Probiotics in Inflammatory Bowel Disease: Is It All Gut Flora Modulation?

620 COMMENTARY

Inflammatory bowel disease by week 10. No change in faecal micro- Gut: first published as 10.1136/gut.2003.032219 on 13 April 2004. Downloaded from ...... flora occurred as a result of this subcutaneous administration of L salivarius, suggesting a mechanism of action dis- Probiotics in inflammatory bowel tinct from colonic floral modulation. Various indicators of altered immune disease: is it all gut flora modulation? function were seen with decreased tumour necrosis factor (TNF) and S Ghosh, D van Heel, R J Playford IL-12 levels being obtained from sple- nocytes that had been stimulated by ...... Salmonella typhimurium. In contrast, transforming growth factor (TGF)-b Understanding probiotic action may permit modulation of the levels were maintained. Such systemic immune system, both locally and systemically anti-inflammatory activity is counter- intuitive to a simplistic model of gut here is considerable public, media, Lactobacilli are a major constituent of flora modulation, and leads to specula- and scientific interest in ‘‘natural’’ the intestinal microflora and are fre- tion about the molecules involved in products, including probiotics, in quently used as probiotics, often in the driving immunomodulation. T 6 modulating intestinal inflammation health food industry. Among the The findings of the current study are and health.1 Intestinal microflora are Lactobacillus genus, different species of not the first to suggest that probiotics intimately involved in the generation of bacteria induce distinct mucosal cyto- have more than a local anti-inflamma- immunocompetent cells and tuning the kine profiles in the gut immune system tory effect by modulating the flora. For 7 example, Lactobacillus casei or Lacto- balance between T helper 1 (Th1) and of BALB/c mice. For example, an bacillus bulgaricus reduced the inflamma- Th2 immunity during the development increase in the Th2 cytokines interleukin of the gut associated immune system. (IL)-10 and IL-4 was observed in mice tory response induced by coculture of It is now generally accepted that the fed Lactobacillus delbrueckii subspecies bacteria with mucosal explants from intestinal bacterial flora contributes Bulgaricus and Lactobacillus casei whereas, Crohn’s disease affected intestinal tis- significantly to the pathogenesis of in contrast, a significant induction of sue. In this study, a significant reduc- inflammatory bowel disease (IBD) along the Th1 cytokines IL-2 and IL-12 was tion of proinflammatory cytokines such with mucosal immune dysregulation observed with Lactobacillus acidophilus.It as TNF was noted.13 Such anti-inflam- and genetic susceptibility. Considerable is therefore important that notice is matory effect might even be systemic, research is focused on modifying the taken of which specific bacteria are as shown by the bacteria CpG DNA intestinal flora with probiotic bacteria being used. experiments discussed later. to attenuate inflammatory activity Various knockout, transgenic, and Probiotics may also influence mucosal and prevent relapses in ulcerative coli- adoptive transfer murine and rodent cell-cell interactions and cellular ‘‘stabi- http://gut.bmj.com/ tis, Crohn’s disease, and pouchitis. models of IBD have been generated lity’’ by actions such as enhancement of Although both Lactobacillus species and and the requirement for bacterial col- intestinal barrier function by modulat- Bifidobacterium species are frequently onisation to induce a IBD phenotype is ing cytoskeletal and tight junctional used, the optimum use of probiotics in virtually universal, despite the complex- protein phosphorylation. For example, IBD requires greater understanding of ities of the immune network.8 In con- live probiotics such as Lactobacillus acid- their effects on the immune system. trast, in the IL-10 knockout mice model ophilus or Streptococcus thermophilus protect in vitro intestinal epithelial cell A rationale for the use of probiotics in of colitis, probiotic therapy with Lacto- on September 28, 2021 by guest. Protected copyright. IBD stems from reports of dysbiosis in bacillus species and Bifidobacterium spe- lines (HT29, Caco-2) from pathogen the intestinal flora in ulcerative colitis, cies has been shown to be effective in invasion and adhesion by enteroinvasive Crohn’s disease, and pouchitis, either by reducing inflammation.910 In animal Escherichia coli.14 Similarly, the non- conventional anaerobic culture or by models, probiotic therapy may prevent pathogenic E coli strain Nissle 1917 analysis using molecular probes. It is relapses of colitis, as shown by treat- inhibited adhesion and invasion of however unclear whether such altera- ment with Lactobacillus GG in HLA-B27 intestinal epithelial cell line (intestine tions in intestinal flora drives the transgenic rats after antibiotic treat- 407) by adherent invasive E coli strains inflammation or is a consequence of it. ment.11 It is therefore clear that not all isolated from patients with Crohn’s The practical application of probiotic bacteria have the same actions on gut disease.15 strategy has been especially encouraged immune function. Separating them into In ‘‘the age of the genome’’, it is not by the positive results of a trial in its use ‘‘good’’ and ‘‘bad’’ bacteria, a marketing surprising that much time and atten- for the prevention and treatment of strategy often used in the commercial tion has been spent on studying the pouchitis.23 The multispecies probiotics industry, is however a gross oversimpli- importance of the detailed bacterial used pose special challenges in identify- fication, and takes no account of host DNA sequences in these effects. Bac- ing precise mechanism of action, differences as a contributory factor. terial DNA contains non-methylated although alterations in faecal flora have In this issue of the Gut, researchers CpG motifs which bind to toll-like been demonstrated.4 Despite some posi- from Cork, Ireland,12 challenge the con- receptor 9 (TLR-9). TLR-9 signalling is tive trials, generalisation from pouchitis ventional hypothesis of mechanisms dependent on the adaptor protein to their use for all forms of IBD appears of probiotic efficacy by administering MyD88. Such immunostimulatory DNA somewhat premature, however, as for Lactobacillus salivarius subcutaneously to sequences (ISS-DNA or CpG DNA) of example, a trial of administration of IL-10 knockout mice [see page 694]. bacterial origin have been shown to Lactobacillus GG after surgical resection The anti-inflammatory effect of subcu- reduce inflammation in rodent IBD for Crohn’s disease proved ineffective in taneous administration was not specific models such as DSS induced colitis, preventing relapse.5 Further studies are as it was also seen in a murine model of hapten induced colitis in BALB/c mice, therefore required in ulcerative colitis arthritis. Non-viable bacteria could not and the IL-10 knockout mice model of and Crohn’s disease before firm recom- be tested as the group receiving heat colitis. This reduction in inflamma- mendations may be made. treated L salivarius had 100% mortality tion was accompanied by inhibition of

