896 COMMENTARIES

Crohn’s disease this observation has not been replicated ...... by other investigators, and there is no evidence of increased frequency of

Crohn’s disease in the offspring of Gut: first published as 10.1136/gut.2004.055889 on 10 June 2005. Downloaded from Does Mycobacterium avium subspecies mothers versus fathers with Crohn’s disease. Even if transmission of viable paratuberculosis cause Crohn’s MAP occurs, a plausible mechanism of tissue injury and induction of chronic disease? intestinal inflammation has not been proposed. Even advocates of the theory R Balfour Sartor that MAP causes Crohn’s disease con- cede that infection, if present, consists ...... of a low bacterial load and that no histochemical evidence of acid fast Reassessing this persistent theory in light of advances in molecular staining in Crohn’s disease tissues is microbial detection and genetic pathogenesis of disease seen. This could be explained by a paucibacillary infection with an obligate imilarities between chronic idio- Data from this study help address intracellular, cell wall deficient bacterial 19 pathic granulomatous ileocolitis some of the controversies that have form. In this setting, inflammation Sand mycobacterial infections have fuelled the vigorous debate between and tissue injury must be mediated by been noted since the original descrip- committed advocates and confirmed a cell mediated immune response. tions of the clinical syndrome now sceptics that is receiving increasing However, a cellular immune response called Crohn’s disease.1–4 Interest in a attention in the scientific literature, lay to MAP has not been documented in 20 possible infectious origin of this disorder press, and internet chat rooms. The Crohn’s disease patients, despite was renewed in 1989 when Chiodini arguments in favour or opposed to this increased serological responses to MAP et al cultured apparently identical theory (table 1) have some merit but antigens in the same patients. Another Mycobacterium avium subspecies paratu- many are flawed by incomplete data and serious flaw in the MAP pathogenesis of berculosis (MAP) from three patients lack of rigorous reflection. There is no Crohn’s disease theory is the observa- with Crohn’s disease.5 This controversy doubt that a potential source of zoonotic tion that these patients respond to 21 increased in intensity following the infection exists, with widespread MAP chronic immunosuppressive therapies detection of the specific DNA insertion infections in the dairy herds of Europe, and acquired immunosuppressive infec- sequence, IS900, of MAP in relatively North America, and Australia,13 14 excre- tions decrease disease activity as CD4 T 22 high numbers of patients with Crohn’s tion of MAP in milk from infected cell counts fall. In contrast, M tubercu- disease relative to ulcerative colitis and cows,15 relative resistance of intracellu- losis massively proliferates with anti- normal controls,6 and is now raging as lar MAP to widely used pasteurisation tumour necrosis factor or steroid treat- several different groups have detected techniques,16 and recovery of viable ment and M avium intracellulare thrives this organism in the food chain7 and MAP from the water supplying Los in the intestine as CD4 counts fall in water supply,8 proposed maternal-fetal Angeles.8 Moreover, the vast majority human immunodeficiency virus http://gut.bmj.com/ transmission in human milk,9 reported of studies using diverse techniques have infected patients. It is possible that long term responses to antimycobacter- detected MAP DNA or cultured this intracellular cell wall deficient MAP ial antibiotic combinations,10 and even organism in higher frequency from may not replicate well despite immuno- cultured viable M paratuberculosis in tissues of patients with Crohn’s disease suppression, but this issue has never blood samples of Crohn’s disease than from those with ulcerative colitis been studied by in vitro investigation or patients.11 and other disorders, although the in animals with Johne’s disease. In the

Additional data to support an associa- reported frequency of recovery in both study by Autschbach et al, corticosteroid on October 1, 2021 by guest. Protected copyright. tion of MAP with Crohn’s disease is Crohn’s disease and ulcerative colitis therapy was associated with lower MAP provided by Autschbach and collea- have ranged from 0% to 100%.17 These detection rates.12 gues12 in this issue of Gut (see page results are consistent with two possibil- The most irrefutable evidence that a 944). This carefully performed and well ities: either MAP infection could cause microbial agent causes a disease is long controlled study used nested polymerase Crohn’s disease in a subset of patients term remission of clinical manifesta- chain reaction (PCR) to detect the IS900 that are either selectively exposed to this tions and an altered natural history of insertion element of MAP in 52% of organism or who are genetically suscep- disease following clearance of the infec- Crohn’s disease resected tissues versus tible to infection or, alternatively, this tion. In vitro sensitivity analyses show 2% of ulcerative colitis and 5% of mostly relatively common dietary organism that clinical isolates of MAP are not non-inflammatory control tissues. This may selectively colonise (or a dead responsive to traditional anti-M tubercu- study provides novel data regarding the organism selectively lodge in) the ulcer- losis agents, and therefore lack of effi- prevalence of MAP in various pheno- ated mucosa of Crohn’s disease patients cacy with isoniazid, ethambutol, and types of Crohn’s disease by showing but not initiate or perpetuate intestinal rifampicin treatment for two years with slightly higher detection of IS900 DNA inflammation. Molecular fingerprints a three year follow up23 does not detract in colonic (66.7%) compared with distal show that genotypes of bovine and from this theory. However, reports of ileal (40.5%) tissues and decreased human isolates are not similar but efficacy of combinations of clarithromy- detection rates with corticosteroid use. instead indicate that human and ovine cin or azithromycin, rifabutin, and a In addition, these authors reported (sheep) strains are more closely variety of other agents in 58–82% of weak associations with perianal involve- related.18 Maternal/fetal transmission of Crohn’s disease patients10 24 are also not ment and a shorter duration of disease MAP has been proposed following cul- definitive due to the uncontrolled nat- but no correlation with patient sex, age ture of MAP from breast milk of two ure of these studies, the small number at diagnosis, stricturing versus penetrat- patients with Crohn’s disease.9 of patients treated, the variable treat- ing phenotype, or presence of granulo- However, the frequency of positive ment regimens, and the fact that these mas. cultures in human milk is uncertain, antibiotics, particularly clarithromycin,

www.gutjnl.com COMMENTARIES 897

Table 1 Arguments for and against a Mycobacterium avium subspecies MAP analogous to Helicobacter pylori in paratuberculosis (MAP) causation of Crohn’s disease peptic ulcer disease, gastritis, and gastric cancer, where host genetics and micro-

