at weaning, reaching the lowest level on the third day post-weaning(Reference Lange, Martinsson and Lönnroth46). The onset of diarrhoeal disease is significantly correlated with the low plasma AF activity(Reference Lange and Lönnroth5).
Lönnroth & Lange(Reference Lönnroth, Lange and Skadhauge1) found that AF production is stress sensitive. In rats, experimentally induced stress or injection of adrenocorticotropic hormone caused a rapid decrease of AF content in both plasma and pituitary gland(Reference Lönnroth and Lange47). According to Lange & Lönnroth(Reference Lange and Lönnroth5) the physiological drop in AF activity at weaning can be inhibited if piglets are fed an AF-inducing diet.
Intestinal fluid accumulation can be caused not only by bacterial enterotoxins or neurohormones but also even by hyperosmotic solutions. Intestinal secretion induced by the intake of solutions enriched with particular sugars, such as mannose and sorbitol, and amino acids such as glycine and alanine, triggered AF production in rat and pig pituitary glands(Reference Lönnroth and Lange47). The concentrations of monosaccharides and amino acids used were low, not able to induce clinical and subclinical diarrhoea. As shown in Table 1, piglets fed a diet with optimised sugar and amino acid content had increased AF activity in plasma, reduced diarrhoea incidence and improved growth performance: daily weight gain increased from 11·3 to 33·8 %; meanwhile, post-weaning diarrhoea incidence decreased by 59·09 % at least(Reference Göransson, Martinsson and Lange48). An AF-inducing diet, made up of a milk substitute enriched with sugars and amino acids, was shown to be an effective growth promoter in calves, but only if reared in poor hygienic conditions(Reference Lange and Lönnroth5).
Table 1 Effects of antisecretory factor (AF)-inducing diet on piglet growth performance, plasma AF content and post-weaning diarrhoea (PWD) incidence
ADG, average daily gain.
* Farms 1–5 from Göransson et al. (Reference Göransson, Martinsson and Lange48); farm 6 from Ulgheri et al. (Reference Ulgheri, Grilli and Rossi110).
Wheat and barley kernels subjected to a new patented hydrothermal process achieve a determined content of sugars and amino acids that, once fed to animals or human subjects, triggers AF endogenous secretion. As shown in Table 2, the hydrothermal process increases the glucose level from 0·3 mg/g to up to 5·3 mg/g while for fructose, sucrose and maltose the concentration values are raised up from 0·3, 8·4 and 0 to 3·1, 65·7 and 9·0 mg/g, respectively. The concentrations of specific amino acids such as lysine, histidine, glutamic acid, tryptophan and isoleucine were also increased by the hydrothermal process(Reference Björck, Bosaeus and Ek9).
Table 2 Content of sugars and amino acids in cereals before and after the hydrothermal process (from Björck et al. (Reference Björck, Bosaeus and Ek9))
We recently assessed the effects of two different concentrations of SPC, added to a wheat–barley-based diet, on the growth performance of weaned piglets. We found that 6 % of SPC supplementation improved daily weight gain and feed conversion rate of piglets compared with the 3 % supplemented diet and the control group (see Table 1).
Eggs are rich in AF, localised mainly in the yolk; according to Lange et al. (Reference Lange, Lönnroth and Martinsson49) the large amount of AF in the yolk could provide the chicken with a kind of protection against gastrointestinal diseases until the animal's capability to actively secrete endogenous AF is restored. Indeed the blood concentration of AF in chickens decreased from the time of hatching and started increasing at age 21 d. As in pigs, AF concentration in chicken blood is stress sensitive.
AF-rich egg yolk powder, from hens fed SPC, has proved to abolish diarrhoea in children affected by both acute and prolonged diarrhoea(Reference Zaman, Mannana and Lange12) and to improve the symptoms in patients affected by inflammatory bowel diseases (IBD)(Reference Eriksson, Shafazand and Jennische50, Reference Eriksson, Shafazand and Jennische51).
The exact mechanism of action of AF at the cellular level remains unclear. Laurenius et al. (Reference Laurenius, Wängberg and Lange13) suggested that the hydrothermal processing of the cereals might cause an unmasking of epitopes, possible ligands for binding sites in the intestinal mucosa.
Antisecretory factor and gut health
In a healthy small intestine, villi absorb nutrients while crypts secrete water and electrolytes. The main constituents of intestinal secretion are mucus, enzymes, water, Na+, Cl− , 
and IgA.
Intestinal secretion is necessary to obtain a chyme with a concentration of nutrients, water and electrolytes optimal for absorption processes and it also might have a function in the homeostatic mechanism such as the control of fluid and acid–base balance in the body(Reference Wapnir and Teichberg52). In pathophysiological conditions, intestinal secretion protects the mucosa by washing away and diluting harmful substances(Reference Lundgren53).
