The Role of Cdx Genes in the Mammalian

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Cdx genes structures (forestomach epithelium)

...... occurring ectopically at more posterior Gut: first published as 10.1136/gut.2003.038240 on 10 September 2004. Downloaded from sites.11 While initially defined as adenomatous polyps, critical histological ana- The role of Cdx genes in the lysis together with studies on timed development of the polyps established mammalian gut an interesting process of so-called intercalary growth.13 It was concluded that F Beck the function of Cdx2 was to direct the ‘‘default state’’ forestomach endoderm ...... towards a caudal phenotype and that Cdx genes are important in pattern formation during the lower levels of expression of Cdx2 in the developing distal intestine of Cdx2+/2 development of the gut and may well contribute to the balance heterozygotes lead to reversion to a between differentiation and cell renewal in the mature intestine more anterior phenotype (that is, to forestomach epithelium). Subsequently, rganisation of multicellular ani- the time of its initiation at the stage of intercalary growth around the gut mals involves the action of genes hindgut invagination throughout devel- lesions results in ‘‘filling in’’ of tissue Othat impart ‘‘positional informa- opment and adult life.5 Cdx1 is not types at the discontinuity between the tion’’. All vertebrates are built on a expressed in the early definitive gut ectopic gastric and surrounding colonic segmental pattern that is most obviously endoderm but appears at postsomite epithelia and an orderly succession of expressed by the appearance of somites stages just before transition of the histologically normal epithelia charac- during embryonic development. multilayered intestinal endoderm to a teristic of cardia, corpus, antrum, and A common feature of genes that single layered epithelium at 14 days small intestine, in that order, appeared impart individual identity (and there- post coitum (dpc).6 Cdx4 is expressed all around the forestomach epithelium fore positional information) to specific in the earliest hindgut invagination7 but at the junction with colonic epithelium. segments is the possession of a ‘‘homeo- little is known of its distribution after The molecular basis of the intercalary box’’ DNA binding motif coding for a 10 dpc. growth is unclear but probably involves consensus sequence of 60–63 amino It is important to note that in addition local intercellular communication. acids that acts as a transcriptional to their role in the gut, Cdx genes are Clonal analysis experiments have regulator of ‘‘downstream’’ genes. The active at multiple other sites during been performed to establish the origin most widely researched homeobox early development where, inter alia, of the secondarily generated tissue genes are the so-called homeotic selec- they regulate the extent of ecto/meso- between ectopic forestomach and colo- tor genes of the Antp-type (the defining dermal expression of Hox genes and nic epithelia.14 Y chromosome painting gene is named Antennapaedia). In the thus also indirectly influence non- in wild-type/Cdx22/2 chimaeric* mice fruit fly Drosophila, these are situated on intestinal anteroposterior patterning.8 of opposite sex indicated that once chromosome 3 as part of the HOM However, this review is principally con- differentiated to express Cdx2, host cluster. HOM-C genes are strongly con- cerned with their role in the gut. colonic epithelium can only contribute http://gut.bmj.com/ served during evolution and in mam- Much insight into the function of a small intestinal epithelium to the sec- mals have been replicated to appear on gene may be obtained by studying the ondarily generated tissue while the Cdx2 separate chromosomes in four para- morphology of mice in which it has mutant chimaeric cells give rise to the logous complexes called Hox clusters. been inactivated by homologous recom- succession of gastric-type tissue but They are expressed principally in devel- bination.9 Knockout of the Cdx1 gene10 never to small intestinal morphology. oping ectodermal and mesodermal tis- produced anterior homeotic shifts (that These findings have interesting impli- sues and in general terms are is, a posterior structure such as a cations in the context of intestinal on September 25, 2021 by guest. Protected copyright. responsible for segmental specification vertebra or rib exhibiting the morphol- regeneration. of the dermatomes, musculoskeletal, ogy of a more anterior element) of the No information concerning the role of and nervous systems.1 However, Hox axial skeleton but no gut abnormalities. Cdx4 in gut development is currently genes are not expressed in the greater Inactivation of Cdx2 on the other hand available as knockout or RNAi knock- part of the gut endoderm but in their produced not only axial homeotic shifts down experiments have not been per- place, mammalian members of the Para- in heterozygotes (one affected allele) formed and no spontaneous mutants 2 Hox genes —an ‘‘evolutionary sister’’ of but was also found to prevent tropho- are available. the Hox clusters—seem to play an blast maturation and consequently blas- Having shown that Cdx2 is necessary important role in gut patterning. tocyst implantation if present in the for the establishment of the midgut Members of this group are Pdx1 which homozygous (both alleles affected) phenotype, we must now ask whether is required for the correct development form. Most importantly in the context it is sufficient to convert the ‘‘default’’ 3 of the pancreas and duodenum and of this commentary, Cdx2+/2 embryos stomach phenotype to that of colon. The three homologues of the Drosophila gene exhibited multiple polyps in the caecum question has been investigated by mak- Caudal which in mammals are called and adjacent ileum and proximal colon ing transgenic mice in which Cdx2 is 11 12 Cdx1, Cdx2, and Cdx4. In addition to their (that is, in the midgut). Histological expressed in the stomach (which in own unique domains, the Cdx genes examination showed the presence of wild-type mice is Cdx2 negative). This exhibit significant topographical overlap forestomach epithelium in the sub- is achieved by introducing a Cdx2 of expression during development, as stance of the polyps and this was expressing ‘‘transgene’’ into the genome well as in the adult, and it is reasonable interpreted as evidence of an anterior to assume that in such areas a degree of homeotic shift involving the gut endo- *Chimaeric mice are mosaics produced by compensation may occur in the event of derm analogous to the mesodermal injecting genetically distinct cells into recipient single gene deficiency. shift involving the axial skeleton. In embryos at the blastocyst stage so that the Cdx2 is expressed in the endoderm of other words, disturbance of ‘‘positional resultant mice are a mixture of the donor and the entire postgastric epithelium4 from information’’ has resulted in anterior recipient cell lines.