Pros bial virulence factors determine Gut: first published as 10.1136/gut.2004.055889 on 10 June 2005. Downloaded from (1) Clinical and pathological similarities between Johne’s and Crohn’s diseases34 immune responses that mediate clinical (2) Presence in food chain (milk, meat) and water supplies78 5633 disease in a small minority of patients (3) Increased detection of MAP in Crohn’s disease tissues by culture, PCR, FISH (4) Positive blood cultures of MAP in Crohn’s disease patients11 exposed to a widespread infectious (5) Increased serological responses to MAP in Crohn’s disease patients20 34 agent? Are we repeating the mistake of 9 (6) Detection of MAP in human breast milk by culture and PCR H pylori where the scientific establish- (7) Progression of cervical lymphadenopathy to distal ileitis in a patient with MAP infection35 (8) Therapeutic responses to combination antituberculosis therapy that include macrolide antibiotics10 24 ment resisted a new theory that chal- Cons lenged established paradigms of peptic (1) Differences in clinical and pathological responses in Johne’s and Crohn’s diseases4 ulcer disease until overwhelming clin- 36 (2) Lack of epidemiological support of transmissible infection ical evidence made such resistance (3) No evidence of transmission to humans in contact with animals infected with MAP (4) Genotypes of Crohn’s disease and bovine MAP isolates not similar18 untenable? Well designed clinical, (5) Variability in detection of MAP by PCR (0–100% in Crohn’s disease and ulcerative colitis tissues)8 microbiological, and mechanistic experi- and serological testing37 ments are urgently needed to defini- (6) No evidence of mycobacterial cell wall by histochemical staining tively settle this still unresolved debate. (7) No worsening of Crohn’s disease with immunosuppressive agents or HIV infection (8) No documented cell mediated immune responses to MAP in patients with Crohn’s disease20 To establish a causal relationship (9) No therapeutic response to traditional antimycobacterial antibiotics23 between MAP and Crohn’s disease, we need to determine if clearance of MAP MAP, Mycobacterium avium subspecies paratuberculosis; PCR, polymerase chain reaction; FISH, selectively changes the natural history fluorescent in situ hybridisation; HIV, human immunodeficiency virus. of disease in an infected subset of patients, perform definitive investiga- tions of cellular immune responses to have a broad spectrum of activity Crohn’s disease is that defective func- this organism in Crohn’s disease and against commensal enteric bacteria. tion of this gene results in ineffective control patients, determine if NOD2/ Moreover, these studies and a yet to be clearance of intracellular MAP infection CARD15 and other microbial signalling published ongoing controlled trial in and in decreased luminal a defensin pathways influence intracellular MAP Australia using these agents in Crohn’s secretion that permits increased muco- infection and clearance, and review disease patients are flawed by not sal adherence and epithelial invasion of results of ongoing large multi-institu- assessing IS900 DNA in biopsy speci- ingested organisms. Defective clearance tional studies to detect MAP in shared mens by PCR and serological responses of intracellular MAP by innate immune coded tissues by various molecular and to MAP before and after therapy, so that cells, including macrophages, could culture methods. These studies need to clinical results can be correlated with explain the seemingly paradoxical ther- be designed and conducted by estab- the presence of tissue MAP and its apeutic response of some Crohn’s lished investigators who bring no pre- clearance with treatment. Selective disease patients to granulocyte-macro- determined biases to this contentious responses in those patients with detec- phage colony stimulating factor.31 topic. If MAP is responsible for a subset tible MAP colonisation that clear infec- However, the phenotypic information http://gut.bmj.com/ of Crohn’s disease, public health mea- tion with antibiotic treatment would provided by Autschbach and collea- sures must be implemented to eliminate strongly imply a causal relationship of gues12 argues against an association of the source of infection in our food chain the infection. NOD2/CARD15 with MAP infection, as and food processing practices must be Our evolving molecular understand- MAP was more commonly detected in modified. In addition, the medical com- ing of gene/environmental interactions colonic than ileal disease and was not munity must develop ways to efficiently offers an opportunity to reassess the more frequently found in early onset or and cost effectively screen for MAP MAP causation theory of Crohn’s dis- stricturing disease.12 Likewise, patients infection and develop methods to effi- on October 1, 2021 by guest. Protected copyright. ease in a new light. NOD2/CARD15 is an with extensive ileocolitis responded bet- ciently clear this organism from infected intracellular receptor for muramyl ter to macrolide antibiotics and rifabu- tissues, possibly through a combination dipeptide (MDP), the smallest immuno- tin than did those with isolated ileal of effective antibiotics and immuno- logically active component of bacterial disease.10 These results directly contrast stimulants that enhance innate clear- peptidoglycan. Ligation of MDP by with the strong association of NOD2/ ance responses. If there is no evidence of NOD2/CARD15 activates nuclear factor CARD15 polymorphisms with early a causal association of MAP and Crohn’s kB. This pathway may contribute to onset ileal Crohn’s disease with a disease, we need to direct resources to clearance of intracellular bacterial infec- stricturing phenotype.32 other avenues of research. This contro- tion25 and secretion of a defensins by Crohn’s disease certainly has environ- versy has persisted far too long and Paneth cells, which constitutively mental and host genetic influences that needs to be expeditiously resolved. express NOD2/CARD15.26 The three interact to cause clinically evident dis- most common polymorphisms of this ease. It is equally clear that MAP is Gut 2005;54:896–898. gene are found in 25–35% of Caucasian widely present in our food chain and doi: 10.1136/gut.2004.055889 27 Crohn’s disease patients and lead to that the DNA of this organism can be Correspondence to: Dr R Balfour Sartor, UNC defective nuclear factor kB activation by recovered from the intestine of Crohn’s Department of Medicine/Division of MDP.28 29 Expression of the common disease patients. Although existing data Gastroenterology and Hepatology, CB #7032, truncation mutation of NOD2/CARD15 do not compellingly implicate MAP as a Room 7309 Biomolecular Bldg (MBRB), Chapel is associated with defective clearance of causal agent in Crohn’s disease, neither Hill, NC 27599-7032, USA; [email protected] invasive salmonella infection in epithe- do they definitively exclude this possi- Conflict of interest: None declared. lial cells.25 In addition, NOD2/CARD15 bility. We must determine whether MAP mutations in Crohn’s disease are asso- infection causes human disease, which REFERENCES ciated with diminished mucosal a is unlikely in my opinion, or whether 30 1 Crohn BB, Ginzburg L, Oppenheimer GD. defensin expression. Thus an attractive this environmental contaminant inno- Regional ileitis: a pathologic and clinical entity. explanation linking NOD2/CARD15 to cently lodges in ulcerated mucosa. Is JAMA 1932;99:1323–9.