www.gutjnl.com COMMENTARY 621 proinflammatory cytokine and chemo- and Listeria species. Bacteriocins are (Leu1007fsinsC) or two missense muta- Gut: first published as 10.1136/gut.2003.032219 on 13 April 2004. Downloaded from kine production and suppression of synthesised in ribosomes as prepeptides tions (Gly908Arg and Arg702Trp). These induction of matrix metalloproteinases before being released extracellularly, alterations are associated with increased in the colon.16 and their genetic locus in Lactobacillus risk of development of Crohn’s disease Further evidence of the central role salivarius has been identified.20 Pro- and result in defective induction of of bacterial DNA has come from novel duction of different classes of bacterio- nuclear factor kB (NFkB) activation by experiments where both intragastric cins confers a competitive survival bacterial peptidoglycan and muramyl and subcutaneous administration of advantage in colonisation and therefore dipeptide (MDP). MDP induced activa- probiotic and E coli DNA attenuated these molecules are most relevant tion of NFkB in mononuclear cells is the severity of DSS induced colitis.17 The within the intestinal flora, but their absent in patients with Crohn’s disease form that this DNA takes appears systemic effects require further study. homozygous for the Leu1007fsinsC crucial, as methylated probiotic DNA, Importantly, the production and activity mutation.27 28 It is therefore interesting calf thymus DNA, and DNAse treated of bacteriocin is not affected by spray to speculate that NOD2 mutations asso- probiotics were ineffective. drying which may facilitate commercial ciated with Crohn’s disease result in Given this complexity, do we need live preparation.21 defective sensing of some bacteria bacteria, dead bacteria, or just the DNA? There has recently been much interest which may precipitate inappropriate Unfortunately, the data are confusing in the function of dendritic cells (DC) diffuse activation of NFkB and inflam- and sometimes contradictory. TLR-9 and in controlling gut immune activity. DC mation through non-NOD2 mechan- the adaptor protein MyD88 appear act as the switch that determines the isms. Repeating the experiments with 13 essential in signalling, and in their delicate balance between Th1 and Th2 Crohn’s mucosal explants from presence even non-viable bacteria can immunity, as well as tolerance (Th3). patients with homozygous, heterozy- signal. In TLR-9 deficient mice, unlike Therefore, it is likely that the DC gous, and double heterozygous NOD2 TLR-2 or TLR-4 deficient mice, intragas- phenotype and state of activation deter- mutations, and appropriate controls tric c irradiated (that is, non-viable) mine whether initiation of immunity or cocultured with Lactobacilli, may provide probiotics had no effect on DSS induced tolerance takes place and DC are likely interesting data using TNF readouts. colitis. Mice deficient in MyD88 did to play a central role in mediating the Our attempts to understand how not respond to c irradiated probiotics.17 effect of probiotic bacteria and deter- bacteria modulate the immune system The immune modulatory function of mining the type of immune response will undoubtedly yield novel therapeu- DNA has also been demonstrated in a that occurs. Different species of lacto- tic targets and therapeutic agents. study of peripheral blood mononu- bacillus exert different DC activation Irrespective of whether or not dysbiosis clear cells from healthy donors where patterns and the complexity of such is reproducibly confirmed in IBD, we Bifidobacterium genomic DNA caused interactions is exemplified by demon- need to consider probiotic therapy in induction of secretion of the anti- stration that Lactobacillus reuteri, a poor terms of specific molecules modulating inflammatory IL-10.18 Given the high inducer of IL-12, is capable of inhibiting defined targets in the gut mucosal and http://gut.bmj.com/ GC content of Bifidobacterium chromoso- DC activation by other Lactobacillus systemic immune system, and move mal DNA, it will be of interest to assess species.22 The threshold of bacterial the effect of its subcutaneous adminis- concentration necessary to induce cyto- Probiotic Antimicrobial peptides Pathogenic tration in the IL-10 knockout model of kine production may be different for bacteria bacteria colitis. proinflammatory cytokines IL-12/TNF The immune modulatory properties and anti-inflammatory/regulatory cyto- PAMPs of the various probiotic bacteria may kine IL-10, permitting fine modulation differ, and this becomes problematic for of the immune response.22 on September 28, 2021 by guest. Protected copyright. the use of multispecies preparations. Evidence of probiotic strains affecting PRRs Furthermore, not all immunostimula- Th1/Th2 immune balance also comes Dendritic cells tory oligonucleotides have the palindro- from experiments in which stimulation mic CpG motif. In one study, chro- of macrophages with Lactobacillus rham- Naïve mosomal DNA was purified from nine nosus GG induced mRNA expression of TH cells strains of Lactobacillus delbrueckii sub- the chemokines CCL2, CCL3, CCL5, species Bulgaricus and six strains of CCL7, CCL19, CCL20, CXCL8, CXCL9, 23 Streptococcus thermophilus derived from and CXCL10. Such a Th1 pattern of yoghurt starter cultures. Only DNA from chemokine induction could explain the TH1 TReg TH2 L bulgaricus NIAI B6 induced significant proposed antiallergenic properties of proliferation of mice Peyer’s patch and this probiotic strain and may benefit splenic B cells although it did not Th2 oriented ulcerative colitis. Interes- contain a palindromic CpG motif.19 It is tingly, studies of oral administration of IFN-γ IL-10 IL-4 therefore clear that ‘‘the devil is in the these bacteria suggest that they may TNF TGF-β IL-5 detail’’ and extrapolation across DNA affect the systemic immune response. sequences and bacterial species may For example, oral administration of Figure 1 Pathogen associated molecular provide false impressions. Lactobacillus rhamnosus GG to healthy patterns (PAMPs) derived from bacteria In addition to indirectly influencing volunteers for five weeks affected the (including probiotics) are recognised by pattern recognition receptors (PRRs, such as Toll-like gut flora and stimulating immune res- systemic cellular immune response to receptors). Initiation of dendritic cell (DC) 24 ponses, the probiotic strains themselves intestinal microorganisms. maturation starts after ligation of PRRs, which produce antimicrobial peptides. Bac- What about the host? Identification also requires adaptor proteins such as MyD88 teriocin production is often associated of NOD2 mutations associated with for signalling. Type of PAMPs determines the with probiotic strains, and Lactobacillus Crohn’s disease provides further support selective priming of DCs for production of TH1, salivarius cultures produce a broad spec- for the central role of bacteria in the TH2, and TReg lymphocyte polarising factors. 25 26 Different PAMPs ligate to specific trum bacteriocin that exhibits activity pathogenesis. Three NOD2 muta- corresponding PRRs. IL, interleukin; TNF, against a range of microorganisms such tions located near or in the leucine rich tumour necrosis factor; TGF-b, transforming as Bacillus, Staphylococcus, Enterococcus, repeats involve a frameshift mutation growth factor b.

www.gutjnl.com 622 COMMENTARY away from a simplistic concept of re- 4 Venturi A, Gionchetti P, Rizzelo F, et al. Impact on 17 Rachmilewitz D, Katakura K, Karmeli F, et al. Gut: first published as 10.1136/gut.2003.032219 on 13 April 2004. Downloaded from the composition of the faecal flora by a new Toll-like receptor 9 signalling mediates the anti- populating the intestinal flora with probiotic preparation: preliminary data on inflammatory effects of probiotics in murine ‘‘friendly’’ bacteria. Bacteria are only maintenance treatment of patients with ulcerative experimental colitis. Gastroenterology ‘‘friendly’’ in the context of the desired colitis. Aliment Pharmacol Ther 1999;13:1103–8. 2004;126:520–8. immune modulation required for a 5 Prantera C, Scribano ML, Falasco G, et al. 18 Lammers KM, Brigidi P, Vitali B, et al. Lactobacillus GG did not prevent endoscopic Immunomodulatory effects of probiotic specific disease. The environment to recurrence at one year nor reduced the severity of bacteria DNA: IL-10 and IL-10 response in which the immune system is exposed recurrent lesions. Gut 2002;51:405–9. human peripheral blood mononuclear cells. to determines DC phenotype and state 6 Ahrne S, Nobaek S, Jeppsson B, et al. The normal FEMS Immunol Med Microbiol lactobacillus flora of healthy human rectal and 2003;38:165–72. of activation and eventually drives the oral mucosa. J Appl Microbiol 1988;85:88. 19 Kitazawa H, Watanabe H, Shimosato T, et al. balance between effector and regulatory 7 Perdigon G, Galdeano CM, Valdez JC, et al. Immunostimulatory oligonucleotide CpG-like T cells (fig 1). Understanding probiotic Interaction of lactic acid bacteria with the gut motif exists in Lactobacillus delbrueckii ssp. bulgaricus NIAI B6. Int J Food Microbiol action may permit modulation of the immune system. Eur J Clin Nutr 2002;56(suppl 4):S21–6. 2003;85:11–21. immune system, both locally and sys- 8 Mizoguchi A, Mizoguchi E, Bhan AK. Immune 20 Flynn S, van Sinderen D, Thornton GM, et al. temically. The article by Sheil and networks in animal models of inflammatory bowel Characterization of the genetic locus responsible for the production of ABP-118, a novel colleagues12 is the right step towards disease. Inflamm Bowel Dis 2003;9:246–59. 9 Madsen KL, Doyle JS, Jewell LD, et al. bacteriocin produced by the probiotic bacterium stimulating further research where Lactobacillus species prevents colitis in interleukin Lactobacillus salivarius subsp. Salivarius UCC118 Microbiology 2002;148:973–984. immunologists, microbiologists, and 10 gene-deficient mice. Gastroenterology 21 Gardiner GE, O’Sullivan E, Kelly J, et al. 1999;116:1107–14. gastroenterologists can collaborate. Comparative survival rates of human-derived 10 McCarthy J, O’Mahony L, O’Callaghan L, et al. probiotic Lactobacillus paracasei and L. Gut 2004;53:620–622. Double blind placebo-controlled trial of two salivarius strains during heat treatment and probiotic strains in interleukin 10 knockout mice doi: 10.1136/gut.2003.034249 spray drying. Appl Environ Microbiol and mechanistic link with cytokine balance. Gut 2000;66:2605–12. 2003;52:975–980...... 22 Christensen HR, Frokiaer H, Pestka JJ. Lactobacilli 11 Dieleman LA, Goerres MS, Arendis A, et al. Authors’ affiliations differentially modulate expression of cytokines Lactobacillus GG prevents recurrence of colitis in and maturation surface markers in murine S Ghosh, D van Heel, R J Playford, HLA-B27 transgenic rats after antibiotic dendritic cells. J Immunol 2002;168:171–8. Gastroentrology Section, Division of Medicine, treatment. Gut 2003;52:370–6. 23 Veckman V, Miettinen M, Matikainen S, et al. Imperial College Faculty of Medicine, 12 Sheil B, McCarthy J, O’Mahony L, et al. Is the Lactobacilli and Streptococci induce inflammatory Hammersmith Hospital, London, UK mucosal route of administration essential for chemokine production in human macrophages probiotic function? Subcutaneous administration that stimulates Th1 cell chemotaxis. J Leukoc Biol is associated with attenuation of murine colitis 2003;74:395–402. Correspondence to: Professor S GhoshImperial and arthritis. Gut 2004;53:694–700. 24 Schultz M, Linde HJ, Lehn N, et al. College London, Hammersmith Hospital, 13 Borruel N, Carol M, Casellas F, et al. Increased Immunostimulatory consequences of oral Ducane Rd, London W12 0NN, UK; mucosal tumour necrosis factor alpha production administration of Lactobacillus rhamnosus strain [email protected] in Crohn’s disease can be downregulated ex vivo GG in healthy volunteers. J Dairy Res by probiotic bacteria. Gut 2002;51:659–64. 2003;70:165–73. 14 Resta-Lenert S, Barrett KE. Live probiotics protect 25 Ogura Y, et al. A frameshift mutation in NOD2 REFERENCES intestinal epithelial cells from the effects of associated with susceptibility to Crohn’s disease. infection with enteroinvasive Escherichia coli Nature 2001;411:603–6. http://gut.bmj.com/ 1 Ghosh S, Playford RJ. Bioactive natural (EIEC). Gut 2003;52:988–97. 26 Hugot JP, et al. Association of NOD2 leucine-rich compounds for the treatment of gastrointestinal 15 Boudeau J, Glasser AL, Julien S, et al. Inhibitory repeat variants with susceptibility to Crohn’s disorders. Clin Sci (Lond) 2003;104:547–6. effect of probiotic Escherichia coli strain Nissle disease. Nature 2001;411:599–603. 2 Gionchetti P, Rizzello F, Helwig U, et al. 1917 on adhesion to and invasion of intestinal 27 Inohara N, et al. Host recognition of bacterial Prophylaxis of pouchitis onset with probiotic epithelial cells by adherent-invasive E.coli strains muramyl dipeptide mediated through NOD2: therapy: a double-blind, placebo-controlled trial. isolated from patients with Crohn’s disease. implications for Crohn’s disease. J Biol Chem Gastroenterology 2003;124:1202–9. Aliment Pharmacol Ther 2003;18:45–56. 2003;278:5509–12. 3 Mimura T, Rizzello F, Helwig U, et al. Once daily 16 Rachmilewitz D, Karmeli F, Takbayashi K, et al. 28 Girardin SE, Travassos LH, Herve M, et al. high dose probiotic therapy (VSL#3) for Immunostimulatory DNA ameliorates Peptidoglycan molecular requirements allowing