www.gutjnl.com COMMENTARY 1395 of wild-type mouse embryos under the cad homologues although clearly Cdx1 modifying gene expression for thera- control of a promoter that specifically does not compensate for Cdx2 in the peutic purposes. Gut: first published as 10.1136/gut.2003.038240 on 10 September 2004. Downloaded from 11 causes expression of Cdx2 in the sto- knockout model of the latter. The Gut 2004;53:1394–1396. mach. The answer appears to be ‘‘yes’’ overlap of effects may only be partial doi: 10.1136/gut.2003.038240 as Silberg and colleagues,15 using cis and this would explain differences in regulatory elements of the Hnf3c pro- proliferating cell distribution and dis- Correspondence to: Professor F Beck, moter to drive ectopic expression of Cdx2 tribution of Paneth cells that exist Department of Biochemistry, University of Leicester, Adrian Building, University Rd, in the stomach, described the presence between the two transgenic models. Leicester LE 1 7RH, UK; [email protected] of alcian blue positive intestinal-type Mammalian cad homologues and in goblet cells in the gastric mucosa of particular Cdx2 are multifunctional transgenic mice as well as induced genes and the picture presented above expression of intestine specific genes in is simplistic. In the mature gut, Cdx1 is REFERENCES the relevant areas. Interestingly, Mutoh expressed principally in intestinal crypts 1 Burke AC, Nelson CE, Morgan BA, et al. and colleagues16 produced similar while Cdx2 is demonstrable in cells Hox genes and the evolution of vertebrate axial morphology. Development results using the promoter of the non- clothing the villi where it regulates 1995;121:333–46. + + catalytic b-subunit gene of rat H /K production of many gut enzymes, such 2 Brooke NM, Garcia-Fernandez J, Holland PW. ATPase to drive the Cdx2 transgene. This as lactase-phlorizin hydrolase.19 Apart The ParaHox gene cluster is an evolutionary sister of the Hox gene cluster. Nature group described complete replacement from its importance in pattern forma- 1998;392:920–2. of gastric mucosal cells in the body of tion during the development of the gut, 3 Offield MF, Jetton TL, Labosky PA, et al. PDX-1 is the stomach at postnatal day 37 by Cdx2 in conjunction with Cdx1 may well required for pancreatic outgrowth and goblet cells, enteroendocrine cells, and contribute to the balance between dif- differentiation of the rostral duodenum. Development 1996;122:983–95. absorptive cells expressing alkaline ferentiation and cell renewal in the 4 Beck F, Erler T, Russell A, et al. Expression of Cdx- phosphatase together with the estab- mature intestine and recently it has 2 in the mouse embryo and placenta: possible lishment of the proliferative zone at the been suggested that in this respect it is role in patterning of the extra-embryonic membranes. Dev Dyn 1995;204:219–27. base of the glands rather than at the controlled by PTEN/phosphatidylinositol 5 James R, Kazenwadel J. Homeobox gene isthmus. As parietal cell function is 3 kinase signalling and tumour necrosis expression in the intestinal epithelium of adult established postnatally, it is reasonable factor a signalling via nuclear factor kB mice. J Biol Chem 1991;266:3246–51. 20 6 Meyer BI, Gruss P. Mouse Cdx-1 expression to assume that the Cdx2 transgene was dependent pathways. during gastrulation. Development expressed in differentiated cells rather Furthermore, Cdx2 functions as a 1993;117:191–203. than in stem cells; the H+/K+ ATPase tumour suppressor in the adult 7 Gamer LW, Wright CV. Murine Cdx-4 bears promoter being activated with the onset mouse.21 22 In one study, Cdx2 /2 mice striking similarities to the Drosophila caudal gene + in its homeodomain sequence and early of proton pump function. This suggests developed adenocarcinomata in the dis- expression pattern. Mech Dev 1993;43:71–81. that either some parietal cells retain tal colon in response to low doses of 8 Chawengsaksophak K, de Graaff W, Rossant J, characteristics of stem cells—unlikely azoxymethane compared with a signifi- et al. Cdx2 is essential for axial elongation in mouse development. Proc Natl Acad Sci U S A but possible as some parietal cells high cantly smaller effect of the same dose on 2004;101:7641–5. up in the gastric glands may not have wild-type controls. The tumours differed 9 Capecchi MR. Altering the genome by undergone a terminal differentiative histologically as well as in geographical homologous recombination. Science 1989;244:1288–92. event and may retain proliferative situation from non-neoplastic hetero- 10 Subramanian V, Meyer BI, Gruss P. Disruption of http://gut.bmj.com/ 17 potential —or else Cdx2 expression topic gastric polyps found in the peri- the murine homeobox gene Cdx1 affects axial directly or indirectly causes ‘‘dediffer- caecal region of these animals.21 skeletal identities by altering the mesodermal entiation’’ of parietal cells and their Another group used Apc mutant/Cdx2 / expression domains of Hox genes. Cell + 1995;83:641–53. establishment as intestinal stem cells 2 compound mice. They found that 11 Chawengsaksophak K, James R, Hammond VE, in the basal region of the mucosa levels of both Apc and Cdx2 were et al. Homeosis and intestinal tumours in Cdx2 whence they have migrated. This in significantly lower in the distal colon mutant mice. Nature 1997;386:84–7. 12 Tamai Y, Nakajima R, Ishikawa T-O, et al. turn might cause the overlying normal and this was correlated with the devel- Colonic hamartoma development by anomolous gastric mucosa to disappear in favour of opment of adenomatous polyps in the duplication in Cdx2 knockout mice. Cancer Res on September 25, 2021 by guest. Protected copyright. the newly established intestinal pheno- distal colon whereas these lesions were 1999;59:2965–70. 13 Beck F, Chawengsaksophak K, Waring P, et al. type. Another possibility is that the predominantly present in the small Reprogramming of intestinal differentiation 22 protein pump promoter used may be intestine in mice with Apc lesions only. and intercalary regeneration in Cdx2 mutant active at low levels in cells other than The authors conclude that reduced Cdx2 mice. Proc Natl Acad Sci U S A fully differentiated oxyntic cells expres- expression is important in colonic 1999;96:7318–23. 14 Beck F, Chawengsaksophak K, Luckett J, et al. A sing the proton pump. Clearly, these are tumorigenesis. Both studies reported study of regional gut endoderm potency by speculative suggestions that require lower apoptotic rates in the colonic analysis of Cdx2 null mutant chimaeric mice. Dev rigorous investigation. mucosa thus allowing greater survival Biol 2003;255:399–406. 18 15 Silberg DG, Sullivan J, Kang E, et al. Cdx2 Subsequently, Mutoh and colleagues of azoxymethane compromised cells or ectopic expression induces gastric intestinal have expressed a Cdx1 transgene in the of cells with loss of heterozygosity at metaplasia in transgenic mice. Gastroenterology mouse stomach, again driven by the H+/ Apc. 2002;122:689–96. + 16 Mutoh H, Hakamata Y, Sato K, et al. Conversion K ATPase b-subunit promoter, and Clearly, much remains to be done of gastric mucosa to intestinal metaplasia in their study is described in this issue of before the role of cad homologues in Cdx2-expressing transgenic mice. Biochem Gut (see page 1416). Surprisingly per- mammalian gut development and func- Biophys Res Commun 2002;294:470–9. 17 Willems G, Lehy T. Radioautographic and haps, they again demonstrated intest- tion is fully elucidated. It is certain that quantitative studies on parietal and peptic inal metaplasia similar in many but not they are critically important during cells in the mouse. Gastroenterology all respects to that seen with Cdx2.As development in defining gut pattern 1975;69:416–26. Cdx1 knockout has no apparent effect on and that in the adult they contribute 18 Mutoh H, Sakurai H, Satoh K, et al. Cdx1 induced 10 intestinal metaplasia in the transgenic mouse differentiation of the midgut, the trans- to the complex mechanisms of cell turn- stomach: comparative study with Cdx2 transgenic differentiating effect of Cdx1 transgene over and phenotypic differentiation of mice. Gut 2004;53:1416–23. expression in the stomach is somewhat stem cells. Future challenges include 19 Troelson JT, Mitchelmore C, Spodsberg N, et al. Regulation of lactase-phlorizin hydrolase gene unexpected. A possible explanation may elucidation of upstream and down- expression by the caudal related homeobox lie in the overlap of function between stream genes and the possibility of protein Cdx2. J Biochem 1997;322:833–8.