www.gutjnl.com 898 COMMENTARIES

2 Dalzeil TK. Chronic intestinal enteritis. BMJ 15 Grant IR. Mycobacterium paratuberculosis and 27 Hugot JP, Chamaillard M, Zouali H, et al. 1913;2:1068–9. milk. Acta Vet Scand 2003;44:261–6. Association of NOD2 leucine-rich repeat variants 3 Greenstein RJ. Is Crohn’s disease caused by a 16 Lund BM, Gould GW, Rampling AM. with susceptibility to Crohn’s disease. Nature mycobacterium? Comparisons with leprosy, Pasteurization of milk and the heat resistance of 2001;411:599–603.

tuberculosis, and Johne’s disease. Lancet Infect Mycobacterium avium subsp. paratuberculosis: a 28 Girardin SE, Boneca IG, Viala J, et al. Nod2 is a Gut: first published as 10.1136/gut.2004.055889 on 10 June 2005. Downloaded from Dis 2003;3:507–14. critical review of the data, Int J Food Microbiol general sensor of peptidoglycan through muramyl 4 Chiodini RJ. Crohn’s disease and the 2002;77:135–45. dipeptide (MDP) detection. J Biol Chem mycobacterioses: a review and comparison of two 17 Sartor RB. Microbial influences in inflammatory 2003;278:8869–72. disease entities. Clin Microbiol Rev bowel disease: Role in pathogenesis and 29 Bonen DK, Ogura Y, Nicolae DL, et al. Crohn’s 1989;2:90–117. clinical implications. In: Sartor RB, Sandborn WJ, disease-associated NOD2 variants share a 5 Chiodini RJ, Van Kruiningen HJ, Thayer, WR, et eds. Kirsner’s inflammatory bowel diseases, 6th signaling defect in response to lipopolysaccharide al. Possible role of mycobacteria in inflammatory edn. Amsterdam: Elsevier Publishers, and peptidoglycan. Gastroenterology bowel disease. I. An unclassified Mycobacterium 2004:138–62. 2003;124:140–6. species isolated from patients with Crohn’s 18 Motiwala AS, Strother M, Amonsin A, et al. 30 Wehkamp J, Harder J, Weichenthal M, et al. disease. Dig Dis Sci 1984;29:1073–9. Molecular epidemiology of Mycobacterium avium NOD2 (CARD15) mutations in Crohn’s disease 6 Sanderson JD, Moss MT, Tizard ML, et al. subsp. paratuberculosis: evidence for limited are associated with diminished mucosal Mycobacterium paratuberculosis DNA in Crohn’s strain diversity, strain sharing, and identification alpha-defensin expression. Gut disease tissue. Gut 1992;33:890–6. of unique targets for diagnosis, J Clin Microbiol 2004;53:1658–64. 7 Millar D, Ford J, Sanderson J, et al. IS900 PCR to 2003;41:2015–26. 31 Dieckgraefe BK, Korzenik JR. Treatment of active Crohn’s disease with recombinant human detect Mycobacterium paratuberculosis in retail 19 Chiodini RJ, Van Kruiningen HJ, Thayer WR, granulocyte-macrophage colony-stimulating supplies of whole pasteurized cows’ milk in et al. Spheroplastic phase of mycobacteria factor. Lancet 2002;360:1478–80. England and Wales. Appl Environ Microbiol isolated from patients with Crohn’s disease. J Clin 32 Ahmad T, Tamboli CP, Jewell D, et al. Clinical 1996;62:3446–52. Microbiol 1986;24:357–63. 8 Mishina D, Katsel P, Brown ST, et al. On the relevance of advances in genetics and 20 Olsen I, Wiker HG, Johnson E, et al. Elevated etiology of Crohn disease. Proc Natl Acad Sci U S A pharmacogenetics of IBD. Gastroenterology antibody responses in patients with Crohn’s 1996;93:9816–20. 2004;126:1533–49. disease against a 14-kDa secreted protein 9 Naser SA, Schwartz D, Shafran I. Isolation of 33 Hulten K, El-Zimaity HM, Karttunen TJ, et al. purified from Mycobacterium avium subsp. Mycobacterium avium subsp paratuberculosis Detection of Mycobacterium avium subspecies paratuberculosis. Scand J Immunol from breast milk of Crohn’s disease patients. paratuberculosis in Crohn’s diseased tissues by in 2001;53:198–203. Am J Gastroenterol 2000;95:1094–5. situ hybridization. Am J Gastroenterol 21 Hanauer SB, Feagan BG, Lichtenstein GR, et al. 10 Gui GP, Thomas PR, Tizard ML, et al. Two-year- 2001;96:1529–35. outcomes analysis of Crohn’s disease treated with Maintenance infliximab for Crohn’s disease: the 34 Naser SA, Hulten K, Shafran I, et al. Specific rifabutin and macrolide antibiotics. J Antimicrob ACCENT I randomised trial. Lancet seroreactivity of Crohn’s disease patients against Chemother 1997;39:393–400. 2002;359:1541–9. p35 and p36 antigens of M. avium subsp. 11 Naser SA, Ghobrial G, Romero C, et al. Culture 22 James SP. Remission of Crohn’s disease after paratuberculosis. Vet Microbiol of Mycobacterium avium subspecies human immunodeficiency virus infection. 2000;77:497–504. paratuberculosis from the blood of patients with Gastroenterology 1988;95:1667–9. 35 Hermon-Taylor J, Barnes N, Clarke C, et al. Crohn’s disease. Lancet 2004;364:1039–44. 23 Thomas GA, Swift GL, Green JT, et al. Controlled Mycobacterium paratuberculosis cervical 12 Autschbach F, Eisold S, Hinz U, et al. High trial of antituberculous chemotherapy in Crohn’s lymphadenitis, followed five years later by prevalence of Mycobacterium avium subspecies disease: a five year follow up study. Gut terminal ileitis similar to Crohn’s disease. BMJ paratuberculosis IS900 DNA in gut tissues from 1998;42:497–500. 1998;316:449–53. individuals with Crohn’s disease. Gut 24 Shafran I, Kugler L, el-Zaatari FA, et al. Open 36 Sandler RS, Loftus EV. Epidemiology of 2005;54:944–9. clinical trial of rifabutin and clarithromycin inflammatory bowel diseases. In: Sartor RB, 13 Jubb TF, Galvin JW. Effect of a test and control therapy in Crohn’s disease. Dig Liver Dis Sandborn WJ, eds. Kirsner’s inflammatory bowel program for bovine Johne’s disease in Victorian 2002;34:22–8. diseases, 6th edn. Philadelphia: WB Saunders, dairy herds 1992–2002. Aust Vet J 25 Hisamatsu T, Suzuki M, Reinecker HC, et al. 2003:245–62. 2004;82:228–32. CARD15/NOD2 functions as an anti-bacterial 37 Bernstein CN, Blanchard JF, Rawsthorne P, et al. 14 Raizman EA, Wells SJ, Godden SM, et al. The factor in human intestinal epithelial cells. Population-based case control study of distribution of Mycobacterium avium ssp. Gastroenterology 2003;124:993–1000. seroprevalence of Mycobacterium