maintaining remission in recurrent or refractory experimental and spontaneous murine colitis. detection by Nod1 and Nod2. J Biol Chem on September 28, 2021 by guest. Protected copyright. pouchitis. Gut 2004;53:108–14. Gastroenterology 2002;122:1428–41. 2003;278:41702–8.

Crohn’s disease especially in CD which is characterised ...... by both active fibrogenesis (that is, ECM production and deposition), leading, for example, to stricture formation, and by Fistulising Crohn’s disease: MMPs gone enhanced fibrolyis (that is, breakdown and removal of ECM), such as occurs in awry fistula formation. While fibroblasts and myofibroblasts, and to a minor degree D Schuppan, T Freitag endothelial and epithelial cells, produce the intestinal ECM, many more cell ...... types are involved in ECM breakdown Broadening our understanding of the role of matrix by releasing a broad spectrum of enzymes that can degrade essentially metalloproteinases in Crohn’s disease every ECM component, such as the various collagens, non-collagenous istulae are a common complication fistulae rarely heal, while surgical resec- (glyco-) proteins, glycosaminoglycans, of Crohn’s disease (CD), and their tion is effective but frequently does not and proteoglycans. The most important most common perianal manifesta- prevent local recurrence or fistulising of these enzymes are the matrix metal- F 1 tion is present in 14–38% of CD patients disease at other sites. Remodelling of loproteinases (MMPs), a structurally in referral populations.1 Despite the extracellular matrix (ECM) is a key related class of at least 20 zinc depen- advances in conservative treatment, event in chronic bowel inflammation,23 dent proteases.45 MMPs are classified

www.gutjnl.com COMMENTARY 623 according to their primary specificities MMP-9 as key players in fistulae of CD blasts/myofibroblasts can secrete atypi- Gut: first published as 10.1136/gut.2003.032219 on 13 April 2004. Downloaded from and structural features into interstitial and other causes. At first glance the cal chemokines attracting fibrolytic collagenases (MMP-1, MMP-8, MMP- occurrence of fistulae, a manifestation inflammatory cells on the one hand, 13, MMP-18), gelatinases (degrading of enhanced fibrolytic activity, in a and have a fibrolytic and migratory denatured and basement membrane fibrostenotic intestinal disease may phenotype themselves on the other.19 collagens: MMP-2, MMP-9), stromely- appear paradoxical. However, severe MMP-3, which has a broad substrate sins (degrading a broad spectrum of inflammation which always leads to specificity and which acts as a proacti- ECM substrates: MMP-3, MMP-7, release and activation of MMPs by cells vator (in addition to plasmin), at least of MMP-10, MMP-11), elastases (MMP- of the inflammatory infiltrate can drive MMP-1, MMP-2, and MMP-9, has been 12), and membrane-type (transmem- both processes: fistulae will form when identified as a particularly aggressive brane) MMPs (with broad substrate there is no rapid compensatory fibro- enzyme in intestinal inflammation3 specificities: MMP-14, MMP-15, MMP- genic response to fill up the defect, which favours invasiveness.20 This does 16, MMP-17, MMP-24, MMP-25). perhaps favoured by a too quick re- not exclude an important contribution As uncontrolled MMP activity would epithelialisation or re-endothelialisa- by MMP-12, MMP-13, MMP-14, and virtually lead to dissolution of organs, tion; fibrosis will develop when the MMP-19 which are expressed in MMP activity is strictly controlled, not fibrogenic response is strong and quick, inflamed intestine21 but were not con- only at the transcriptional and transla- leading to scar formation. Both pro- sidered in this study. tional levels, but also by the require- cesses can obviously coexist in close Can we derive therapeutic concepts ment of proteolytic activation of the proximity. What are the factors deter- from this and related studies on intest- zymogens and by several protease inhi- mining these divergent pathways? inal inflammation? A drug that effec- bitors. Proteolytic activation occurs by Recent studies demonstrated that at tively prevents emerging or eliminates the plasminogen activator-plasmin cas- least early activation of MMPs is neces- chronic fistulae would be most wel- cade and by certain active MMPs them- sary to allow for a fibrogenic come. Anti-inflammatory, antibiotic, selves, such as MMP-3, MMP-10, MMP- response,14 15 either by destroying base- and immunosuppressive approaches 14, and MMP-24. The most important ment membranes which have a role in can lead to long term fistula control or physiological inhibitors are the tissue maintaining cellular quiescence and even closure in 25–50% of patients, inhibitors of metalloproteinases (TIMP), differentiation in the gut16 with subse- roughly doubling the placebo response mainly TIMP-1, which blocks almost all quent mesenchymal activation, or by which is unsatisfactory.1 Anti-tumour MMPs, and TIMP-2, which is specific allowing mesenchymal cell migration necrosis factor a (TNF-a) strategies can for MMP-2. A fifth level of control is and proliferation by removal of con- lead to good short term and occasionally localisation of secreted MMPs either at straining ECM. long term results in cases that are cell membrane compartments that are Thus the cellular source and temporo- refractory to these therapies but treat- involved in local ECM proteolysis (for spatial pattern of MMP release and ment is costly and may have grave side 1