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20 Kim S, Domon-Dell C, Wang Q, et al. PTEN and 21 Bonhomme C, Duluc I, Martin E, et al. The 22 Aoki K, Tamai Y, Horiike S, et al. Colonic TNF-alpha regulation of the intestinal-specific Cdx2 homeobox gene has a tumour suppressor polyposis caused by mTOR-mediated Gut: first published as 10.1136/gut.2003.038240 on 10 September 2004. Downloaded from Cdx-2 homeobox gene through a PI3K, PKB/Akt, function in the distal colon in addition to a chromosomal instability in Apc+/Delta716 and NF-kappaB-dependent pathway. homeotic role during gut development. Gut Cdx2+/2 compound mutant mice. Nat Genet Gastroenterology 2002;123:1163–78. 2003;52:1465–71. 2003;35:323–30.

Irritable bowel syndrome WHAT HAPPENS TO ANIMALS ...... WHEN THE SERT GENE IS KNOCKED OUT OR IS DECREASED BY DISEASE? Is there a SERT-ain association with SERT deficient mice display increased anxiety related behaviours based on IBS? increased serotonergic neurotransmis- sion resulting in desensitised and down- 11 M Camilleri regulated 5-HT1A and 5-HT2A or 5-HT2C receptors.12 Gastrointestinal motility is ...... also abnormal in SERT knockout mice.13 The homozygous short genotype of SERT-P may be a candidate Adaptive changes occur in the subunit composition of enteric 5-HT3 receptors gene for diarrhoea predominant irritable bowel syndrome in in these knockout mice. Such changes women are reflected in altered 5-HT3 receptor affinity and desensitisation in the n this issue of Gut, Yeo and collea- the synaptic cleft and thus termination response of the receptor to 5-HT 14 gues1 report on the association of its action. The approved gene symbol released from enteroendocrine cells. Ibetween a functional polymorphism for SERT is SLC6A4 (solute carrier An intriguing recent observation sug- in the serotonin transporter gene and family 6 member 4); this abbreviation gests that experimental colitis alters diarrhoea predominant irritable bowel is used in other papers and this may be 5-HT signalling by increasing 5-HT syndrome (D-IBS) in women (see page confusing to readers. Human SERT is availability while decreasing 5-HT reup- 15 1452). In a study of 194 North American encoded by a single gene on chromo- take. The authors speculated that female participants with D-IBS in a some 17q11 and is composed of 14–15 altered 5-HT availability might contri- clinical trial programme and 448 female exons. Transcriptional activity of the bute to the dysmotility of inflammatory controls, there was an association SERT gene, SERT availability, and ulti- bowel disease, possibly due to desensi- between the homozygous short geno- mately 5-HT reuptake is modulated by a tisation of 5-HT receptors. type of SERT-P (serotonin reuptake polymorphic repetitive element unique transporter gene) and the D-IBS pheno- to humans and simian primates, the HAVE OTHER STUDIES ADDRES- type, with an odds ratio of 2.25 (95% 5-HTT gene linked polymorphic region SED THE ASSOCIATION OF THIS confidence interval 1.51–3.31). The fact (5-HTTLPR) upstream of the transcrip- POLYMORPHISM AND IBS? http://gut.bmj.com/ that the confidence interval does not tion start site. Additional variations There have been five studies that cross the value of 1 suggests that the have been described in the 59 untrans- explored the association of SERT association is statistically significant, lated region (59UTR), in intron 2, and and the authors suggest that the 39UTR (reviewed by Reif and Lesch5). SERT-P may be a candidate gene for Neurotransmitter transporters are D-IBS in women. channel-like proteins that are involved To place these interesting observa- in chemical signalling in the brain and tions in perspective, the reader may periphery; in fact they are considered to on September 25, 2021 by guest. Protected copyright. wish to address the following. What is do the heavy lifting in neurotransmitter SERT? What is the theoretical conse- inactivation.56SERT in the gut is similar quence of a polymorphism of the SERT- to that in the brain of the same species.7 P gene? What happens to animals when To control 5-HT actions in the gut and the SERT gene is knocked out? Have limit 5-HT receptor desensitisation, both other studies addressed the association neurones and crypt epithelial cells of this polymorphism and IBS? Are synthesise SERT proteins.89 there any pitfalls in the interpretation of such association studies? WHAT IS THE THEORETICAL CONSEQUENCE OF A Figure 1 Ligand-receptor interaction at WHAT IS SERT? POLYMORPHISM OF THE SERT-P synapse and neurotransmitter reuptake by Serotonin (5-hydroxtryptamine, 5-HT) GENE? presynaptic terminal. Differences in the function is secreted in copious amounts from gut In elegant functional studies, Lesch et al of the transporter protein are genetically enteroendocrine cells and serves as a showed that, compared with the homo- determined and influence the degree of activation of postsynaptic membrane receptors. critical messenger for gastrointestinal zygous long genotype, polymorphic For example, s/s homozygous polymorphism 23 fluid secretion and gut motility. homozygous short and heterozygous of the SERT-P gene would be expected to There are seven subclasses of serotoner- SERT-P were associated with reduced reduce the function of SERT, and increasing the gic receptors, differentiated on the basis function of the transporter protein in a effect of endogenously released 5-HT to activate of structure, molecular mechanism, and lymphoblastoid cell line.10 Figure 1 illus- receptors (for example, 5-HT3 or 5-HT4) function.4 In contrast with the remark- trates the consequence of a less effective receptors on cholinergic neurones to induce colonic contractions or propulsive colonic able diversity of 5-HT receptors, only a reuptake process for 5-HT, as might motility and potentially lead to symptoms of single protein, the 5-HT transporter (or occur in a patient with an s/s homo- diarrhoea predominant irritable bowel SERT), mediates reuptake of 5-HT from zygous or l/s heterozygous polymorphism. syndrome.