paratuberculosis in the environment surrounding 26 Lala S, Ogura Y, Osborne C, et al. Crohn’s paratuberculosis in patients with Crohn’s disease http://gut.bmj.com/ Minnesota dairy farms. J Dairy Sci disease and the NOD2 gene: a role for paneth and ulcerative colitis. J Clin Microbiol 2004;87:2959–66. cells. Gastroenterology 2003;125:47–57. 2004;42:1129–35.

Colitis beyond inherent basic nutrition’’.3 ...... Bacteria associated with probiotic activ- ity are most commonly lactobacilli, on October 1, 2021 by guest. Protected copyright. bifidobacteria, and streptococci but Probiotics and barrier function in colitis other non-pathogenic bacteria such as some strains of Escherichia coli and P Gionchetti, K M Lammers, F Rizzello, M Campieri microorganisms such as the yeast Saccharomyces boulardii have been used ...... in IBD. Probiotic administration may exert a protective effect in colitis by Encouraging results have been obtained with probiotics in several preventing mucosal barrier disruption and influencing the extent experimental animal models of IBD.4–7 of mucosal injury In humans, probiotics are effective in the prevention of pouchitis onset and 8–10 here is strong evidence of a role for undiscovered due to technical limita- relapse. Results in ulcerative colitis the indigenous flora in driving tions, it was hypothesised that modula- are promising, both in prevention of inflammatory responses in inflam- tion of an abnormal microflora in these relapse and treatment of mild to mod- T 11–13 matory bowel disease (IBD) in geneti- patients by introducing high titres of erate attacks. Results in Crohn’s cally predisposed individuals.1 For years, ’’protective’’ bacteria might overwhelm disease are not yet clear because of researchers have tried in vain to identify the ’’aggressive’’ strain(s) and inhibit its conflicting data and the limited number a specific pathogen as the cause of these deleterious effects. On this basis, pro- of well performed studies.14–16 chronic intestinal inflammatory disor- biotic treatment was proposed as a Efforts are being made by many ders but the possibility that one or more therapeutic approach.2 researchers to unravel the precise bacterial agents are responsible cannot Probiotics are defined as ‘‘living mechanisms by which probiotic bacteria be ruled out. Considering the implica- organisms which, on ingestion in cer- and their metabolic products (short tions of a pathogen in IBD, as yet tain numbers, exert health benefits chain fatty acids, vitamins) exert their