During gastrointestinal diseases, regulatory mechanisms of secretion are altered and inflammation and diarrhoea, caused by either infectious or non-infectious aetiologies, are the result of changes in fluid and electrolyte transport in the small and/or large intestine(Reference Fordtran54).
Enteric pathogens that cause the most severe diarrhoea diseases are rotavirus, Vibrio cholera, Shigella spp., Salmonella spp. and enterotoxigenic E. coli (Reference Petri, Miller and Binder55). Intestinal hypersecretion caused by V. cholera toxin and E. coli labile toxin (LT) has been studied in depth in experimental animals. CT-induced diarrhoea has been considered a prototype of enterotoxin-caused diarrhoea, because it does not cause histological changes in the intestine despite substantial rates of net fluid secretion(Reference Field, Fromm and al-Awqati56). Both CT and LT are heterohexameric molecules which consist of a single A-subunit and five identical B-subunits(Reference Fan, O'Neal and Mitchell57). Despite their similarities, CT and LT induce secretion by different mechanisms at the level of receptors and transductional pathways involved in the secretory response(Reference Griffiths and Critchley58, Reference Mourad, O'Donnell and Dias59). Both CT and E. coli LT attach to GM1-ganglioside via the B-subunit and the binding itself triggers the toxin internalisation. Inside the cell, the A-subunit catalyses ADP-ribosylation of the Gs-subunit of adenylate cyclase, increasing mucosal cAMP level(Reference Moss and Vaughan60). CT and cAMP stimulate active Cl− secretion by activating or inserting Cl− channels into the apical membrane of the crypt cells and inhibit electroneutral NaCl absorption by decreasing the activity of Na+/H+ and Cl− / exchange in villous cells, without interfering with the glucose-stimulated Na absorption(Reference Petri, Miller and Binder55). Studies with fish tetrodotoxin (TTX) showed that CT interacts with the enteric nervous system, inducing the secretion of neurohormones such as 5-hydroxytryptamine and vasoactive intestinal peptide(Reference Cooke61, Reference Cassuto, Fahrenkrug and Jodal62).
LT is the major virulence factor of enterotoxigenic E. coli; it was found that LT binds to several brush-border galactoproteins only weakly recognised by CT, and it was suggested that binding of LT to this additional receptors might have a role in intestinal secretion pathophysiology(Reference Mourad, O'Donnell and Dias59, Reference Holmgren and Svennerholm63). Recent studies demonstrated that, beyond its ability to bind to different glycolipids on the surface of enterocytes, LT binds to LPS on the surface of E. coli (Reference Horstman and Kuehn64). As a result of the LT–LPS surface interaction, more than 95 % of secreted LT is found associated with E. coli outer membrane vesicles, rather than being secreted in a soluble form. LT is able to mediate the internalisation of intact outer membrane vesicles into host cells(Reference Kuehn and Kesty65). In addition, competitive binding assays and microscopy with fluorescently labelled enterotoxigenic E. coli vesicles revealed that GM1 and LPS binding sites are distinct since the binding process can occur at the same time(Reference Horstman and Kuehn64). In cultured human intestinal cells, a third binding substrate for LT is the sugar residues of the receptor molecule of human blood group A antigen(Reference Holmner, Askarieh and Okvist66). According to Mourad et al. (Reference Mourad, O'Donnell and Dias59), LT induces secretion without involving 5-hydroxytryptamine, whose receptors' antagonist had no effect on LT-induced fluid and electrolyte secretion.
C. difficile toxin A has a marked pro-inflammatory effect on the intestinal mucosa, causing fluid accumulation, inflammation and tissue damage. It binds to glycosphingolipid receptors and is internalised in the enterocytes(Reference Lönnroth, Lange and Jennische67). Inside the cell, toxin A exerts enzymic activity, by catalysing glucosylation, triggering the secretion of multiple inflammatory mediators as substance P, histamine, NO and PG(Reference Kelly and La Mont68).
Rotavirus infection is one of the leading causes of infantile gastroenteritis. The virus infects the mature enterocytes that line the small intestine and, consequently, it impairs activities of intestinal disaccharides and Na+-solute symports coupled with water transport. Rotavirus protein NSP4 induces a Ca ion-dependent chloride secretion, disrupts Na+-dependent GLUT1 transporter-mediated reabsorption of water, causes maldigestion of carbohydrates and probably activates the Ca ion-dependent secretory reflexes of the enteric nervous system(Reference Lorrot and Vasseur69). The malabsorption of nutrients in the intestine is associated with inhibited water reabsorption and can lead to diarrhoea and malnutrition(Reference Lorrot and Vasseur69).