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properties at all known cellular loca- IBS, all 7 tions and, in accordance with the Gut: first published as 10.1136/gut.2003.038240 on 10 September 2004. Downloaded from groups C-IBS D-IBS Controls 20 90 general receptor theory, Bellini et al I/I proposed that it is conceivable that 80 I/s similar alterations in 5-HT uptake effi- s/s ciency may also occur at the intestinal 70 level. SERT was found to be expressed on platelet membranes of 12 D-IBS 60 female patients at a low density 50 (decreased Bmax) as well as to display a low degree of affinity (increased Kd) 40 at its ligand binding site compared with 21

% of patients 12 healthy female volunteers. They 30 also suggest a possible correlation 20 between the reduced capacity of serotonin reuptake and the severity of D-IBS 10 symptoms.21 However, the association with genotype was not formally 0 explored. The fifth report is the paper in the 1 Turkey Turkey Turkey Turkey current issue of Gut. Yeo and colleagues N Amer N Amer observed a significant odds ratio of Mid-West US Mid-West US Mid-West US Mid-West US having the s/s genotype in D-IBS patients relative to controls in North Figure 2 Proportion of different SERT-P genotypes in patients with different irritable bowel disease America. This observation contrasts (IBS) phenotypes and healthy controls; note that the diarrhoea predominant (D-IBS) patients in with the significant association with Turkey have predominantly the l/s genotype in contrast with none of the 18 patients with the l/l genotype. Note also that controls in the two series from North America have very similar the l/s genotype in Turkey; however, distributions but the proportions differ from controls in Turkey, suggesting significant racial from the function studies performed in variation. Finally, note that in the series of Yeo et al from North America, there are relatively more vitro,10 both l/s and s/s genotypes would D-IBS patients with the s/s genotype compared with controls but the actual proportion of the three be predicted to produce SERT molecules genotypes in D-IBS was very similar. C-IBS, constipation predominant irritable bowel syndrome. with reduced function, and therefore result in higher synaptic levels of 5-HT. On the other hand, the odds ratio polymorphisms and IBS; results from polymorphisms was not significant (0.7; provides statistical evidence of an asso- the three genetic association studies are 95% confidence interval 0.4, 1.2). This ciation although it does not prove a summarised in fig 2. study included a post hoc analysis to disorder in the function of the SERT or The first report was a study from determine whether clinically meaning- that it is causatively related to the Turkey in 54 IBS patients (18 with D- ful associations of the three candidate development of D-IBS. In fact, the IBS) and 91 healthy controls. Pata et al genotypes could have been detected proportion of the l/l, l/s, and s/s geno- http://gut.bmj.com/ examined possible associations between with at least 80% power. Thus it was types in D-IBS were virtually equivalent SERT polymorphisms and the different reported that the sample size of the in the 194 patients from North clinical patterns of IBS. They observed study could detect a difference in pre- America.1 that the l/s genotype was present in 18 valence of wild type (l/l) versus poly- In summary, these data show that the D-IBS patients with a frequency of 88% morphic (l/s or s/s) genotypes of 12% for distribution of the l/s genotype seems whereas none of the patients had the l/l all lower FGID, and 19–20% for IBS-C very heterogeneous in different popula- genotype. Overall, the distribution of and IBS-D. On the other hand, the study tions. A polymorphism which is asso- on September 25, 2021 by guest. Protected copyright. SERT polymorphisms were similar in identified that combinations of poly- ciated with a disorder in one population healthy subjects and the whole group of morphisms were associated with high may not be in another ethnic group. 16 IBS patients. somatic symptom scores: a2C Del 322– This is a problem that is increasingly The second report by Kim et al at the 325 with SERT-P (odds ratio 5.0 (95% being appreciated in behavioural genet- Mayo Clinic, which studied people confidence interval 1.11, 22.22)).17 This ics. This may explain, at least in part, a residing in the Mid-West in the USA, suggests that the SERT-P genotype may number of apparently contradictory was recently published in Gut.17 The predispose to somatisation or other findings gathered on specific genetic study explored the distributions of complex behavioural traits (including variations in different studies. genetic polymorphisms for SERT-P, a2C fibromyalgia, anxiety, and depression, 1 Del 322–325, and a2A21291 (CRG) as discussed by Yeo and colleagues ) and INTERPRETATION OF GENE- polymorphisms in 274 patients with that studies of interactions between ASSOCIATION STUDIES lower functional gastrointestinal disor- candidate genes that modify motor, There are several potential pitfalls that ders (FGID) and 120 controls. The sensory, or behavioural functions would need to be considered in the interpreta- distribution of SERT polymorphisms be of interest. tion of these studies. was not significantly different between A third report from Korea18 showed the lower FGID patients versus controls no differences in genotype distributions Ethnic differences (an observation that replicated the between IBS, IBS subgroups, and Difficulties in interpretation of popula- result in the whole IBS group studied healthy controls. tion based association studies arise due in Turkey) or between the subset with The fourth report from Italy was to ethnic differences in SERT-P allele the phenotype of D-IBS (n = 128) versus centred around the function of platelet frequencies which may also explain controls. In the final analysis of 90 SERT. SERT is found in peripheral some conflicting results. The frequency C-IBS patients reported by Kim et al, sites, including platelets.19 As the SERT of the l/l genotype was 6% in Japanese the odds ratio for SERT-P l/s or s/s protein displays the same molecular subjects,22 23 5% in Koreans,18 34% in

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European-Americans,10 and 24% in Genetic dissections of complex 7 Masson J, Sagn C, Hamon M, et al. 16 Neurotransmitter transporters in the central Turks. This variation in background diseases are extremely complex nervous system. Pharmacol Rev Gut: first published as 10.1136/gut.2003.038240 on 10 September 2004. Downloaded from prevalence of a genotype clearly influ- Investigations of gene-gene and gene- 1999;51:439–64. ences the statistical power to detect a environment interactions in humans 8 Chen J-X, Pan H, Rothman TP, et al. Guinea pig 5-HT transporter: cloning, expression, genotype related difference. and animals are revealing genes that distribution, and function in intestinal sensory may underlie behavioural variations. reception. Am J Physiol 1998;275:G433–8. However, studies also lead to the notion 9 Wade PR, Chen J, Jaffe B, et al. Localization and Racial homogeneity of control and expressed by Reif and Lesch that a function of a 5-HT transporter in crypt epithelia of disease groups: the gastrointestinal tract. J Neurosci In the current paper by Yeo and collea- probabilistic rather than deterministic 1996;16:2352–64. impact of the genes can be identified on 10 Lesch KP, Bengel D, Heils A, et al. Association of gues,1 there were no differences in the the phenotypic expression of these anxiety-related traits with a polymorphism in the proportion with the l/l genotype in serotonin transporter gene regulatory region. behavioural or functional disorders.5 To D-IBS patients and controls. In contrast, Science 1996;274:1527–31. understand the probabilistic impact or 11 Li Q, Wichems C, Heils A, et al. Reduction in the they observed differences in the distri- contribution of each genotypic variation density and expression, but not G-protein bution of s/s