example, by binding to ECM receptors activation probably explain these diver- effects. None the less, this approach is http://gut.bmj.com/ that mediate cellular locomotion gent pathologies. As mesenchymal cells rational as TNF-a is a potent inducer of through the matrix67) or on certain (fibroblasts, myofibroblasts, and to a MMP-3 in intestinal myofibroblasts.3 ECM components, such as collagens. lesser extent endothelial cells) and Based on this and other studies, four Interestingly, it is preferentially the inflammatory cells (mainly macro- novel strategies aimed at inhibiting inactive proforms of MMPs that bind phages, monocytes, and neutrophils) intestinal inflammation and remodel- to collagen which become released from are the major sources of MMPs and ling in inflammatory bowel disease in the ECM on proteolytic activation.89 TIMPs, there must be a differently tuned general, or in fistulising CD in particu- This illustrates that both homeostasis interplay of these cells driving either lar, merit further exploration. These are on September 28, 2021 by guest. Protected copyright. and proteolytic remodelling of the ECM fistula or fibrosis. Kirkegaard et al the use of: (1) inhibitors of growth are highly sophisticated and tightly observed that acute fistulising inflam- factors or cytokines, such as epidermal regulated processes, and that simply mation in CD and other aetiologies is growth factor, hepatocyte growth factor, measuring upregulation or downregula- characterised by high expression of insulin-like growth factor (IGF-I and tion of MMPs and their inhibitors will MMP-3 and MMP-9 coupled with high IGF-II), fibroblast growth factor (aFGF, give a very incomplete picture of ECM activity of MMP-2 and MMP-9 in bFGF), interleukin 1, and stem cell remodelling at best. In this line, no inflammatory cells, while in chronic factor that induce MMPs in epithelial characteristic difference is found fistulae, MMP-9 almost disappeared and mesenchymal cells22; (2) synthetic between procollagen mRNA levels as a but MMP-3 expression was maintained, inhibitors of kinases that trigger MMP measure of fibrogenesis2 and MMP and with a shift to mesenchymal, mainly production, especially p38 kinase23; (3) TIMP mRNA levels as a measure of myofibroblastic, cells.13 The finding that synthetic MMP inhibitors that were fibrolysis10–12 in inflamed colonic speci- TIMPs remained low in all fistula speci- shown to ameliorate experimental coli- mens from patients with CD and ulcera- mens is of interest as TIMP-1 in tis in mice24; and (4) inhibitors of tive colitis. This was initially unexpected particular is a major determinant of integrins (that is, cellular receptors for as CD often leads to stenosis due to fibrogenesis, as exemplified in liver ECM proteins that can bind to and/or excess ECM deposition while fibrosis is fibrosis.17 Low expression of TIMP-1 is induce certain MMPs).6 25–28 Inhibition uncommon in ulcerative colitis. But it also characteristic of osteoarthritis, the of growth factors/cytokines and kinases comes as no surprise when one con- hallmark of which is unopposed MMP always carries the risk of side effects siders the complex regulation of fibro- activation in the joint leading to because of their importance in normal lysis in the transmural inflammation of destruction of cartilage and bone, some- tissue regeneration and of their ubiqui- CD. what reminiscent of fistula formation in tous expression in other cells and The paper by Kirkegaard and collea- the gut.18 Also, it is osteoarthritis that organs. Inhibition of MMPs appears gues13 in this issue of Gut brings our has led to a novel concept of chronic more promising, especially as the first understanding of the role of MMPs in fibrolytic inflammation that is driven by generation of broad spectrum inhibitors CD a step further [see page 701]. Using altered synovial fibroblasts, the equiva- which have multiple side effects are complementary techniques, they identi- lent of which could be myofibroblasts in becoming replaced by orally available fied and investigated MMP-3 and chronic fistulising diseases. These fibro- subtype specific agents.18 However, the

www.gutjnl.com 624 COMMENTARY Gut: first published as 10.1136/gut.2003.032219 on 13 April 2004. Downloaded from necessity for cell-type specific targeting 2 Matthes H, Herbst H, Schuppan D, et al. Cellular 16 Fritsch C, Simon-Assmann P, Kedinger M, et al. localization of procollagen gene transcripts in Cytokines modulate fibroblast phenotype and (for example, to MMP-3 expressing inflammatory bowel dieseases. Gastroenterology epithelial-stroma interactions in rat intestine. myofibroblasts of fistulae) remains an 1992;102:431–42. Gastroenterology 1997;112:826–38. unsolved problem. Therefore, it is attrac- 3 Pender SL, Tickle SP, Docherty AJ, et al. A major 17 Benyon RC, Arthur MJ. Extracellular matrix role for matrix metalloproteinases in T cell injury degradation and the role of hepatic stellate cells. tive to target integrins that promote in the gut. J Immunol 1997;158:1582–90. Semin Liver Dis 2001;21:373–84. MMP-expression as many of these 4 Brinckerhoff CE, Matrisian LM. Matrix 18 Baker AH, Edwards DR, Murphy G. receptors are cell-type specific. Thus metalloproteinases: a tail of a frog that became a Metalloproteinase inhibitors: biological actions prince. Nat Rev Mol Cell Biol 2002;3:207–14. and therapeutic opportunities. J Cell Sci MMP-1, MMP-2, and MMP-3, MMP- 5 Nagase H, Woessner JF. Matrix 2002;115:3719–27. 14, MMP-1 and MMP-3, and MMP-9 metalloproteinases. J Biol Chem 19 Buckley CD. Why does chronic inflammatory joint are induced and activated by integrins 1999;274:21491–4. disease persist? Clin Med 2003;3:361–6. 6 Dumin JA, Dickeson SK, Stricker TP. Pro- 20 Mercapide J, Lopez De Cicco R, Castresana JS, a2b1, a4b1, a6b1, and avb3, respec- collagenase-1 (matrix metalloproteinase-1) binds et al. Stromelysin-1/matrix metalloproteinase-3 tively, which are predominantly found the alpha(2)beta(1) integrin upon release from (MMP-3) expression accounts for invasive on activated (myo-) fibroblasts.6 25–28 keratinocytes migrating on type I collagen. J Biol properties of human astrocytoma cell lines. Chem 2001;276:29368–74. Int J Cancer 2003;106:676–82. Such inhibitors have (in part) been 7 Yu Q, Stamenkovic I. Localization of matrix 21 Salmela MT, MacDonald TT, Black D, et al. developed, and orally available agents metalloproteinase 9 to the cell surface provides a Upregulation of matrix metalloproteinases in a are in phase I and II clinical studies (for mechanism for CD44-mediated tumor invasion. model of T cell mediated tissue injury in the gut: Genes Dev 1999;13:35–48. analysis by gene array and in situ hybridisation. example, for metastatic tumour dis- 8 Schuppan D, Ruehl M, Somasundaram R, et al. Gut 2002;51:540–7. 29 ease). It remains to combine the most Matrix as a modulator of hepatic fibrogenesis. 22 Liu Z, Klominek J. Regulation of matrix promising of these agents, alone or in Semin Liver Dis 2001;21:351–72. metalloprotease activity in malignant combination, with established therapies 9 Ruehl M, Somasundaram R, Farndale R, et al. mesothelioma cell lines by growth factors. Thorax Pro-matrix-metalloproteinase-2 (MMP-2) is bound 2003;58:198–203. in preclinical and clinical studies, for our to liver collagens via collagenous consensus 23 Lee HS, Miau LH, Chen CH, et al. Differential role patients with complicated inflammatory mitives and released from extracellular matrix of p38 in IL-1alpha induction of MMP-9 and bowel disease. stores by activation. (abstract). Gastroenterology MMP-13 in an established liver myofibroblast cell 2002;122(suppl 1):A653. line. J Biomed Sci 2003;10:757–65. 10 Gu¨nther U, Schuppan D, Matthes H, et al. 24 Di Sebastiano P, di Mola FF, Artese L, et al. ACKNOWLEDGEMENTS Fibrogenesis and fibrolysis in collagenous colitis: Beneficial effects of Batimastat (BB-94), a matrix T Freitag was supported by the ELAN Fonds patterns of procollagen type I and IV, matrix- metalloproteinase inhibitor, in rat experimental metalloproteinase-1, and TIMP-1 gene colitis. Digestion 2001;63:234–9. of the University of Erlangen-Nuernberg. expression. Am J Pathol, 155:493–503. 25 Lauer-Fields JL, Sritharan T, Stack MS, et al. Gut 2004;53:622–624. 11 Heuschkel RB, MacDonald TT, Monteleone G, Selective hydrolysis of triple-helical substrates by et al. Imbalance of stromelysin-1 and TIMP-1 in matrix metalloproteinase-2 and -9. J Biol Chem doi: 10.1136/gut.2003.034207 the mucosal lesions of children with inflammatory 2003;278:18140–5. bowel disease. Gut 2000;47:57–62. 26 Pender SL, Salmela MT, Monteleone G, et al...... 12 von Lampe B, Barthel B, Coupland SE, et al. Ligation of alpha4b1 integrin on human Authors’ affiliations Differential expression of matrix intestinal mucosal mesenchymal cells metalloproteinases and their tissue inhibitors in selectively up-regulates membrane type-1 D Schuppan, T Freitag, Department of colon mucosa of patients with inflammatory bowel matrix metalloproteinase and confers a