www.gutjnl.com COMMENTARIES 899 beneficial effects. Several mechanisms permeability to macromolecules and 4 Fabia R, Ar’rajab A, Johansson M-L, et al. The effect of exogenous administration of have been proposed to account for toxins. Recently it has been shown that Lactobacillus reuterii R2LC and oat fiber on acetic probiotic action. These include: (a) a mixture of probiotic bacteria, in acid-induced colitis in the rat. antagonistic activity against pathogenic addition to decreasing proinflammatory Scand J Gastroenterol 1993;28:155–62. Gut: first published as 10.1136/gut.2004.055889 on 10 June 2005. Downloaded from bacteria by competing for binding cytokines—which increase intestinal 5 Madsen KL, Tavernini MM, Doyle JSG, et al. Lactobacillus sp prevents development of 17 18 sites ; (b) stimulation of mucosal permeability (interferon c) and induce enterocolitis in interleukin-10 gene-deficient mice. defence at the level of both immune a cascade of inflammatory events (inter- Gastroenterology 1999;116:1107–14. and epithelial function, by increasing leukin (IL)-1b, tumour necrosis factor a, 6 Schultz M, Veltkamp C, Dieleman LA, et al. 19 Lactobacillus plantarum 299V in the treatment sIgA production, decreasing proin- IL-8) resulting in mucosal injury by and prevention of spontaneous colitis in flammatory and increasing anti-inflam- invading immune cells (neutrophils, interleukin-10 deficient mice. Inflamm Bowel Dis matory cytokine levels,20–26 and inducing tissue macrophages, dendritic cells20–26— 2002;8:71–80. 7 Shibolet O, Karmeli F, Eliakim R, et al. Variable production of protective substances by reinforce barrier function by secretion response to probiotics in two models of the epithelium, such as antimicrobial of soluble factors that enhance barrier experimental colitis in rats. Inflamm Bowel Dis peptides (bacteriocins) and hydrogen integrity and by regulation of tight 2002;8:399–408. peroxide, mucins,17 27 and heat shock 20 21 8 Gionchetti P, Rizzello F, Venturi A, et al. Oral junctions (TJ). TJ are dynamic bacteriotherapy as maintenance treatment in 28 proteins, respectively; and (c) inhibi- structures which represent the major patients with chronic pouchitis: a double-blind, tion of bacterial translocation and rein- barrier within the paracellular pathway placebo-controlled trial. Gastroenterology forcement of barrier function20 21 by and regulate in a rapid and coordinated 2000;119:305–9. 9 Mimura T, Rizzello F, Helwig U, et al. Once daily mechanisms that have yet to be estab- way paracellular permeability. Many high dose probiotic therapy for maintaining lished in detail. pathogenic bacteria modulate intestinal remission in recurrent or refractory pouchitis. Gut The paper presented in this issue of permeability by alteration of TJ.30 2004;53:108–14. 29 10 Gionchetti P, Rizzello F, Venturi A, et al. Gut describes the effect of modulation Probiotics prevent Salmonella induced Prophylaxis of pouchitis onset with probiotic of the microflora by Lactobacillus casei on alteration of the distribution of the TJ therapy: a double blind, placebo controlled trial. disease course and extent in the trini- protein zonula occludens 1 and Gastroenterology 2003;124:1202–9. 11 Kruis W, Fric P, Pokrotnieks J, et al. Maintaining trobenzene sulphonic acid (TNBS) Salmonella induced enhanced permeabil- remission of ulcerative colitis with Escherichia Coli model of experimental colitis (see page ity by increasing transepithelial resis- Nissle 1917 is as effective as with standard 955). The elegance of this study lies in tance and decreasing mannitol flux.20 21 mesalazine. Gut 2004;53:1617–23. the experimental set up, which allowed 12 Venturi A, Gionchetti P, Rizzello F, et al. Impact Maintenance of epithelial barrier on the faecal flora composition of a new probiotic controlled mucosal colonisation with function is essential for the preservation preparation. Preliminary data on maintenance indigenous flora and selected bacterial of mucosal integrity. Altered TJ struc- treatment of patients with ulcerative colitis (UC) strains after removal of the native flora intolerant or allergic to 5-aminosalicylic acid (5 ture in ulcerative colitis results in ASA). Aliment Pharmacol Ther 1999;13:1103–8. 31 by antibiotics, and in the straightfor- impaired barrier function. Increased 13 Fedorak RN, Gionchetti P, Campieri M, et al. ward hypothesis and performance of the permeability in IBD is observed during VSL#3 probiotic misture induces remission in study; TNBS exerted a direct toxic effect the active phase of the disease and patients with active ulcerative colitis. Gastroenterology 2003;124:A377. on epithelial cells and disrupted the therefore reinforcement of barrier func- 14 Guslandi M, Mezzi G, Sorghi M, et al. intestinal barrier, and the effect of L tion, together with their immune mod- Saccharomyces boulardii in maintenance casei on impaired barrier function and ulatory and metabolic properties, may treatment of Crohn’s disease. Dig Dis Sci 2000;45:1462–4. bacterial translocation in the TNBS http://gut.bmj.com/ be central in the mechanism of action of 15 Prantera C, Scribano ML, Falasco G, et al. model was examined. Colonisation of probiotic bacteria. Further investiga- Ineffectiveness of probiotics in preventing bacterial strains, measure of bacterial tions on how probiotics regulate the TJ recurrence after curative resection for Crohn’s translocation, neutrophil tissue influx, disease: a randomised contraolled trial with complex and influence intestinal perme- Lactobacillus GG. Gut, 2002;51;405–9.. and degree of inflammation were eval- ability are warranted. 16 Campieri M, Rizzello F, Venturi A, et al. uated. Combination of antibiotic and probiotic treatment Gut 2005;54:898–900. The data from this study suggest that is efficacious in prophylaxis of post-operative doi: 10.1136/gut.2004.060640 recurrence of Crohn’s disease: a randomised