and l/s between controls coupling, of serotonin receptors (5-HT1A) in 5-HT in a semi quantitative manner requires (17.2% and 47.8%, respectively) and transporter knock-out mice: gender and brain studies with large sample sizes. Such region differences. J Neurosci D-IBS (31.4% and 31.9%, respectively). studies are laborious and difficult, par- 2000;20:7888–95. Regrettably, the authors did not provide Li Q ticularly in IBS where there is no 12 , Wichems CH, Ma L, et al. Brain region- critically important data on the racial specific alterations of 5-HT2A and 5-HT2C diagnostic test. receptors in serotonin transporter knockout mice. derivation of patients and controls J Neurochem 2003;84:1256–65. participating in this study. They 13 Chen JJ, Li Z, Pan H, et al. Maintenance of included a larger number of controls to CONCLUSION serotonin in the intestinal mucosa and ganglia of A hypothesis proposed by Blakely26 is mice that lack the high-affinity serotonin attempt to suppress confounding effects transporter: Abnormal intestinal motility and the such as population stratification and not that IBS is equivalent to SERT expression of cation transporters. J Neurosci admixture. The proportions in predomi- dysfunction but that SERT dysfunction 2001;21:6348–61. may present with a gastrointestinal 14 Liu MT, Rayport S, Jiang Y, et al. Expression and nantly European-Americans of the s/s function of 5-HT3 receptors in the enteric neurons and l/s genotypes of 19% and 49%, phenotype or as a behavioural disorder. of mice lacking the serotonin transporter. respectively,10 and 20% and 49%,17 sug- Anxiety disorders are commonly Am J Physiol 2002;283:G1398–411. gests that the controls in the paper by observed to be comorbid with IBS.27 28 15 Linden DR, Chen JX, Gershon MD, et al. Serotonin availability is increased in mucosa of 1 Yeo and colleagues were similar to two SERT dysfunction may contribute to guinea pigs with TNBS-induced colitis. other independent control cohorts. This behavioural and functional gut disor- Am J Physiol 2003;285:G207–16. is reassuring as the 448 control DNA ders; however, as previously noted by 16 Pata C, Erdal ME, Derici E, et al. Serotonin transporter gene polymorphism in irritable samples used by Yeo et al were obtained others, the influence of a single poly- bowel syndrome. Am J Gastroenterol from three different commercial morphism on continuously distributed 2002;97:1780–4. sources. In contrast, the study by Kim traits is likely to be small in humans. 17 Kim HJ, Camilleri M, Carlson PJ, et al. Association of distinct a2A adrenoceptor and et al which did not show an association Much work is needed to assess the serotonin-transporter polymorphisms associated between SERT-P polymorphisms and influence of genetic variation of SERT with constipation and somatic symptoms in D- IBS (n = 128) or other IBS phenotype, in the manifestations and response to functional gastrointestinal disorders. Gut 29 2004;53:829–37. therapy of irritable bowel syndrome http://gut.bmj.com/ drew patients (n = 276) and controls 18 Lee DY, Park H, Kim WH, et al. Serotonin (n = 120) from the same geographical before we can be certain of the role of transporter gene polymorphism in healthy adults region in the Mid-West of the USA SERT genetics in D-IBS. Acquired and patients with irritable bowel syndrome. Korean J Gastroenterol 2004;43:18–22. and provided detailed information changes in SERT mRNA in animal 30 19 Lesch KP, Wolozin BL, Murphy DL, et al. Primary about the sex and race of participants. models of colitis and in patients with structure of the human platelet serotonin uptake 31 Thus female participants predominated ulcerative colitis and IBS suggest that site: Identity with the brain serotonin transporter. there specific molecular alterations J Neurochem, 2003;60, 2319–22. in both lower functional gastrointes- 20 Kenakin T. Drug efficacy at G protein-coupled deserve further study. tinal disorder patients (82%) and con- receptors. Ann Rev Pharmacol Toxicol on September 25, 2021 by guest. Protected copyright. trols (79%), and European Americans Gut 2004;53:1396–1399. 2002;42:349–79. 21 Bellini M, Rappelli L, Blandizzi C, et al. Platelet were 89% of controls and 97% of doi: 10.1136/gut.2004.039826 serotonin transporter in patients with diarrhea- patients; there were 6% Asian and 3% predominant irritable bowel syndrome both Hispanic among healthy participants. Correspondence to: Professor M Camilleri, before and after treatment with alosetron. CENTER, Mayo Clinic, Charlton 8-110, 200 Am J Gastroenterol 2003;98:2705–11. Control for ethnic differences is criti- First St SW, Rochester 55905, USA; camilleri. 22 Katsuragi S, Kunugi H, Sano A, et al. Association cally important in appraising disease [email protected] between serotonin transporter gene associations between genotype and polymorphism and anxiety-related traits. Biol phenotype. Psychiatry 1999;45:368–70. REFERENCES 23 Kumakiri C, Kodama K, Shimizu E, et al. Study of the association between the serotonin transporter 1 Yeo A, Boyd P, Lumsden S, et al. Association gene regulatory region polymorphism and Interpretation of data from between a functional polymorphism in the personality traits in a Japanese population. surrogate measurements serotonin transporter gene and diarrhoea Neurosci Lett 1999;263:205–7. Although the structure and biochemis- predominant irritable bowel syndrome in women. 24 Klimek V, Roberson G, Stockmeier CA, et al. Gut 2004;53:1452–8. Serotonin transporter and MAO-B levels in try of SERT molecules at different sites 2 Ormsbee HS III, Fondacaro JD. Action of monoamine nuclei of the human brainstem are are similar within the same species, it is serotonin on the gastrointestinal tract. Proc Soc normal in major depression. J Psychiatry Res unclear whether the results obtained in Exp Biol Med 1985;178:333–8. 2003;37:387–97. 3 Gershon MD. Review article: roles played by 5- 25 Belous AR, Ramamoothy S, Blakely RD, et al. binding studies of SERT in platelets hydroxytryptamine in the physiology of the bowel. Decrease in the platelet level of 43 kDa really reflect the function of enteric Aliment Pharmacol Ther 1999;13:15–30. immunoreactive fraction of serotonin transporting SERT. For example, binding studies of 4 Kim D-Y, Camilleri M. Serotonin: a mediator of protein correlates with depressive symptoms in the brain-gut connection. Am J Gastroenterol patients with somatoform disorders. Voprosy SERT in brainstem nuclei may be 2000;95:2698–709. Meditsinskoi Khimii 1999;45:256–62. normal24 in diseases such as major 5 Reif A, Lesch KP. Toward a molecular architecture 26 Blakely RD. Physiological genomics of depression whereas other studies sug- of personality. Behav Brain Res 2003;139:1–20. antidepressant targets: keeping the periphery in 6 Blakely RD, Bauman AL. Biogenic amine mind. J Neurosci 2001;21:8319–23. gest that platelet SERT function is transporters: regulation in flux. Curr Opin 27 Walker EA, Katon WJ, Jemelka RP, et al. abnormal in depression.25 Neurobiol 2000;10:328–36. Comorbidity of gastrointestinal complaints,

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depression, and anxiety in the Epidemiologic 29 Camilleri M, Atanasova E, Carlson PJ, et al. Am J Physiol Gastrointest Liver Physiol

Catchment Area (ECA) Study. Am J Med Serotonin transporter polymorphism 2003;285:G207–16. Gut: first published as 10.1136/gut.2003.038240 on 10 September 2004. Downloaded from 1992;92:26S–30. pharmacogenetics in diarrhea-predominant 31 Coates MD, Mahoney CR, Linden DR, et al. 28 Woodman CL, Breen K, Noyes R jr, et al. irritable bowel syndrome. Gastroenterology Molecular defects in mucosal serotonin The relationship between irritable bowel 2002;123:425–32. content and decreased serotonin reuptake syndrome and psychiatric illness. 30 Linden DR, Chen JX, Gershon MD, et al. transporter in ulcerative colitis and irritable bowel A family study. Psychosomatics Serotonin availability is increased in mucosa of syndrome. Gastroenterology 1998;39:45–54. guinea pigs with TNBS-induced colitis. 2004;126:1657–64.