Medicine I, University of Erlangen-Nu¨rnberg, disease. Gut 2000;47:63–73. migratory phenotype. Am J Pathol http://gut.bmj.com/ Germany 13 Kirkegaard T, Hansen A, Bruun E, et al. 2000;157:1955–62. Expression and localisation of matrix 27 Chen CC, Chen N, Lau LF. The angiogenic metalloproteinases and their natural inhibitors in factors Cyr61 and connective tissue growth Correspondence to: Professor fistulae of patients with Crohn’s disease. Gut factor induce adhesive signaling in primary D SchuppanDepartment of Medicine I, 2004;53:701–9. human skin fibroblasts. J Biol Chem University of Erlangen-Nu¨rnberg, Ulmenweg 14 Swiderski RE, Dencoff JE, Floerchinger CS, et al. 2001;276:10443–52. 18, 91054 Erlangen, Germany; detlef. Differential expression of extracellular matrix 28 Rolli M, Fransvea E, Pilch J, et al. Activated [email protected]–erlangen.de remodeling genes in a murine model of integrin alphavbeta3 cooperates with bleomycin-induced pulmonary fibrosis. metalloproteinase MMP-9 in regulating Am J Pathol 1998;152:821–8. migration of metastatic breast cancer cells. on September 28, 2021 by guest. Protected copyright. REFERENCES 15 Ammarguellat FZ, Gannon PO, Amiri F, et al. Proc Natl Acad Sci U S A Fibrosis, matrix metalloproteinases, and 2003;100:9482–7. 1 Sandborn WJ, Fazio VW, Feagan BG, et al. AGA inflammation in the heart of DOCA-salt 29 Tucker GC. Alpha v integrin inhibitors and technical review on perianal Crohn’s disease. hypertensive rats: role of ET(A) receptors. cancer therapy. Curr Opin Invest Drugs Gastroenterology 2003;125:1508–30. Hypertension 2002;39:679–84. 2003;4:722–31.

Banner duodenum.1 For many years this puta- ...... tive factor remained elusive. However, recent studies indicate that a peptide hormone, hepcidin, may play a crucial Hepcidin: what every regulatory role in normal iron homeostasis, haemochromatosis, and the gastroenterologist should know anaemia of chronic disease.2 Hepcidin was identified not from A P Walker, J Partridge, S K Srai, J S Dooley studies of iron homeostasis but from investigation of novel antimicrobial ...... peptides in body fluids. Krause and The circulating peptide hepcidin is important in the normal colleagues3 screened human blood ultra- filtrate (a source of the antimicrobial response to iron overload, the pathogenesis of peptides defensins) for small cysteine haemochromatosis, and possibly the anaemia of chronic disease rich peptides. A 25 amino acid peptide with a mass of 2.7 kDa was found, n health, the body content of iron is the toxic effects of excess. Early experi- containing a remarkable eight disul- maintained within fairly narrow lim- ments provided tantalising evidence for phide bonded cysteine residues. It Iits to provide sufficient iron for a humoral regulatory factor. Serum showed antimicrobial activity against synthesis of ferroproteins essential for obtained from iron repleted rats inhib- some bacteria and the yeast Saccha- oxygen transport and catalysis yet avoid ited iron absorption in normal rat romyces cerevisiae. Quantitative reverse

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transcription-polymerase chain reaction erythropoietin, decreased survival of The medical importance of hepcidin is Gut: first published as 10.1136/gut.2003.032219 on 13 April 2004. Downloaded from showed predominant expression in erythrocytes, impaired iron absorption, emphasised by recent studies in human liver. The peptide was called LEAP-1 and retention of iron in macrophages. iron overload. Homozygous mutations (liver-expressed antimicrobial peptide).3 The net effect would be to decrease the of hepcidin have been demonstrated in The cDNA sequence predicted an 84 iron available to erythroid precursor two families with juvenile haemochro- amino acid pre-propeptide with two cells.89 Two patients with severe iron matosis.13 This disorder is characterised cleavage sites. The first predicts cleavage refractory anaemia associated with by a rapid rate of iron loading, usually of the N terminal endoplasmic reticu- hepatic adenoma showed spontaneous causing presentation before the age of lum targeting signal sequence (amino resolution of anaemia after removal of 30 years. There is a high frequency of acids 1–24). The second consensus the tumour.9 The adenomatous tissue endocrine and cardiac iron overload, cleavage site for prohormone conver- expressed inappropriately high levels of and failure of early diagnosis and treat- tases would yield the central prodomain hepcidin mRNA. On the basis of these ment may be fatal.14 Juvenile haemo- (amino acids 25259) plus the 25 amino observations, it was proposed that hep- chromatosis is genetically hetero- acid C terminal peptide (also called cidin may be important in the patho- geneous with a second form mapping hepcidin-25; amino acids 60–84). A genesis of anaemia of chronic disease.9 to chromosome 1q21. Whether this slightly truncated 20 amino acid peptide Analysis of urinary hepcidin excretion second gene will encode another protein was also found (hepcidin 20; amino in patients with anaemia due to chro- in the hepcidin-HFE pathway remains acids 65–84). Independently, Park and nic infection or severe inflammatory to be resolved.15 The potential interac- colleagues4 isolated the same cysteine disease provided some confirmatory tion between hepcidin and HFE has also rich peptide from human urine and evidence of increased hepcidin levels been suggested by the description of named it hepcidin, reflecting its hepatic in response to inflammation in haemochromatosis associated with one expression and bactericidal properties. humans.10 In these patients, hepcidin mutated allele of both hepcidin and In addition to hepcidin-20 and hepci- excretion correlated with serum ferritin HFE, termed ‘‘digenic inheritance’’.16 din-25, a minor 22 amino acid form was concentration which, like hepcidin, The classical HFE related form of the found (hepcidin-22; amino acids 63– increases in response to both elevated disease accounts for more than 90% of 84). The antimicrobial activity of urinary body iron stores and inflammation. well characterised haemochromatosis hepcidin was inhibited by 100–150 mM Interestingly, injection of erythropoie- patients in the UK.17 In HFE related NaCl,4 similar to the normal range for tin into mice was shown to decrease haemochromatosis and in Hfe knockout plasma sodium (,135–145 mM). dramatically liver hepcidin expression. mice, there is failure to upregulate Whether circulating plasma hepcidin Therefore, the efficacy of erythropoietin hepcidin levels despite hepatic iron plays a significant antimicrobial role in in non-renal anaemias such as cancer overload. This indicates that the HFE vivo therefore remains unclear. and autoimmune diseases may result protein is involved in regulation of hep- Experiments in mice have shown that from both stimulation of erythropoie- cidin levels in response to iron. Disrup- hepcidin plays a role in iron regulation. sis and inhibition of hepcidin expres- tion of the normal hepcidin response to Pigeon et al identified mouse liver sion, leading to increased plasma iron body iron stores may contribute to the http://gut.bmj.com/ hepcidin mRNA because it was induced levels.11 pathogenesis of iron overload seen in by carbonyl iron overload.5 The mouse Regulation of hepcidin expression has HFE related haemochromatosis.18–20 has two hepcidin genes in tandem been investigated in cultured cells. Recognition of liver produced hepci- (probably arising from a duplication Surprisingly, iron loading of primary din as an iron regulatory factor has event), located adjacent to the upstream human hepatocytes with either ferric challenged the hypothesis that the con- stimulatory factor 2 (Usf2) transcription ammonium citrate or diferric transferrin trol of iron absorption in relation to 5 10 factor gene. Mice that lack hepatic decreased hepcidin mRNA levels. This body stores is mediated in duodenal on September 28, 2021 by guest. Protected copyright. hepcidin expression, due to targeted is in contrast with experiments in mice crypts. Hepcidin has revived the concept disruption of the adjacent Usf2 gene, in which dietary or parenteral iron that some regulation may be mediated developed predominant iron loading of loading increased hepcidin mRNA by the liver. Rats switched from a parenchymal cells of the liver and levels.5 This suggested that during iron control to an iron deficient diet showed pancreas with relative sparing of the overload in the whole animal, a signal changes in hepcidin expression in close macrophage rich spleen.6 Similar pat- from non-parenchymal cells may induce temporal relationship with changes in terns of iron overload in Hfe knockout hepatocytes to express hepcidin.10 duodenal iron transporter expression. and Usf2 (hepcidin deficient) knockout Mouse liver hepcidin mRNA is induced This suggested that hepcidin may act mice suggested that hepcidin and Hfe not only by iron overload but also by directly on villus enterocytes, pointing may function in the same regulatory treatment with bacterial lipopolysac- to the liver as a regulatory site.21 pathway. Hepcidin was proposed to be a charide, hinting that iron and inflam- Certainly, siderosis in Kupffer cells, negative regulator (that is, ‘‘inhibitor’’) matory cytokines may have a common often on a background of mild iron of iron absorption in the duodenum and signalling pathway.5 Hepcidin mRNA in deposition in hepatocytes, is a familiar of iron release from macrophages.6 This primary human hepatocytes was feature of common liver diseases such as was supported by demonstration that induced either by medium conditioned hepatitis C infection and alcoholic liver transgenic mice constitutively expres- by monocytes exposed to lipopolysac- disease.22 Whether increased concentra- sing liver hepcidin died within a few charide or by interleukin 6 (IL-6).10 At tion of hepcidin, perhaps in response to hours of birth with decreased body iron the level of regulation by transcription inflammation, has a role in the hepatic levels and severe microcytic hypochro- factors, the promoters of human and siderosis observed in viral and alcoholic mic anaemia.7 mouse hepcidin genes contain at least liver disease remains to be demon- Further observations implicate hepci- one functional binding site for CCAAT/ strated. din in the anaemia of chronic disease. enhancer binding protein a (C/EBPa), a To date, investigation of hepcidin in This is the most common anaemia of transcription factor highly expressed in various clinical conditions has been hospitalised patients, often associated adult liver which appears to stimulate impeded by the lack of anti-hepcidin with infection, cancer, and autoimmune hepcidin expression. Hepatocyte nuclear antibodies and suitable assays. Recently, diseases. There is a diminished res- factor 4a (HNF4a) appears to reduce a western blot densitometric assay was ponse of erythroid precursors to hepcidin expression.12 reported using an antibody raised