Shigellosis is one of the major causes of diarrhoea-related morbidity and mortality in developing countries, transmitted through contaminated food and water as well as through person-to-person contact(Reference Kotloff, Winickoff and Ivanoff70, Reference Baldi, Bianco and Nardone71). Effects of Shigella spp. infection are inflammation, mucosal ulceration and bleeding of the gastrointestinal tract(Reference Baldi, Bianco and Nardone71).
The AF protein and its derivatives seem to be the most potent inhibitor of intestinal hypersecretion of various aetiologies. Indeed, less than 1 pmol of human recombinant AF was shown to inhibit CT-induced fluid secretion in a rat model(Reference Johansson, Jennische and Lange4). Torres et al. (Reference Torres, Jennische and Lange72) suggested that AF is released as a response to the hypersecretion itself, rather than to the causative agents. Grøndahl et al. (Reference Grøndahl, Sorensen and Unmack73) tested different AF-derived peptides on pig intestinal loops challenged with different secretagogues including CT, LT and 5-hydroxytryptamine and found that AF inhibited intestinal fluid accumulation in the proximal small intestine of CT-treated loops while there was no effect in the distal part.
In vivo experiments conducted with rats demonstrated that repeated exposure to CT causes a long-lasting over-expression of AF in the intestinal mucosa able to counteract the C. difficile toxin A-induced signal, thereby preventing intestinal inflammation and fluid secretion(Reference Lönnroth, Lange and Jennische67). The CT-induced tolerance to C. difficile toxin A might be mediated by the enteric nervous system(Reference Lönnroth, Lange and Jennische67). C. difficile toxin A causes the secretion of substance P that triggers the release of a wide range of chemical mediators by the mucosal enterocytes(Reference Goyal and Hirano74). In the gut, AF is highly expressed in the submucosal T-lymphocytes, suggesting a sort of immunological memory. The antisecretory activity of the AF protein and its derived peptides is usually estimated by an in vivo assay based on the graded inhibition of CT-induced secretion in the intestinal loop model(Reference Lange and Lönnroth5, Reference Lange6).
In toxin-challenged experimental animals, the AF antisecretory effect differs with the type of toxin and AF administration route. Human recombinant AF administrated to rats, via the penis dorsal vein or directly into the lumen of an intestinal loop, just before or 1 h after the C. difficile toxin A challenge, reduced mucosal damage and fluid accumulation by 60–70 %(Reference Johansson, Jennische and Lange4). Intravenous injection of recombinant AF counteracted the intestinal hypersecretion but not the intestinal epithelial damage caused by okadaic acid(Reference Johansson, Jennische and Lange4).
The in vivo assay used to evaluate AF activity in plasma is based on the graded inhibition of CT-induced hypersecretion in ligated loops of the rat small intestine(Reference Lange6). As observed by Johansson et al. (Reference Johansson, Lönnroth and Jonson75), this method is reliable and reproducible, it is labour intensive, technically difficult and, furthermore, it requires a large number of experimental animals.
Polyclonal antibodies, raised against linear epitopes of AF and its derived peptides, were used to investigate the possible changes in the protein structure responsible for its biological activities(Reference Jennische, Johansson and Hansson76). These antibodies were used to screen the presence of conformational variants of the antisecretory protein in the pig CNS, though they were not able to discriminate between the active and inactive AF form, a great inconvenience since most of the AF present in plasma is in an antisecretory inactive state(Reference Jennische, Johansson and Hansson76).
A first step in the development of an in vitro assay, able to detect the AF active forms, has recently been made with the realisation of an ELISA test. This in vitro assay has been developed using monoclonal antibodies raised against human placenta AF(Reference Johansson, Lönnroth and Jonson75). The efficacy of the ELISA test, compared with the in vivo assay (ligated rat intestinal loop), was assessed by analysing AF activity in plasma samples from volunteers who ate the hydrothermally processed cereals. The plasma AF values obtained by ELISA determination correlated well with AF activity as measured by bioassay with the intestinal loops (r 0·85)(Reference Johansson, Lönnroth and Jonson75).
Antisecretory factor and inflammatory bowel disease
IBD is a complex disorder, characterised by chronic inflammation of the gastrointestinal tract, abdominal pain or discomfort, associated with altered bowel habits(Reference Ferguson, Shelling and Browning77). The two main types of IBD are ulcerative colitis and Crohn's disease. Ulcerative colitis is characterised by chronic mucosal inflammation and epithelial dysfunction in the large intestine, which causes open sores, ulcers and bleeding(Reference Fiasse, Denis and Dewit78). The main symptoms of ulcerative colitis are inflammation and ulcers in the top layer lining of the large intestine and frequent bloody diarrhoea. Crohn's disease can affect all layers of the intestine in any area of the gastrointestinal tract, from the mouth to the anus, but it most commonly affects the ileum. The swelling extends deep into the lining of the affected organ, causing pain and frequent bowel movement.