Ulcerative colitis addressed. Available studies mainly ...... investigated the effects of pre-illness diet and subsequent development of UC. Russel et al performed a case control Diet and relapsing ulcerative colitis: study in recently diagnosed cases with UC.3 They showed a positive correlation take off the meat? between consumption of soft drinks and chocolate and UC. In another study, a H Tilg, A Kaser high fat intake was associated with an increased risk for UC whereas a negative ...... correlation with vitamin C and fruit consumption was found.4 A third study Dietary factors such as red meat, high protein intake, and alcohol also showed a positive correlation with are associated with relapse in ulcerative colitis, probably fat intake and development of UC.5 All mediated via hydrogen sulphide production. of these findings however may also be an expression of modern lifestyle involving other risk factors for the develop- ajor advances in the understand- may open the perspective of lifestyle ment of IBD. ing of the aetiopathogenesis and modification in the treatment of UC. As Mgenetics of inflammatory bowel discussed in detail by the authors, these disease (IBD) have been accompanied effects may be related to sulphur and HYDROGEN SULPHIDE: BAD by an increase in the therapeutic arma- sulphate contents of food. Most impor- MALODOROUS GAS mentarium, including immunosuppres- tantly, to continue to prove this impor- RESPONSIBLE FOR RELAPSES IN sants and anticytokine drugs. Whereas tant work, in a next step an intervention UC? patients are in many cases highly study is needed before it can be con- Mercaptides such as sodium hydrogen motivated to use prescribed drugs in cluded that sulphur and sulphate rich sulphide (one of the main malodorous http://gut.bmj.com/ those chronic disorders, they would be foods definitely increase the risk of compounds in human flatus) are redu- even more willing to change lifestyle relapse. However, caution with regard cing agents that help maintain anaer- and dietary habits so they could actively to interpretation of the data of Jowett et obic conditions in the colonic lumen. influence the course of their disease. al is also warranted. Habitual diet was They are produced in the human large Therefore, one of the most common assessed only once and therefore might intestine by bacterial reduction of diet- questions physicians treating patients not a priori reflect food intake over the ary inorganic sulphate and sulphite and with IBD are asked is whether changing entire observation period, especially the by fermentation of sulphur amino acids. diet could positively affect the course of period immediately preceding relapse. The acute toxicity of hydrogen sulphide on September 25, 2021 by guest. Protected copyright. their disease. So far, and this has been Furthermore, based on energy intake appears to result from inhibition of especially true for patients with ulcera- estimates by physical activity level cytochrome oxidase leading to mucosal tive colitis (UC), our answer had been (PAL), 23% of patients over- or under- damage, loss of barrier function, and ‘‘we do not know and there are no reported their food intake. When these histological changes resembling UC. special recommendations’’. patients were excluded from the analy- Hence the colonic mucosa has developed This may now change as Jowett and sis, only high meat intake (particularly a very effective means of detoxifying colleagues1 in this issue of Gut present red and processed meat) and high hydrogen sulphide.6 interesting and clinically novel data alcohol consumption were associated Mainly exogenous sources contribute studying the role of dietary factors on with relapse while all other associations to the colonic pool of sulphur, such as the clinical course of UC (see page were no longer statistically significant. red meat, cheese, milk, fish, nuts, and 1479). In this prospective cohort study, Provocatively, therefore, it may well be eggs, and as preservatives found in com- they investigated the effects of habitual speculated that other constituents of mercial breads, beers, many alcoholic diet on relapses of disease. Impressively, meat besides sulphur compounds (see drinks, sausages, and dried fruits. Faecal 96% of patients (n = 191) completed the below) might play a role in relapsing sulphide levels increase after consump- study. Dietary factors such as red and UC. tion of increasing amounts of meat,78 processed meat, protein, and alcohol, as providing evidence that meat is an well as sulphur and sulphate intake PRE-ILLNESS DIET AND important substrate for sulphide gen- were positively associated with relapses. SUBSEQUENT DEVELOPMENT OF eration by bacteria in the human large Even though dietary factors in this UC intestine. study were less important than mea- As the rising incidence of IBD in the last It has been proposed that sulphide sures of prior disease activity in deter- decades coincides with profound toxicity may be important in the patho- mining the risk of relapse,2 clinically this changes in diet pattern, various life- genesis of UC.910The initial evidence in information is extremely valuable as it style/dietary aspects have been this regard was demonstration that

www.gutjnl.com 1400 COMMENTARY experimental exposure of colonic tissue So, how to increase faecal butyrate beneficial effects via modulating short to sulphide causes inhibition of butyrate levels? In animal and human studies, chain fatty acid metabolism. Gut: first published as 10.1136/gut.2003.038240 on 10 September 2004. Downloaded from use (see below), a defect similar to that ingestion of resistant fibre has resulted in an increase in the population of observed in mucosal biopsies obtained FISH OIL SUPPLEMENTATION/ from UC patients.11 UC patients have Bifidobacillus and Lactobacillus in the ESSENTIAL FATTY ACIDS: NOT significantly higher luminal concentra- colon and an increase in faecal butyrate EFFECTIVE IN THE TREATMENT tions of hydrogen sulphide than con- concentrations. Administration of oat AND MAINTENANCE OF trols, and disease activity correlates with bran over three months to UC patients sulphide production rates.12 Hydrogen in remission (corresponding to 20 g REMISSIONINUC Prostaglandin E and leukotriene B4 sulphide induces hyperproliferation of dietary fibre) has recently been shown 2 are metabolites of arachidonic acid via colonic mucosa and this effect is antag- to result in increased faecal butyrate the cyclooxygenase pathway and the onised by butyrate.13 Treatment with levels and in this pilot study no relapses 22 lipoxygenase pathway, respectively. 