L casei administration exerts a protective controlled study vs mesalazine. Gastroenterology on October 1, 2021 by guest. Protected copyright. effect by preventing barrier disruption ...... 2000;118:A781. by TNBS, as translocation of bacteria to 17 Mack DR, Michail S, Wei S, et al. Probiotics Authors’ affiliations inhibit enteropathogenic E. coli adeherence in mesenteric lymph nodes was signifi- P Gionchetti, K M Lammers, F Rizzello, vitro by inducing intestinal mucin gene cantly reduced in rats colonised with M Campieri, Policlinic S Orsola, University of expression. Am J Physiol 1999;276:G941–50. standard rat flora and L casei compared Bologna, Department of Internal Medicine and 18 Bernet MF, Brassart D, Neeser JR, et al. with rats colonised with only standard Gastroenterology, Bologna, Italy Lactobacillus acidophilus LA1 binds to cultured human intestinal cell lines and inhibits cell rat flora. L casei intervention influenced attachment and cell invasion by enterovirulent further extension of mucosal injury Correspondence to: Dr P Gionchetti, Policlinic bacteria. Gut 1994;35:483–9. induced by TNBS, but not the nature Sant’Orsola, Department of Internal Medicine 19 Gewirtz AT, Liu Y, Sitaraman SV, et al. Intestinal epithelial pathobiology: past, present and future. of the colonic lesions (transmural and Gastroenterology, University of Bologna, Via Massarenti 9, 40138 Bologna, Italy; Best Pract Res Clin Gastroenterol lesions were similar in depth compared [email protected] 2002;16:851–67. with TNBS colitis), and resulted in 20 Madsen K, Cornish A, Soper P, et al. Probiotic Conflict of interest: None declared. bacteria enhance murine and human intestinal lower myeloid peroxidase activity, a epithelial barrier function. Gastroenterology measure of tissue neutrophil infiltra- 2001;121:580–91. tion. REFERENCES 21 Otte J-M, Podolsky DK. Functional modulation of enterocytes by gram-positive and gram-negative The exact mechanisms by which 1 Guarner F, Casellas F, Borruel N, et al. Role of microorganisms. Am J Physiol Gastrointest Liver probiotics can influence barrier function microecology in chronic inflammatory bowel Physiol 2004;286:G613–26. remain to be elucidated. It is known diseases. Eur J Clin Nutr 2002;56:S34–8. 22 Lammers KM, Helwig U, Swennen E, et al. Effect that certain lactobacilli adhere to muco- 2 Campieri M, Gionchetti P. Probiotics in of probiotic strains on interleukin 8 production by inflammatory bowel disease: new insight to HT29/19A cells. Am J Gastroenterol sal surfaces, inhibit attachment of pathogenesis or a possible therapeutic 2002;97:1182–6. pathogenic bacteria, and enhance secre- alternative? Gastroenterology 23 Ulisse S, Gionchetti P, D’Alo S, et al. Expression tion of mucins.17 18 27 These properties 1999;116:1246–9. of cytokines, inducible nitric oxide synthase, and 3 Schaafsma G. State of the art concerning matrix metalloproteinases in pouchitis: effects of may be instrumental in improving probiotic strains in milk products. IDF Nutr Newsl probiotic treatment. Am J Gastroenterol mucosal barrier function and decreasing 1996;5:23–4. 2001;96:2691–9.

www.gutjnl.com 900 COMMENTARIES

24 Jijon H, Backer J, Diaz H, et al. DNA from 27 Willemsen LE, Koetsier MA, van Deventer SJ, et barrier injury induced by exposure to probiotic bacteria modulates murine and human al. Short chain fatty acidsstimulate epithelial trinitronbenzene sulphonic acid. Gut epithelial and immune function. Gastroenterology mucin 2 expression through differential effects on 2005;54:955–9. 2004;126:1358–73. prostaglandin E(1) and E(2) production by 30 Fasano A, Nataro JP. Intestinal epithelial tight

25 Hart AL, Lammers KM, Brigidi P, et al. Modulation intestinal myofibroblasts. Gut 2003;52:1442–7. junctions as targets for enteric bacteria- Gut: first published as 10.1136/gut.2004.055889 on 10 June 2005. Downloaded from of human dendritic cell phenotype and function by 28 Petrof EO, Kojima K, Ropeleski MJ, et al. derived toxins. Adv Drug Deliv Rev probiotic bacteria. Gut 2004;53:1602–9. Probiotics inhibit nuclear factor-kappaB and 2004;56:795–807. 26 Lammers KM, Brigidi P, Vitali B, et al. induce heat shock proteins in colonic epithelial 31 Schmitz H, Barmeyer C, Fromm H, et al. Immunomodulatory effects of probiotic bacteria cells through proteasome inhibition. Altered tight junction structure contributes to DNA: IL-1 and IL-10 response in human Gastroenterology 2004;127:1474–87. the impaired epithelial barrier function in peripheral blood mononuclear cells. FEMS 29 Llopis M, Antolı´n M, Guarner F, et al. Mucosal ulcerative colitis. Gastroenterology Immunol Med Microbiol 2003;38:165–72. colonisation with Lactobacillus casei mitigates 1999;116:301–9.