IBD is currently considered as a bio-psychosocial disorder with disturbances of motor function, heighted gut sensitivity, and possibly CNS disturbances(Reference Levy, Olden and Naliboff79). There is a genetic component in the incidence of IBD, since approximately 20 % of individuals with one form of IBD have a blood relative with IBD(Reference Fiasse, Denis and Dewit78). Studies with twins showed that 36 % of monozygotic twins share the disease, compared with only 4 % of dizygotic twins(Reference Jess, Riis and Jespersgaard80). The highest incidence rates and prevalence of IBD have been reported from northern Europe, the UK and North America, though the rates are beginning to stabilise. On the contrary, rates continue to rise in low-incidence areas such as southern Europe, Asia and in most developing countries(Reference Loftus, Silverstein and Sandborn81). Curative treatment for IBD has yet to be established; up until now therapeutic focus is on alleviating symptoms(Reference Eriksson, Shafazand and Jennische50).
Björck et al. (Reference Björck, Bosaeus and Ek9) evaluated the effect of an increased endogenous AF secretion on the clinical outcome in patients suffering from IBD. Patients with long-lasting ulcerative colitis or Crohn's disease received a diet containing either placebo cereals or SPC, without changing food habits and conventional medical treatment(Reference Björck, Bosaeus and Ek9). The outcomes were evaluated by analysing objective and subjective data. Every patient kept a diary to daily record the number of bowel movements and stool consistency, and clinical improvement or clinical deterioration was plotted as percentages on a visual analogue scale score and expressed as percentage of change. The study demonstrated that a diet supplemented with SPC was able to induce an increase of AF in the plasma of patients suffering from IBD. An increased AF level in plasma was associated with an improvement of patient welfare, reduced frequency of bowel movement and solid stools. Rectal biopsies analysed by immunohistochemistry showed that SPC supplementation induced a larger number of AF-positive cells compared with biopsies from the patients receiving the placebo cereals. However, a correlation was found between AF content in plasma and biopsy cell staining. No effects were observed on C-reactive protein in plasma. The AF values in plasma declined slowly after the end of the experiment.
The influence of orally administrated AF, provided by egg yolk with a high AF content, was evaluated in a randomised double-blind study(Reference Eriksson, Shafazand and Jennische50). The study involved twenty patients with acute attacks of ulcerative colitis receiving AF or placebo treatment as a supplement to the conventional therapy usually performed. The ‘AF therapy’ was shown to have a positive effect on the inflammatory reaction of colonic mucosa. Rectal biopsy analysis evidenced a reduction in the relative number of neutrophils and eosinophil granulocytes in the AF group while no effect on intestinal secretion was observed(Reference Eriksson, Shafazand and Jennische50). The study concluded that AF, used as a supplement to conventional pharmacological and nutrition therapies, can help to alleviate inflammation as shown by rectal biopsies(Reference Eriksson, Shafazand and Jennische50). Passive administration of AF by the means of an AF-rich egg yolk powder, followed by active induction through a diet supplemented with SPC, induced the regression of Crohn's disease in a patient with long-standing disease, resected colon and not responsive to conventional pharmacological treatment(Reference Eriksson, Shafazand and Jennische51). However, Ekesbo et al. (Reference Ekesbo, Nilsson and Sjölund82) reported no additional beneficial effects of SPC diet supplementation in a placebo-controlled diet study conducted with patients affected by irritable bowel syndrome, which received placebo cereals or active AF-inducing cereals. It was noted that general conditions in both groups, measured by a visual analogue scale score, improved regardless of which diet was consumed, probably because of a good fibre amount of the diets, which may have had a bulking effect and so reduce diarrhoea(Reference Ekesbo, Nilsson and Sjölund82). However, in the patients with the most severe diarrhoea symptoms, the SPC supplementation increased AF blood levels and led to an improvements in symptoms(Reference Ekesbo, Nilsson and Sjölund82).
Antisecretory factor and short bowel syndrome disease
Short bowel syndrome (SBS) can appear after intestinal surgery or radiation injury, or can result from loss of function due to vascular insufficiency. Major outcomes are malnutrition and dehydration caused by malabsorption of fluid, electrolytes, minerals and nutrients(Reference Tilg83). SBS is often associated with intestinal failure requiring parenteral nutrition, impairment of health-related quality of life and morbidity(Reference Tilg83–Reference Nightingale85).
Lange et al. (Reference Lange, Bosaeus and Jennische86) performed a study on patients with resected intestine and found that the extent of AF activity and the ability to maintain a high level of plasma AF after SPC treatment are significantly correlated with the length of the remaining small intestine. The study found that a length of at least 100 cm of small intestine is required to induce an improvement of AF activity by dietary means and the presence or absence of the large intestine appeared to not affect AF activity.