5-aminosalicylates and bismuth subsa- were observed. Alternative strategies of Increased levels of prostaglandin E licylates has been shown to reduce delivering short chain fatty acids to the 2 and leukotriene B4 are found in active hydrogen sulphide production in the inflamed colon are by providing a UC. Diets containing high levels of n-3 colonic lumen.12 14 Apart from the direct substrate, a ‘‘prebiotic’’, for short chain fatty acids such as eicosapentaenoic acid toxicity of hydrogen sulphide, it has fatty acid production by colonic bacteria, and docosahexaenoic acid are known to been speculated that thiols may react or directly delivering probiotics to the modify leukotriene production. Dietary with sulfhydryl containing compounds intestinal lumen. fish oil supplementation has improved to form persulfides, which may alter patients with other inflammatory dis- protein function as well as antigenicity, PREBIOTICS IN UC: EFFECTIVE VIA orders, such as rheumatoid arthritis. which could theoretical lead to a chronic BUTYRATE INDUCTION? Five placebo controlled double blind immune mediated process, as known in Prebiotics are defined as non-digestible 6 studies have addressed this question in UC. In summary, there is evidence in food ingredients that beneficially affect UC.29–33 Despite reduced levels of leuko- the literature that hydrogen sulphide the host by selectively stimulating the triene B4, some histopathological may play a role in UC and on the other growth or activity of bacterial species improvement, and a tendency towards hand certain foods such as meat cause already present in the gut. A germinated a steroid sparing effect, no overall an increase in colonic levels of hydrogen barley foodstuff (GBF) which contains convincing clinical benefit of dietary sulphide. hemicellulose rich fibre and glutamine fish oil supplementation for 4– rich protein has been shown to attenu- 12 months was seen in the treatment FAECAL BUTYRATE: KEY ate inflammation in DSS, trinitroben- of patients with active UC. Most of these zene sulphonic acid (TNBS), and HLA- METABOLITE IN UC trials involved treatment of active dis- B27 transgenic animal models of coli- 31 Short chain fatty acids, including buty- ease whereas one study failed to tis.23 In DSS colitis, GBF suppressed rate, proprionate, and lactate, are gen- demonstrate any benefit in maintenance significantly serum interleukin 6 levels erated in the colon as result of bacterial therapy. A recent randomised controlled and mucosal STAT-3 expression. These fermentation of dietary fibre by luminal trial again failed to demonstrate any effects may be caused by increased efficacy of essential fatty acid supple- bacteria such as Bifidobacterium, 24 faecal butyrate production. GBF has http://gut.bmj.com/ Eubacterium, and Lactobacillus species. mentation in the maintenance of remis- been demonstrated to improve disease 34 Roediger et al demonstrated significant sion in UC. Therefore, despite some activity in a small pilot study in patients inhibition of butyrate but not glucose modest effects of n-3 polyunsaturated with active UC.25 In a controlled small oxidation by hydrogen sulphide in the fatty acids in the treatment of active study investigating 18 patients with mild to moderate disease, essential fatty ascending colon, splenic flexure, and in active UC, patients were treated with the rectosigmoid region.15 A direct anti- acids combination therapies have no baseline anti-inflammatory treatment benefit in maintaining remission in UC. inflammatory effect for butyrate, the with or without GBF. GBF therapy most extensively studied of the short resulted in a significantly better out- on September 25, 2021 by guest. Protected copyright. chain fatty acids, may be attributable to come and was associated with increased ANTIOXIDANTS AND OXIDATIVE its inhibition of nuclear factor kB, thus faecal concentrations of Bifidobacterium STRESS preventing the transcription of pro- and Eubacterium limosum.26 Another Oxidative stress is believed to play a key 16 inflammatory cytokines. In this study, important study in the area of prebiotic role in the pathogenesis of IBD as butyrate also attenuated dextran sul- and dietary fibre has been performed by intestinal inflammation is accompanied phate sodium (DSS) induced colitis. a Spanish collaborative group.27 In this by excessive production of reactive oxy- Furthermore, butyrate has been demon- large study, Plantago ovata seeds (dietary gen species and nitrogen metabolites. strated to reduce colonic permeability fibre 20 g/day) was compared with D’Odorico et al showed increased free by enhancing peroxisome proliferator mesalamine in the maintenance of radical peripheral leucocyte DNA activated receptor c (PPAR-c) activa- remission in patients with UC damage and decreased plasma anti- tion.17 This is of special interest as (n = 105). Treatment failure rate was oxidant defences in both UC and PPAR-c ligands show antineoplastic 40% in the Plantago ovata seed group, Crohn’s disease patients.35 36 N-3 fatty and anti-inflammatory effects in experi- 35% in the mesalamine group, and 30% acids have been shown to increase mental colitis.18 in the Plantaga ovata plus mesalamine antioxidant concentrations, although Patients with active extensive UC group. A significant increase in faecal as mentioned, n-3 polyunsaturated fatty have decreased colonic butyrate oxida- butyrate levels was observed after acids are not effective in the therapy of tion. As remission of disease is asso- Plantago ovata seed administration. The UC. Dietary iron supplementation ciated with normalisation of butyrate same preparation was also shown to increases lipid peroxidation, decreases oxidation, UC mucosa is not intrinsically ameliorate colonic damage in HLA-B27 antioxidant vitamins, and enhances altered in butyrate oxidation.19 Butyrate transgenic rats and this effect was also DSS induced colitis in rats.37 Studies enemas have been shown to be of associated with increased production in investigating the effects of oral iron benefit in the management of distal short chain fatty acids.28 Therefore, most administration in humans are lacking, UC.20 21 of prebiotic products might exert their as well as clinical studies assessing the