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against hepcidin-25, allowing quantita- three antisera on western blots.24 They ACKNOWLEDGMENTS Gut: first published as 10.1136/gut.2003.032219 on 13 April 2004. Downloaded from tion of human hepcidin in serum attributed the 10 kDa band to pro- Research in the authors’ laboratories is samples.23 In this issue of Gut, Kulaksiz hepcidin on the grounds that it was supported by European Commission Grant and colleagues24 describe antisera raised detected by antisera to both the mature QLK6-CT-1999-02237, Wellcome Trust Grant 059762/Z/99/Z, and BBSRC Project Grant 90/ against two unique synthetic peptides hepcidin peptide and to the prodo- D1340. corresponding to the predicted human main. However, the predicted mass hepcidin prodomain (two antisera to the of pro-hepcidin is 6.9 kDa; that of Gut 2004;53:624–627. same peptide) and the mature hepcidin pre-pro-hepcidin is 9.4 kDa (ExPASy doi: 10.1136/gut.2003.030304 C terminal peptide [see page 735]. All programme: http://ca.expasy.org/tools/ ...... three antisera detected immunoreactive peptide-mass.html).27 It might be infor- Authors’ affiliations bands estimated at 10 and 20 kDa on mative to compare the mobility of A P Walker, J Partridge, J S Dooley, Centre western blots of guinea pig and human synthetic peptides versus the bands for Hepatology, Department of Medicine, liver and HepG2 cells. The 10 kDa band observed in human serum in this sys- Royal Free and University College Medical only was detected faintly in human tem.24 Perhaps attention to denatura- School, University College London, London, serum. Immunohistochemistry of tion conditions and accurate verification UK guinea pig and human liver with all of experimentally determined mole- S K Srai, Department of Biochemistry and three antisera showed staining in hepa- cular masses may help to clarify the Molecular Biology, Royal Free and University tocytes with a basolateral expression discrepancies. College Medical School, University College pattern. This confirms and extends ear- Is pro-hepcidin an active player in London, London, UK lier mRNA results, and is consistent iron regulation or merely a precursor with basolateral release of hepcidin into and an indirect measure of hepcidin Correspondence to: Dr A P Walker, Centre for 24 Hepatology, Department of Medicine, Royal the liver sinusoids. potential? Interestingly, a homozygous Free and University College Medical School A new enzyme linked immunosorbent point mutation of the human hepcidin (UCL), Royal Free Campus, Rowland Hill St, assay (ELISA) was developed using the prodomain has been detected, predict- London NW3 2PF, UK; [email protected] prodomain antiserum. The ELISA was ing a threonine to methionine substitu- used to investigate serum levels of pro- tion at amino acid 31 (T31M). This REFERENCES hepcidin immunoreactivity in controls individual had normal serum iron 1 MacDermott RP, Greenberger NJ. Evidence for a and patients with haemochromatosis, indices, normal haemoglobin, and nor- humoral factor influencing iron absorption. renal anaemia, and chronic renal insuf- mal mean corpuscular volume.28 Gastroenterology 1969;57:117–25. ficiency. The mean level of pro-hepcidin However, this residue is not conserved 2 Ganz T. Hepcidin, a key regulator of iron metabolism and mediator of anemia of immunoreactivity detected in the serum in evolution: both wild-type mouse inflammation. Blood 2003;102:783–8. of healthy volunteers was an order of hepcidin genes have a methionine at 3 Krause A, Neitz S, Magert HJ, et al. LEAP-1, a magnitude greater than levels of hepci- the position equivalent to human threo- novel highly disulfide-bonded peptide, exhibits antimicrobial activity. FEBS Lett din detected in human urine.4 Pro- nine 31. The normal phenotype asso-

2000;480:147–50. http://gut.bmj.com/ hepcidin immunoreactivity was ciated with homozygous T31M mutation 4 Park CH, Valore EV, Waring AJ, et al. Hepcidin, increased in chronic renal insufficiency therefore does not resolve the role of a urinary antimicrobial peptide synthesised in the liver. J Biol Chem 2001;276:7806–10. patients who had normal haemoglobin pro-hepcidin. 5 Pigeon C, Ilyin G, Courselaud B, et al. A new levels compared with the healthy con- In summary, hepcidin is a peptide mouse liver-specific gene, encoding a protein trol group, despite erythropoietin treat- hormone with important biological homologous to human antimicrobial peptide hepcidin, is overexpressed during iron overload. ment, which has been observed to effects on iron metabolism. It has J Biol Chem 2001;276:7811–19. decrease hepatic hepcidin expression.11 sequence homology to antimicrobial 6 Nicolas G, Bennoun M, Devaux I, et al. Lack of