Pagoldh et al. (Reference Pagoldh, Eriksson and Heimtun87) conducted a double-blind trial to investigate the effects of diet supplemented with SPC or with non-processed cereals on health-related quality of life in a heterogeneous group of patients affected by SBS of various aetiologies. Patients differed by short bowel length and intestinal surgical techniques. Compared with SPC, the non-processed cereals were more effective in reducing daily faecal volume, especially in patients with ulcerative colitis, probably due to the high content of gel-forming dietary fibre that improved fluid absorption. The surgical techniques influenced the clinical effects of the cereal supplementation(Reference Pagoldh, Eriksson and Heimtun87). Moreover, the effects of the diet supplementation were more evident in the last part of the trial, probably due to a sensitisation process of the mucosa, which was independent from the type of cereals used in the first part of the trial(Reference Pagoldh, Eriksson and Heimtun87). According to the authors, an interesting approach to SBS would be a diet supplementation targeting increased viscosity of the intestinal content that could prolong the transit time and thus improve the uptake of nutrients(Reference Nightingale85).
Antisecretory factor and endocrine diarrhoea
Endocrine diarrhoea is caused by an imbalance of specific hormones which inhibit water and electrolyte absorption and/or increase fluid secretion or stimulate motility. The most important hormones involved in the regulation of intestinal secretion are vasoactive intestinal peptide, serotonin, substance P, PGE2, calcitonin, thyroxin and pancreatic polypeptide hormone(Reference Dodi and Spencer88).
Carcinoid syndrome and medullary thyroid carcinoma can lead to an alteration of the hormone balance. Carcinoid syndrome refers to a range of symptoms that occur as a result of carcinoid tumours. Carcinoid tumours originate in the cells of the neuroendocrine system, and could occur anywhere along the gastrointestinal tract and in the lungs and are characterised by the production of serotonin (5-hydroxytryptamine)(Reference O'Toole, Ducreux and Bommelaer89). The syndrome is characterised by endocrine-induced diarrhoea with flushing and bronchospasm, caused by the tumour's ability to secrete vasoactive peptides(Reference Dodi and Spencer88). Medullary thyroid carcinoma is a neuroendocrine tumour that develops in the thyroid C cells and occurs either as an erratic event or secondary to a germline mutation with an autosomal dominant pattern of inheritance(Reference Kaltsas, Besser and Grossman90). Thyroid C cells are responsible for the production of both calcitonin and carcinoembryonic antigen(Reference Flemming, Lee and Bouvet91). Neuroendocrine tumours may induce severe persistent diarrhoea, sometimes therapy-resistant(Reference Laurenius, Wängberg and Lange13, Reference Wängberg, Westberg and Thylen92). Laurenius et al. (Reference Laurenius, Wängberg and Lange13) investigated the effects of AF therapy, AF-rich egg yolk powder or SPC on patients suffering from endocrine diarrhoea due to midgut carcinoid syndrome or metastasising medullary thyroid carcinoma. The study showed that even though the severely ill patients were optimally medicated, several of them responded positively to the AF therapy(Reference Laurenius, Wängberg and Lange13). Passive intake of AF had positive effects on the frequency of bowel movements, indicating that AF was still active after passing the upper gastrointestinal tract. Compared with the passive AF therapy, the SPC therapy required a larger intake volume, which was difficult to reach in severely ill patients(Reference Laurenius, Wängberg and Lange13). Thus AF-rich egg yolk powder could be the best option for patients who are not able to ingest large amounts of food.
The main clinical outcomes obtained by using the AF-inducing diets in patients affected by irritable bowel syndrome, IBD and SBS are summarised in Table 3.
Table 3 Specially processed cereal (SPC) supplementation and main outcomes in subjects suffering from diarrhoea or bowel diseases
MTC, medullary thyroid carcinoma; AF, antisecretory factor; SBS, short bowel syndrome.
‘Antisecretory factor-therapy’: a tool to reduce childhood diarrhoea?
As previously stated, diarrhoea remains a problem amongst children especially in developing countries, in some cases being even life threatening. Severe malnutrition and infections are the main cause of the children's premature and avertable deaths(Reference Brown, Lifschitz and Nicols93). Diarrhoea can be prevented by improving hygienic conditions, through vaccination and encouraging mothers to breastfeed their infants(Reference Mihrshahi, Oddy and Peat94). Breast milk is easily digestible and contains antibodies that can protect the infant from bacterial and viral infections. Furthermore, it aids the development of the child's immune system. According to Ziyane(Reference Ziyane95), exclusively breastfed infants under 2 months are twenty-five times less likely to die of diarrhoea-related illness than non-breastfed infants. However, in many diarrhoea cases breast-feeding alone does not suffice(Reference Saleemi, Zaman and Akhtar96).