Ulcerative colitis as abnormal permeability8). The mucus ...... layer is abnormal, both in its thickness9 and composition (such as abnormal glycosylation of mucins10–12 and abnorm- Reinforcing the mucus: a new alities of the phospholipid compo- nent13). Many of these abnormalities therapeutic approach for ulcerative are independent of the presence of mucosal inflammation, although colitis? whether they are primary abnormalities or secondary to other processes has P R Gibson, J G Muir never been definitively demonstrated. Why such abnormalities are present— ...... whether autoimmune injury to the epithelium is occurring, whether there Luminal delivery of phosphatidyl rich phospholipids appears to might be a genetic basis for epithelial reduce mucosal inflammatory activity in a high proportion of structure or function, whether there are patients with chronically active ulcerative colitis. The simplicity and luminal factors that might induce apparent safety of this therapeutic approach offers new insights abnormal behaviour of the epithelium, or a combination of any or all of these— into the importance of the mucus barrier in the pathogenesis and has been the basis of much study and treatment of ulcerative colitis speculation without definitive answers. Targeting the epithelial barrier in he treatment of active mucosal for example, disease distribution. The order to reduce the stimulus to inflam- inflammation in ulcerative colitis nature of the inflammatory response in matory events, to enhance healing of actively inflamed mucosa, and to pre- Tremains challenging. Current thera- ulcerative colitis—intense polymorph http://gut.bmj.com/ pies have limited efficacy and may be infiltration and predominant antibody vent relapse have also been the subject of much speculation and study. associated with clinically significant mediated (TH2) responses with less adverse effects. There is room for new prominent T cell activation—is most Approaches have ranged from improv- therapeutic approaches. While nearly all consistent with exposure of the immune ing the regenerative ability of the of our current pharmacological system to large numbers of different epithelium (such as the use of growth 14 15 approaches involve attacking various ‘‘foreign’’ molecules.2 Such events might factors ), to trying to enhance energy immune and inflammatory pathways be anticipated in a situation where substrate supply (using butyrate ene- 16 on October 1, 2021 by guest. Protected copyright. in order to facilitate healing, few in the multiple molecules are able to pass mas ), and to enhancing the mucous 15 17 clinician’s current arsenal are directed through a deficient epithelial barrier. barrier (with trefoil peptides or towards enhancing or protecting the This is in marked contrast with the inhibition of bacterial sulphatases with 18 colonic epithelial barrier. Is this situa- situation in Crohn’s disease where the bismuth ). None of these approaches tion about to change? patchy inflammation involving deeper has yet to be promoted from potential The understanding of the pathogen- layers of the intestinal wall and draining therapy to use in regular practice esis of ulcerative colitis has not con- lymph nodes, together with dominant T because, for example, the theoretical basis was misguided, efficacy was lim- siderably progressed over the last cell activation and TH1-type cytokine decade. The proponents of concepts that profile that characterises the response to ited, or further development was hin- the primary abnormalities lie within a limited number of antigens specifically dered by funding difficulties and immune and inflammatory mechanisms taken up and presented to T cells via commercial realities. Will further atten- have stumbled in attempts to explain follicle associated epithelial cells.23 tion to colonic mucus change this the striking features of ulcerative colitis, Studies that date back more than situation? such as the diffuse nature of the 20 years have demonstrated that the inflammation, its confinement to the colonic epithelium is abnormal in struc- COLONIC MUCUS mucosal compartment, and its distribu- ture and function in patients with The normal colon is lined by a layer of tion in the large bowel. The alternative ulcerative colitis. The epithelium com- mucus that is more than 100 mm thick.9 concept that primary abnormalities lie prises cells that are metabolically abnor- The mucus serves essential functions. It within an abnormal epithelial barrier mal (such as deficient b-oxidation4 or is a lubricant ensuring low friction sits more comfortably with these char- sulphation of phenols5), respond abnor- between moving structures (luminal acteristic features of the disease.1 The mally to stress (as shown by the contents) and the epithelium. This con- barrier has regional differences in response in vitro after its separation ceptually simple but essential protective structure, composition, and function, from the basement membrane67), and function is predominantly the responsi- which offer simple explanations for, have an abnormal cell membrane (such bility of surface acting phospholipids.19

www.gutjnl.com COMMENTARIES 901

They form an oligolamellar lining that contain one saturated (palmitic acid that induce apoptosis turn off the converts the hydrophilic epithelial sur- 16:0 or stearic acid 18:0) and one biosynthesis of PC.28 Most epithelial cell face into a hydrophobic one that inter- unsaturated (oleic acid 18:1or linoleic death in ulcerative colitis appears to 29 30 faces with luminal contents. The tight acid 18:2) fatty acid with PC (that is, PC follow an apoptotic pathway. Thus Gut: first published as 10.1136/gut.2004.055889 on 10 June 2005. Downloaded from packing together of fatty acid chains 16:0/18:1 and PC 18:0/18:2).13 This the increased apoptosis that is occurring provides a good basis for a hydrophobic contrasts with the PC of pulmonary in the epithelium may deplete the pool barrier. Indeed, the regions of the surfactant, dipalmitoylphosphatidylcho- of PC by inhibiting its biosynthesis. gastrointestinal tract with the most line, which contains two saturated fatty PC can be destroyed within the developed hydrophobicity are the stom- acids, palmitic acid (PC 16:0/16:0).19 epithelium, thereby depleting the pool ach and colon, where the potential for The origin of PC in mucus has not of PC available for secretion into mucus, injurious insults from luminal factors been established and more research is or can be destroyed within the mucus are the greatest.20 Most attention in required to understand how, when, and itself. Mucosal phospholipase A2 activ- recent years has been paid to other where these surface active phospho- ity is increased in patients with ulcera- mucous components that subserve com- lipids are synthesised, stored, and tive colitis or Crohn’s disease31 32 and plementary protective functions. Mucins secreted. In animal studies, there is this activity resides in the epithelium.33 are glycoproteins that function to some evidence that PC is primarily Protein kinase C, which is involved in exclude large molecules (polymers with secreted by the jejunum and ileum, several signal transduction pathways a molecular weight .20 000) that are suggesting that PC is delivered to the linked with inflammatory responses not glycoproteins, and to trap other mucus via the lumen.13 In these studies, (see Brown and colleagues34), activates molecules either non-specifically via the contribution of PC produced via the a PC specific phospholipase C in the general ‘‘stickiness’’ or more specifically colonic epithelium appeared to be mini- plasma membrane with subsequent via carbohydrate structures (lectin bind- mal. It seems somewhat surprising and breakdown of PC.35 Indeed, colonic ing sites) that may be similar to those highly unlikely that a local source of PC mucosa from patients with ulcerative on the cell surface.21 Such trapped secretion into the mucosal gel layer is colitis has significantly elevated activity matter can be discarded by the constant not operational. Goblet cells are an of protein kinase C in the particulate removal of mucus. It is no surprise then obvious site for further investigation. fraction compared with that in normal that mucus secretion is increased in the Approaches that have been used to mucosal samples.36 Insertion of fluores- face of threats and this may be understand the role of surface active cent analogues of PC into the plasma mediated, at least in part, by immune phospholipids in the gastric mucosa and membrane of cells followed by activa- events.22 The mucus environment also as pulmonary surfactant could be read- tion of protein kinase C with phorbol supports the presence of other protective ily employed to gain a greater under- esters has been used to follow the proteins and peptides such as secretory standing about the production of movement of PC and its metabolites 23 24 25 IgA, lactoferrin, and trefoil peptides. mucous PC in the colon. A great deal via fluorescent microscopy.37 A similar Of all of these components, it has not has been learnt by the use of special approach could be used to gain a greater been clear what is or are the most probes and stains specific for lipophilic understanding about the fate of PC in important from functional, pathogenic, areas and choline based phospholipids the colonic mucus in ulcerative colitis 20 or treatment points of view. Much of the (see Lichtenberger ), and their applica- and in healthy individuals. glamour and excitement have sur- tion to colonic mucus seems warranted. Once phospholipids enter the mucus http://gut.bmj.com/ rounded the mucin glycoproteins and they remain vulnerable to the action of other secreted proteins within the MUCUS PHOSPHOLIPIDS IN phospholipases, which may be of mucus. However, recent evidence sug- ULCERATIVE COLITIS epithelial origin or derive from mucosa gests that the phospholipid component As outlined above, mucus is abnormal associated bacteria. In the stomach, might be a critical factor that can be in patients with ulcerative colitis. There Helicobacter pylori colonises the mucous readily modulated when the mucous is, however, limited knowledge regard- layer in part by producing phospho- barrier is failing.