These observations are therefore consis- peptides, although whether this func- hepcidin gene expression and severe tissue iron on September 28, 2021 by guest. Protected copyright. overload in upstream stimulatory factor 2 (USF2) tent with renal metabolism and/or tion is conserved in vivo remains to be knockout mice. Proc Natl Acad Sci USA elimination of peptide. Pro-hepcidin demonstrated. Hepcidin is derived by 2001;98:8780–5. was decreased in HFE related haemo- cleavage of a pre-propeptide; questions 7 Nicolas G, Bennoun M, Porteu A, et al. Severe iron deficiency anemia in transgenic mice chromatosis patients (C282Y homozy- remain about the processing and expressing liver hepcidin. Proc Natl Acad Sci USA 24 gous) compared with healthy controls, activity of the peptide products. It has 2002;99:4596–601. consistent with earlier mRNA studies,18 been implicated as a regulator of body 8 Weiss G. Pathogenesis and treatment of and indicating defective hepcidin regu- anaemia of chronic disease. Blood Rev iron stores and mutations have been 2002;16:87–96. lation in HFE related haemochromato- found in some families with juvenile 9 Weinstein DA, Roy CN, Fleming MD, et al. sis. haemochromatosis. Iron balance is Inappropriate expression of hepcidin is Questions and inconsistencies remain probably sensed by Kupffer cells and associated with iron refractory anemia: implications for the anemia of chronic disease. regarding the size and identity of signalled to hepatocytes by cytokines Blood 2002;100:3776–81. hepcidin immunoreactive bands on wes- such as IL-6. Hepcidin expression is 10 Nemeth E, Valore EV, Territo M, et al. Hepcidin, tern blots. The sequence of hepcidin-25 also induced by inflammatory agents a putative mediator of anemia of inflammation, is a type II acute-phase protein. Blood predicts a mass of 2.7 kDa, correspond- giving it a potential role in the anaemia 2003;101:2461–3. ing to the result determined by mass of chronic disease. The reagents and 11 Nicolas G, Viatte L, Bennoun M, et al. Hepcidin, a spectrometry.3 Furthermore, the oxi- assays described24 will permit investiga- new iron regulatory peptide. Blood Cells Mol Dis 2002;29:327–35. dised synthetic hepcidin-25 co-migrates tions of the role of hepcidin in clinical 12 Courselaud B, Pigeon C, Inoue Y, et al. C/EBP with native hepcidin-25 in analytical samples in relation to the siderosis alpha regulates hepatic transcription of hepcidin, high pressure liquid chromatography observed in viral and alcoholic liver an antimicrobial peptide and regulator of iron 25 metabolism. Cross-talk between C/EBP pathway and capillary electrophoresis. On wes- diseases. This peptide is a fascinating and iron metabolism. J Biol Chem tern blots however, serum hepcidin-25 addition to the family of molecules 2002;277:41163–70. immunoreactivity has been detected involved in iron metabolism. Future 13 Roetto A, Papanikolaou G, Politou M, et al. Mutant antimicrobial peptide hepcidin is with an apparent mass of 10 kDa, co- studies of its action and regulation in associated with severe juvenile hemochromatosis. migrating with the synthetic peptide,23 vivo and in vitro should fill in some Nat Genet 2003;33:21–2. possibly reflecting aggregation of hepci- of the tantalising gaps in our under- 14 Kelly AL, Rhodes DA, Roland JM, et al. 26 Hereditary juvenile haemochromatosis: a din monomers. Kulaksiz et al detected standing of this increasingly complex genetically heterogeneous life-threatening iron- bands of 10 kDa and 20 kDa with their field. storage disease. QJM 1998;91:607–18.

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15 Roetto A, Totaro A, Cazzola M, et al. Juvenile 20 Muckenthaler M, Roy CN, Custodio AO, et al. 25 Kluver E, Schulz A, Forssmann WG, et al. Gut: first published as 10.1136/gut.2003.032219 on 13 April 2004. Downloaded from hemochromatosis locus maps to chromosome 1q. Regulatory defects in liver and intestine implicate Chemical synthesis of beta-defensins and Am J Hum Genet 1999;64:1388–93. abnormal hepcidin and Cybrd1 expression in LEAP-1/hepcidin. J Peptide Res 16 Merryweather-Clarke AT, Cadet E, Bomford A, et mouse hemochromatosis. Nat Genet 2002;59:241–8. al. Digenic inheritance of mutations in HAMP and 2003;34:102–7. 26 Hunter HN, Fulton DB, Ganz T, et al. The solution HFE results in different types of 21 Frazer DM, Wilkins SJ, Becker EM, et al. Hepcidin structure of human hepcidin, a peptide hormone haemochromatosis. Hum Mol Genet expression inversely correlates with the expression with antimicrobial activity that is involved in iron 2003;12:2241–7. of duodenal iron transporters and iron absorption uptake and hereditary hemochromatosis. J Biol 17 The UK Haemochromatosis Consortium.A in rats. Gastroenterology 2002;123:835–44. Chem 2002;277:37597–603. simple genetic test identifies 90% of UK patients 22 Turlin B, Deugnier Y. Evaluation and 27 Wilkins MR, Lindskog I, Gasteiger, E, et al. with haemochromatosis. Gut 1997;41:841–4. interpretation of iron in the liver. Semin Diagn Detailed peptide characterisation using 18 Bridle KR, Frazer DM, Wilkins SJ, et al. Disrupted Pathol 1998;15:237–45. PEPTIDEMASS—a World-Wide Web hepcidin regulation in HFE-associated 23 Dallalio G, Fleury T, Means RT. Serum hepcidin in accessible tool. Electrophoresis haemochromatosis and the liver as a regulator of clinical specimens. Br J Haematol 1997;18:403–8. 28 Lee P, Gelbart T, West C, et al. A study of genes body iron homoeostasis. Lancet 2003;122:996–1000. that may modulate the expression of hereditary 2003;361:669–73. 24 Kulaksiz H, Gehrke SG, Janetzko A, et al. Pro- hemochromatosis: transferrin receptor-1, 19 Ahmad KA, Ahmann JR, Migas MC, et al. hepcidin: expression and cell specific localisation ferroportin, ceruloplasmin, ferritin light and heavy Decreased liver hepcidin expression in the Hfe in the liver and its regulation in hereditary chains, iron regulatory proteins (IRP)-1 and -2, knockout mouse. Blood Cells Mol Dis haemochromatosis, chronic renal insufficiency, and hepcidin. Blood Cells Mol Dis 2002;29:361–6. and renal anaemia. Gut 2003;53:735–43. 2001;27:783–802.

Gastrointestinal symptoms the presence of positive features indicat- ...... ing a functional bowel disorder before deciding that the patient is at low risk of significant organic disease. Bringing science to the art of diagnosis These data will provide useful information for the Rome group9 when they P Moayyedi next consider guidelines for the assessment of irritable bowel syndrome (IBS)...... They have highlighted symptoms simi- Which gastrointestinal symptoms are useful in distinguishing lar to the classical Manning criteria that are important in making a positive organic from functional disease? diagnosis of functional gastrointestinal disease but also other characteristics atients and clinicians are becoming organic from functional disease. The such as radiating and frequent pain that increasingly intolerant of diagnostic strength of this study is that it evaluates make IBS more likely. Unfortunately, http://gut.bmj.com/ Puncertainty. This is reflected in the a large relatively unselected group of the model is probably not sufficiently rising demand for endoscopic proce- patients, reducing spectrum and selec- accurate to be used to exclude patients dures1 implicitly suggesting that gastro- tion bias.8 It also divided patients into from investigation in clinical practice. intestinal symptoms are an unreliable those with and without organic disease Using the prevalence of lower gastro- indicator of serious pathology. rather than subdividing the data into intestinal organic disease in the article, Unfortunately, the growing array of different diagnoses. This improves the the model had a positive predictive

tests that are being demanded for power of the study and gives the value of 79% and a negative predictive on September 28, 2021 by guest. Protected copyright. patients are placing further pressures clinician overall information on whether value of 86%. The possibility of missing on already stretched health care bud- the patient is likely to have an organic organic pathology in 14% of patients gets. The natural reaction to this is to disease. would lead most clinicians to opt for evaluate whether we can improve on the Their data suggest that aspects of the investigating even if the model pre- value of the history to diagnose gastro- history are valuable diagnostic tools for dicted a low probability of disease. The intestinal disease. This process was lower gastrointestinal disease. They accuracy of the model may have been started over 30 years ago when identify three ‘‘alarm features’’ that improved if non-gastrointestinal symp- researchers such as Card and collea- were important independent predictors toms such as backache and frequency of gues23and de Dombal and colleagues45 of organic lower gastrointestinal pathol- micturition had been included10 but the evaluated the use of computers to aid ogy. These were age over 50 years, male concern about using models to predict the clinician in making diagnoses in sex, and blood on the toilet paper. They organic disease is emphasised by the patients with upper gastrointestinal also identified five ‘‘non-alarm’’ fea- conflicting messages from other studies. symptoms. The enthusiasm for this tures (frequent abdominal pain, radiat- For example, three papers also found approach faded when data suggested ing pain, pain with loose bowel that rectal bleeding11–13 predicted organic that computer improved diagnostic movements, reflux, and absence of disease whereas two studies14 15 found accuracy of computers was not suffi- diarrhoea) that independently predicted no statistically significant association. cient to prevent investigations.6 The functional bowel disease. The approach Hammer et al found that weight loss and paucity of subsequent research in this of positively diagnosing functional anorexia were not independent predic- field is disappointing as it would be bowel disease with non-alarm symp- tors of disease whereas others13 15 16 useful to know what information com- toms or positively diagnosing organic reported these symptoms were asso- puters were using that enhanced diag- disease with alarm symptoms per- ciated with organic pathology. Finally, nostic acumen. The article by Hammer formed equally well. The interesting Hammer and colleagues7 found that and colleagues7 in this issue of Gut [see observation is that putting these two severe abdominal pain was associated page 666] is therefore refreshing as it pieces of information together improved with a reduced risk of having organic prospectively evaluates a wide range of the accuracy of the model. This is pathology in the univariate analysis. gastrointestinal symptoms to establish intuitive, as a clinician is likely to weigh This observation is supported by one which are useful in distinguishing up the absence of alarm symptoms and study12 whereas another suggested