In a study conducted in Pakistan on children suffering from acute and prolonged diarrhoea episodes, it was found that when given B 221, a medical food based on AF-rich egg yolk powder, the incidence of diarrhoea subsided(Reference Zaman, Mannana and Lange12) (see Table 3). The study, which lasted 3 d, involved 240 children from 6 to 24 months; every 5 h they were randomly given 2 g of B 221 or placebo (regular egg yolk powder), dissolved in rehydration solution. In children suffering from acute diarrhoea (less than 7 d of diarrhoea illness), the AF administration improved frequency and consistency of the stools, the clinical outcome after 3 d was positive for 83 % of the children fed B 221, while the response to placebo was positive in only 54 % of children(Reference Zaman, Mannana and Lange12). In children with prolonged diarrhoea, B 221 treatment resulted in a 91 % successful clinical outcome, compared with the placebo group which only had a 63 % success rate(Reference Zaman, Mannana and Lange12).
As demonstrated by Zaman et al. (Reference Zaman, Mannana and Lange12), B 221 counteracted diarrhoea of different aetiologies, since it was unlikely that the children involved in the study were infected by the same agent. Dosing and mode of administration of B 221 are still yet to be understood, but according to the authors, a higher dosage might be useful in the management of acute diarrhoea in children, especially in developing countries, avoiding the appearance of prolonged diarrhoea, with its threatening consequences(Reference Zaman, Mannana and Lange12).
Svensson et al. (Reference Svensson, Lange and Lönnroth97) found that AF can be induced in maternal milk by hydrothermally processed cereal intake. Furthermore, the AF-inducing diet prevented mastitis, a painful infection and inflammation of the breast tissue, probably due to the anti-inflammatory properties of AF. Thus, SPC intake in lactating women might achieve the double goal of preventing mastitis in the mothers and diarrhoea in children.
Antisecretory factor-inducing foods in Ménière's disease
An increase of AF in plasma has been found to reduce symptoms in patients suffering from Ménière's disease, a disturbance of the inner ear that can affect hearing and balance, characterised by episodes of vertigo, tinnitus and progressive hearing loss(Reference Hanner, Jennische and Lange98). It was first hypothesised that endolymphatic hydrops causes dilation of the cochlear duct affecting the endolymph drainage process. Recent findings suggest that impaired production or resorption of endolymph and/or transport across membranes appear to be major factors in the pathophysiology of Ménière's disease(Reference Hanner, Jennische and Lange98, Reference Hanner, Rask-Andersen and Lange99). Dysfunctions of many structures of the inner ear and of the sympathetic nervous system are likely to be involved in creating the symptoms of this disease.
Experimental evidence about the role of AF as a regulator of fluid transport across the mucosal membranes and its anti-inflammatory properties led Hanner et al. (Reference Hanner, Jennische and Lange98) to perform a clinical pilot study on patients suffering from Ménière's disease, designed to investigate the effect of increased AF plasma concentration on the frequency and extent of vertigo, which are the main symptoms of this disease. The study required that the patients received 1 g SPC/kg body weight per d, divided into two to three dosages over 2–4 weeks. Patients were instructed to keep a diary regarding their subjective symptoms, including frequency, duration of vertigo and fluctuations of hearing. Although the study was performed on a small number of patients, the results were interesting since in about 50 % of patients the attacks of rotatory vertigo diminished or completely disappeared, with reduced final American Academy of Otolaryngology-Head Neck Surgery (AAO-HNS) functional level scale (1995; range 1–6)(Reference Hanner, Jennische and Lange98). Even if there was a positive correlation between plasma AF activity after the treatment and the reduction of vertigo (r 0·65; P = 0·001), the response to the diet was individual and not related to the severity of the symptoms(Reference Hanner, Jennische and Lange98). The findings of the study confirmed that an AF-inducing diet could be useful in some Ménière's disease patients and also reinforced the authors' idea that AF could be involved in inner ear homeostasis. Following the success of the pilot study, Hanner et al. (Reference Hanner, Rask-Andersen and Lange99) performed a double-blind study on a larger number of patients (n 51) for a longer period of time (3 months). Patients were randomly assigned to two groups: a control group which received placebo cereals and the SPC group. In the SPC group, fourteen of the twenty-seven patients experienced a decrease in their symptoms, while in the control group only two participants reported decreased vertigo severity(Reference Hanner, Rask-Andersen and Lange99). Once the double-blind arm of the study was concluded, the participants of the control group were offered the option to receive the active cereal treatment for a further 3 months: twenty-two patients accepted; of these, seventeen had alleviated symptoms. According to Hanner et al. (Reference Hanner, Rask-Andersen and Lange99), the results from the open arm of the study might have been influenced by the expectations of the patients. In both trials the feeding treatment had no effects on the clinical otoneurological status of patients, assessed before and after the treatment by pure tone average and speech audiometry tests. Therefore, the SPC treatment exerted its effect only on the vertigo component of the disease. A clear explanation of the mechanism of action of AF remains unknown; however, many experiments and clinical studies indicate that AF can normalise water and ion transport. It has been shown that AF interacts with the lipid raft protein flotillin-1(Reference Johansson, Jonson and Bosaeus18), and they are both expressed in the inner ear of the rat, as well as the human cochlea and endolymphatic saccus(Reference Jennische, Hanner and Johansson16, Reference Johansson, Jonson and Bosaeus18, Reference Hanner, Jennische and Lange98). Hanner et al. (Reference Hanner, Rask-Andersen and Lange99) suggested that the interaction between AF and flotillin-1 might cause a down-regulation or a reorganisation of aquaporin channels, thus regulating the homeostasis of endolymph.