www.gutjnl.com COMMENTARY 1401 effects of antioxidants in patients with ulcerative colitis. Am J Gastroenterol 22 Hallert C, Bjorck I, Nyman M, et al. Increasing 2000;95:1008–13. fecal butyrate in ulcerative colitis patients by diet: UC. Gut: first published as 10.1136/gut.2003.038240 on 10 September 2004. Downloaded from 6 Levitt MD, Furne J, Springfield J, et al. controlled pilot study. Inflamm Bowel Dis Detoxification of hydrogene sulfide and 2003;9:116–21. methanethiol in the cecal flora. J Clin Invest 23 Goh J, O’Morain CA. Nutrition and adult CONCLUSION 1999;104:1107–14. inflammatory bowel disease. Aliment Pharmacol In summary, this provocative and clini- 7 Richardson CJ, Magee EA, Cummings JH. A new Ther 2003;17:307–20. cally important report by Jowett et al method for the determination of sulphide in 24 Kanauchi O, Suga T, Tochihara M, et al. gastrointestinal contents and whole blood by Treatment of ulcerative colitis by feeding with reopens the topic of diet and relapsing microdistillation and ion chromatography. Clin germinated barley foodstuff: first report of a 1 UC. The findings are well taken and Chim Acta 2000;293:115–25. multicenter open control trial. J Gastroenterol may offer a new perspective for poten- 8 Magee EA, Richardson CJ, Hughes R, et al. Suppl 2002;14:67–72. 25 Mitsuyama K, Saiki T, Kanauchi O, et al. tial intervention by practical lifestyle Contribution of dietary protein to sulphide production in the large intestine: an in vitro and a Treatment of ulcerative colitis with germinated modifications, and as such are eagerly controlled feeding study in humans. Am J Clin barley foodstuff feeding: a pilot study. Aliment awaited by our patients. Despite this Nutr 2000;72:1488–94. Pharmcaol Ther 1998;12:1225–30. excitement, interventional studies are 9 Roediger WEW, Morre J, Babidge W. 26 Kanauchi O, Serizawa I, Araki Y, et al. Colonic sulfide in the pathogenesis and Germinated barley foodstuff, a prebiotic product, now needed, setting the scene for treatment of ulcerative colitis. Dig Dis Sci ameliorates inflammation of colitis through specific dietary recommendations and 1997;42:1571–9. modulation of the enteric environment. 10 Roediger WEW. Decreased sulphur aminoacid J Gastroenterol 2003;38:134–41. for further defining the role of sulphur/ 27 Fernandez-Banares F, Hinojosa J, Sanchez- sulphate which may even lead to novel intake in ulcerative colitis. Lancet 1998;351:1555. Lombrana JL, et al. Randomized clinical trial of therapies. 11 Chapman MA, Grahn MF, Boyle MA, et al. Plantago ovata seeds (dietary fiber) as compared Butyrate oxidation is impaired in the colonic with mesalamine in maintaining remission in mucosa of sufferers of quiescent ulcerative colitis. ulcerative colitis. Am J Gastroenterol ACKNOWLEDGEMENT Gut 1994;35:73–6. 1999;94:427–33. 28 Rodriguez-Cabeza ME, Galvez J, Camuesco D, The work in HT’s laboratory is supported by 12 Pitcher MC, Beatty ER, Cummings JH. The et al. Intestinal anti-inflammatory activity of the Austrian Science Fund (P14641 and contribution of sulphate reducing bacteria dietary fiber (Plantago ovata seeds) in HLA-B27 P15783). and 5-aminosalicylic acid to faecal sulphide in transgenic rats. Clin Nutr 2003;22:463–71. patients with ulcerative colitis. Gut 29 Lorenz R, Weber PC, Szimnau P, et al. Gut 2004;53:1399–1401. 2000;46:64–72. Supplementation with n-3 fatty acids from fish oil doi: 10.1136/gut.2003.035287 13 Christl SU, Eisner HD, Dusel G, et al. Antagonistic in chronic inflammatory bowel disease; a effects of sulphide and butyrate on proliferation of randomised, placebo-controlled, double-blind colonic mucosa: a potential role for these agents ...... cross-over trial. J Intern Med Suppl in the pathogenesis of ulcerative colitis. Dig Dis 1989;225:225–32. Authors’ affiliations Sci 1996;41:2477–81. 30 Stenson WF, Cort D, Rodgers J, et al. Dietary H Tilg, Department of Medicine, Division of 14 Suarez FL, Furne JK, Springfield J, et al. Bismuth Gastroenterology and Hepatology, University supplementation with fish oil in ulcerative colitis. subsalicylate markedly decreases hydrogen Ann Intern Med 1992;116:609–14. Hospital Innsbruck, Innsbruck, Austria sulphide release in the human colon. 31 Hawthorne AB, Daneshmend TK, Hawkey CJ, A Kaser, Brigham and Women’s Hospital, Gastroenterology 1998;114:932–9. et al. Treatment of ulcerative colitis with fish oil Department of Gastroenterology, Harvard 15 Roediger WE, Duncan A, Kapaniris O, supplementation: a prospective 12 month et al. Reducing sulphur compounds of the colon Medical School, Boston, MA, USA randomised controlled trial. Gut impairs colonocyte nutrition: implications for 1992;33:922–928. ulcerative colitis. Gastroenterology 32 Aslan A, Triadafilopoulos G. Fish oil fatty acid Correspondence to: Dr H Tilg, Department of 1993;104:802–9. supplementation in active ulcerative colitis: a Medicine, Division of Gastroenterology and 16 Venkatraman A, Ramakrishna BS, Shaji RV, et al. double-blind, placebo-controlled, cross-over Hepatology, University Hospital Innsbruck, Amelioration of dextrane sulphate colitis by study. Am J Gastroenterol 1992;87:432–37. Anichstrasse 35, 6020 Innsbruck, Austria; butyrate: role of heat shock protein 70 and NF- 33 Greenfield SM, Green AT, Teare JP, et al. A http://gut.bmj.com/ [email protected] kappaB. Am J Physiol Gastrointest Liver Physiol randomized controlled study of evening primrose 2003;285:G177–84. oil and fish oil in ulcerative colitis. Aliment 17 Kinoshita M, Suzuki Y, Saito Y. Butyrate reduces Pharmacol Ther 1993;7:159–66. colonic paracellular permeability by enhancing REFERENCES 34 Middleton SJ, Naylor S, Woolner J, et al. A PPAR gamma activation. Biochem Biophys Res double-blind, randomised, placebo-controlled 1 Jowett SL, Seal CJ, Pearce MS, et al. Influence of Commun 2003;293:827–31. trial of essential fatty acid supplementation in the dietary factors on the clinical course of ulcerative 18 Tanaka T, Kohno H, Yoshitani S, et al. Ligands for maintenance of remission of ulcerative colitis. colitis: a prospective cohort study. 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