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abdominal pain is associated with colo- statistically significantly associated with 2 Card WI, Good IJ. Logical foundations of Gut: first published as 10.1136/gut.2003.032219 on 13 April 2004. Downloaded from 17 medicine. BMJ 1971;i:718–20. rectal disease. disease but when they are combined in a 3 Lucas RW, Card WI, Knill-Jones RP, et al. These inconsistent findings probably model they generally perform very Computer interrogation of patients. BMJ relate to differences in the design and poorly.20 This is particularly true when 1976;ii:623–5. 21 4 Horrocks JC, McCann AP, Staniland JR, et al. populations of the different studies. The the models are tested prospectively or Computer-aided diagnosis: description of an quality of the data collection on gastro- transferred from a hospital to a primary adaptable system, and operational experience intestinal symptoms, sample size, and care setting.22 with 2,034 cases. BMJ 1972;ii:5–9. Science has a great deal to offer the 5 Horrocks JC, de Dombal FT. Computer-aided prevalence of underlying organic disease diagnosis of ‘‘dyspepsia’’. Am J Dig Dis varied between studies. In particular, clinician in helping determine a diag- 1975;20:397–406. Hammer et al had very few colorectal nosis from the patient history. 6 Horrocks JC, de Dombal FT. Diagnosis of Statistical techniques can identify symp- dyspepsia from data collected by a physician’s cancers in their sample and the main assistant. BMJ 1975;iii:421–3. organic disease driving the model was toms and signs that are important to 7 Hammer J, Eslick GD, Howell SC, et al. inflammatory bowel disease. If ulcera- consider and those that do not contri- Diagnostic yield of alarm features in irritable tive colitis was the main disease found, bute to making a diagnosis. The problem bowel syndrome and functional dyspepsia. Gut 2003;53:666–72. then rectal bleeding and absence of pain is that a single study is not sufficient. 8 Moayyedi P, Duffy J, Delaney B. New will predict pathology and weight loss After all, we do not rely on one study to approaches to enhance the accuracy of the may not be an important feature in the definitively tell us that smoking causes diagnosis of reflux disease. Gut 2003;53(suppl iv):55–57. 12 model. It is interesting that the study lung cancer or Helicobacter pylori causes 9 Talley NJ, Stanghellini V, Heading RC, et al. that also had few colorectal cancers distal gastric adenocarcinoma. There is a Functional gastrointestinal disorders. Gut found similar results, whereas the need for a range of diagnostic studies 1999;45(suppl ii):ii37–42. 17 10 Maxton DG, Morris J, Whorwell PJ. More report that evaluated a larger number conducted in different settings using accurate diagnosis of irritable bowel syndrome of colorectal cancers found that features different designs. The common feature by the use of ‘‘non-colonic’’ symptomatology. Gut such as weight loss and abdominal pain of all should be the careful prospective 1991;32:784–6. collection of a detailed symptom assess- 11 Goulston KJ, Cook I, Dent OF. How important is were important. The case mix of disease rectal bleeding in the diagnosis of bowel cancer 23 in the underlying population is likely to ment. When sufficient data have been and polyps? Lancet 1986;ii:261–4. have a large influence on the symptoms accumulated, a picture will emerge on 12 Fijten GH, Starmans R, Muris JWM, et al. what are the important features that Predictive value of signs and symptoms for that computers models will predict are colorectal cancer in patients with rectal bleeding important. identify a patients as having organic in general practice. Fam Pract 1995;12:279–86. Upper gastrointestinal symptoms are disease. The problem that is addressed 13 Majumdar SR, Fletcher RH, Evans AT. How does also has to be amenable to this colorectal cancer present? Symptoms, duration, a good example of this. Hammer et al and clues to location. Am J Gastroenterol found that weight loss, dysphagia, age, approach. It will be difficult to use the 1999;94:3039–45. and sex were not predictors of organic history to guide us in patients with 14 Helfand M, Marton KI, Zimmer-Gembeck MJ, et upper gastrointestinal disease but the al. History of visible rectal bleeding in a primary pathology whereas other researchers care population. JAMA 1997;277:44–8. have found these to be significantly situation with lower gastrointestinal 15 Chak A, Post AB, Cooper GS. Clinical variables http://gut.bmj.com/ associated with the risk of upper gastro- pathology looks more promising. associated with colorectal cancer on colonoscopy: 18 19 Insights provided by diagnostic studies a prediction model. Am J Gastroenterol intestinal cancer. Indeed, it is almost 1996;91:2483–8. inconceivable that male sex and increas- may allow us to avoid invasive tests 16 Zarchy TM, Ershoff D. Which clinical variables ing age would not be risk factors for in some patients and identify others predict an abnormal double-contrast barium ? upper gastrointestinal malignancy given as having a high risk of serious enema result Ann Int Med 1991;114:137–41. colorectal disease that warrants urgent 17 Neugut AI, Garbowski GC, Waye JD, et al. what is known about the epidemiology Diagnostic yield of colorectal neoplasia with of these cancers.1 Hammer et al did not investigation. It is not sufficient to show colonoscopy for abdominal pain, change in find that these were important because the sensitivity and specificity of this bowel habits, and rectal bleeding. on September 28, 2021 by guest. Protected copyright. approach as the ultimate goal is to Am J Gastroenterol 1993;88:1179–83. the majority of their patients had 18 Meineche-Schmidt V, Jørgensen T. ‘‘Alarm gastro-oesophageal reflux and peptic provide more cost effective health care symptoms’’ in patients with dyspepsia: a three- 24 ulcer and these diseases were driving to patients. Randomised controlled year prospective study from general practice. trials will therefore be required to assess Scand J Gastroenterol 2002;37:999–1007. the model. It is not surprising therefore 19 Johanessen T, Petersen H, Kleveland PM, et al. that heartburn symptoms and aspirin whether a more thorough clinical his- The predictive value of history in dyspepsia. use were found to predict pathology tory will reduce costs or improve patient Scand J Gastroenterol 1990;25:689–97. outcomes. 20 Wallace MB, Durkalski VL, Vaughan J, et al. Age whereas alarm symptoms did not. and alarm symptoms do not predict endoscopic Although alarm symptoms may be of Gut 2004;53:627–628. findings among patients with dyspepsia: a more value than is suggested by this doi: 10.1136/gut.2003.032219 multicentre database study. Gut 2001;49:29–34. paper, the overall message is consistent Correspondence to: Professor P Moayyedi, 21 Numans ME, van der Graaf Y, de Wit NJ, et al. with other researchers. Factors that are Gastroenterology Division, McMaster How useful is selection based on alarm symptoms ? found to be predictive of functional or University-HSC 4W8, 1200 Main St West, in requesting gastroscopy Scand J Gastroenterol 2001;36:437–43. organic disease are not very useful Hamilton, Ontario, Canada L8N 3Z5; evansl@ 22 Bytzer P, Møller Hansen J, Schaffalitzky de clinically. The model used by Hammer mcmaster.ca Muckadell, et al. Predicting endoscopic diagnosis et al found it very difficult to distinguish in the dyspeptic patient. The value of predictive score models. Scand J Gastroenterol between groups and as most patients REFERENCES 1997;32:118–25. had organic disease it generally 23 Crean GP, Holden RJ, Knill-Jones RP, et al. A defaulted to assigning patients to the 1 Delaney BC, Moayyedi P. Dyspepsia. Health care database on dyspepsia. Gut needs assessment, 3rd series. Department of 1994;35:191–202. disease group. Others have found the Health, Radcliffe Medical Press, 2003 (http:// 24 Sackett DL, Haynes RB. The architecture of same problem: individual factors may be hcna.radcliffe-oxford.com/dysframe.htm). diagnostic research. BMJ 2002;324:539–41.