New insight on antisecretory factor properties
Brain oedema, haemorrhage, and obstruction or derangement of the blood–brain barrier can lead to increased intracranial pressure (ICP) that can damage brain function, resulting in inflammation, with possible persistent neurological and psychiatric malfunctions(Reference Vink, Young and Bennett100–Reference Säljö, Bolouri and Mayorga103). The extent of brain damage is related to the severity and length of ICP increase(Reference Jennische, Bergström and Johansson104).
In humans, blast-related brain injuries are known to cause brain oedema, vasospasm and intracranial haemorrhage(Reference Armonda, Bell and Vo105). The current therapeutic options applied in counteracting elevated ICP are not indicated at pressure levels lower than 25 mmHg. However, less is known about the medium- and long-term effects of levels of ICP lower than the treatment threshold on physical and psychological functioning of patients(Reference Jantzen106). The effects of exposure to low and mild blast levels on brain injury is increasingly of interest due to the involvement of large numbers of troops in the Afghani war, where soldiers are often subjected to traumatic brain injury without damage to the head(Reference Säljö, Svensson and Mayorga102). It was recently reported that in a rat model low blast overpressure in a shock tube (10–30–60 kPa) impaired cognitive functions, assessed by the Morris water maze, and caused an elevated ICP in a dose-dependent manner(Reference Hoane, Kaplan and Ellis101). The same experiment was repeated with rats fed or not a diet supplemented with 20 % of hydrothermally processed barley(Reference Säljö, Svensson and Mayorga102). The study showed that the inclusion of hydrothermally processed barley in the rats' diet prevented the impairment of cognitive function and drastically reduced the ICP elevation. The peak of ICP was reached 10 h after the rats were exposed to both 30 and 60 kPa, though in rats exposed to 60 kPa there was a first smaller peak after 10 min. At 10 h after the overpressure exposure, the mean ICP in rats of the control group subjected to 30 and 60 kPa was raised by 90 and 145 %, respectively, while in rats fed hydrothermally processed barley the ICP increased by 60 and 50 %, respectively. The feed treatments had no effect on the initial peak, recorded after 10 min from the exposure at 60 kPa. Säljö et al. (Reference Säljö, Bolouri and Mayorga103) suggested that the diet supplementation with hydrothermally processed barley reduced brain oedema but the mechanisms behind this protective effect remain unknown.
In a rat experimental model of herpes simplex encephalitis, induced by type 1 virus (HSV-1), AF-16 peptide was used to suppress the raised ICP(Reference Jennische, Bergström and Johansson104). The intranasal instillation of AF-16 peptide granted a complete rescue of rats receiving 25 μg twice daily from the 4th day after infection. Prevention of neurological malfunctions, usually associated with encephalitis, was found to be dose dependent. In rats receiving 1 μg AF-16 twice daily, starting immediately after the infection, there was a moderate mortality, while in animals of the untreated group the mortality was about 90 %(Reference Jennische, Bergström and Johansson104). The AF-16 peptide reduced the ICP increase and fluctuations, and also abolished the pressure peaks in treated rats. Jennische et al. (Reference Jennische, Bergström and Johansson104) suggested that the AF-16-mediated suppression of increased ICP in infected rats to a non-injurious level might be the cause of abrogated neurological malfunctions.
Summary and conclusions
In recent years, nutrient content has ceased to be the main goal of nutrition, both for man and animals. Food and feeds are now considered as possible tools to improve health and wellness. Well-known foods, such as grains, tomatoes and milk, have been studied for health-promoting activities of nutrients such as β-glucans, lignans, lycopene or conjugated linoleic acid.
In healthy individuals, the AF protein is secreted in plasma and other body tissues, probably in a biologically inactive state. Exposure to bacterial toxins induces activation of plasma AF, probably reflecting a natural defence mechanism against agents causing diarrhoea, thereby contributing to a favourable clinical outcome and disease termination(Reference Lönnroth, Lange and Jennische67). The possibility to stimulate AF secretion, by means of processed cereals, and exploit its positive effects on alleviating symptoms in patients suffering from intestinal chronic inflammatory diseases, diarrhoea or affected by Ménière's disease, has been demonstrated in several studies and, although ‘AF therapy’ cannot be considered a real therapy, its efficacy in reducing symptoms resulted in improvement of patients' life quality. Furthermore, the inclusion of SPC in the diet of lactating women could be exploited in order to reduce intestinal disorders in suckling children, particularly in developing countries, where breast-feeding is highly recommended and the access to anti-diarrhoeal drugs and/or rehydrating solutions is poor. As suggested by Zaman et al. (Reference Zaman, Mannana and Lange12), these active cereals can be used as prophylaxis in highly exposed populations during the diarrhoea season. AF-rich egg yolk gives the opportunity of a direct fast administration of the AF molecule to young children and to individuals with impaired food consumption.
A recent study showed the positive effect of SPC diet supplementation on prevention of the negative effects of exposure to low levels of blast overpressure in rats, where the extent of ICP increase and cognitive function impairment were improved(Reference Säljö, Bolouri and Mayorga103). So far, there are few studies on the effects of blast-related brain injuries and their effects on brain function in the long-term period, but their prevention by dietary tools is an interesting perspective. Indeed, cereals are part of daily food intake in many countries, so it could be easy to improve the consumption of active cereals, particularly by individuals exposed to a high risk of blast-related brain injuries such as soldiers during training sessions or conflict. They also might be helpful for individuals subjected to the risk of ICP increase, as patients affected by brain pathologies such as tumours in conditions of sudden pressure changes, such as during long flights, or exposed to overpressure as divers.
In pig farming, intensive breeding conditions often expose animals to such stress that they have a negative impact on their health and welfare. Indeed, early weaning exposes the piglets to simultaneous nutritional, social and environmental stresses when the immune and the gastrointestinal systems of the piglets have not completely reached functional development. This situation often results in low voluntary feed intake, severe growth reduction and can lead to an impairment in health status, to a major susceptibility towards infections and gut disorders. Weaned piglets frequently show malabsorption syndrome, characterised by increased excretion of carbohydrates and fatty acids in faeces, watery stools and degeneration of the intestinal epithelium. Moreover, opportunistic pathogens such as enterotoxigenic E. coli can take advantage from the poor health condition of piglets affected by non-infectious diarrhoea and cause the post-weaning diarrhoea syndrome.
For a long time, the employment of in-feed antimicrobials has been strategic in reducing the negative effects of premature weaning on piglets' health and growth performance.
Since the complete ban of in-feed antimicrobials in 2006, several nutritional approaches and alternative substances have been proposed but, so far, none of these have been shown to be as effective as antibiotics on growth promotion and disease prevention of food animals.
Among the alternatives proposed, immunomodulating substances are gaining interest, since the correct development of both the innate and acquired immune system of the piglets is strategic in determining the outcomes of a large exposure to antigens and stress conditions, typical of weaning(Reference Gallois, Rothköttera and Baileya107). AF-inducing diets such as those including hydrothermally processed cereals appear to be suitable candidates as replacers of antibiotic growth promoters.
Indeed, AF is present in sows' milk and a correlation between AF concentration in milk and diarrhoea in the offspring has been proved. Several in vitro and clinical studies have found evidence for its potent capability to counteract intestinal fluid imbalance of various aetiologies; meanwhile, recent reports have focused on its immunomodulatory and anti-inflammatory properties. It is noteworthy that SPC, being simple cereal kernels, may have a positive image in the eyes of modern European consumers, who are increasingly sensitive to ethical considerations and whose opinion has a great influence on legislation(Reference Florkowski, Elnagheeb and Huang108). Compared with other feed additives, SPC do not contain any active compound, since their action is to stimulate the animals' capability to produce AF. In addition, they do not pose any environmental harm such as the use of molecules such as ZnO that, even with improved growth promoting and anti-diarrhoeic properties, accumulate in the soil(Reference Jondreville, Revy and Dourmad109). In our field trial, we showed that an AF-inducing diet was effective in improving growth performance and feed conversion rate. It is well known that a more efficient use of nutrients present in the diet leads to decreased nutrition costs and to a reduction of N and phosphate excretion in manure and their accumulation in the soil.
The mechanism of action behind its biological activity has not yet been clarified and, so far, few data are available on pig field trials. Moreover, compared with its use in human nutrition, the proper level of diet supplementation has not been defined and further studies are required.
Acknowledgements
We wish to thank Professor T. Niewold for his availability and his supervision on the in vitro experiments on macrophages cited in the text. We also thank Professor Gianfranco Piva for his advice during the elaboration of this paper.
All authors contributed equally to the preparation of this paper.
The authors have no conflicts of interest, financial or otherwise, to disclose.
This research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors.