ing the phospholipid component of lipases A1, A2, and C, and can extract on October 1, 2021 by guest. Protected copyright. colonic mucus in ulcerative colitis. the host phospholipids for its own pro- PHOSPHOLIPIDS Recently, quantitatively less PC and tective coating.20 H pylori can also gen- Phospholipids, the major lipid compo- LPC were reported in samples taken erate high concentrations of ammonium nents of mucus, are amphiphilic mol- from the rectal mucosa of patients with ion that competes with phospholipids ecules and contain a polar head group ulcerative colitis than from healthy for negatively charged glycoprotein and non-polar hydrocarbon (fatty acid) controls and patients with Crohn’s dis- binding sites.20 It is intriguing to spec- tails. The major classes of phospholipids ease.13 This could be due to reduced ulate about the possible role of a include phosphatidylcholine (PC), phos- production, increased breakdown, or H pylori-like bacterium that might be phatidylethanolamine, phosphatidyl- both. responsible (in part at least) for the inostiol, and phosphatidylserine. In If indeed goblet cells do contribute to breakdown of the colonic mucous bar- colonic mucus, PC and lysophospha- the PC content of colonic mucus, they rier in ulcerative colitis. Large numbers tidylcholine (LPC) are the major might play more than a passive role in of bacteria are found within the species.13 LPC is an intermediate in the PC depletion of mucus. Goblet cell depleted mucous layer of these metabolism of PC but is also produced depletion is a more prominent patholo- patients,38 39 but whether such bacteria after the hydrolysis of PC by phospho- gical feature of ulcerative colitis than for are efficient producers of phospho- lipase A2.26 Orientation of the lipophilic Crohn’s colitis. While it might just lipases is not known. region of the phospholipid and the reflect excessive stimulation of the gob- The net result of reduced biosynthesis nature of the fatty acids characterise let cells to discharge their mucus, the and increased breakdown of phospholi- the hydrophobicity of the mucus gel cells might also be defective in their pids within the mucosa might be a PC layer.20 The fatty acid tails extend into ability to incorporate PC into the mucus, starved system in ulcerative colitis with the lumen to form a ‘‘non-wettable’’ offering another candidate for the pri- subsequent depletion of PC available for resistant layer.20 27 They also extend mary abnormality in ulcerative colitis. mucus. Indeed, the observation that from the mucosal cell side of the mucus Control of PC biosynthesis is a key in plasma phospholipid levels are low in gel.27 In mucus, the PC species typically the apoptotic programme so that agents patients with severe ulcerative colitis

www.gutjnl.com 902 COMMENTARIES supports such a concept.40 If this situa- is somewhat surprising to those used to acid in place of the cellulose), to tion were combined with excessive dealing with randomised controlled determine the appropriate dose, and to destruction of PC within the mucus drug trials. This suggests laxity in define the time course of effect. layer itself, the ability of PC to offer documenting every minor event but Additional questions are raised by the Gut: first published as 10.1136/gut.2004.055889 on 10 June 2005. Downloaded from adequate barrier and lubricant function again does not detract from the efficacy complex nature of the phospholipid might be severely compromised. Could demonstrated. The placebo treated mixture used. For example, is the effect correction of phospholipid deficiency of patients faired badly. A 10% response observed indeed due to the PC compo- the mucosa and/or mucus be of ther- (and remission) rate is at the lower end nent or is the active moiety a minor part apeutic value in ulcerative colitis? of what might be anticipated in most of the phospholipid mixture, such as the trials, except that patients with chroni- unsaturated fatty acid component? The cally active disease, as selected, might question of how this phospholipid mix- PHOSPHOLIPIDS AS A THERAPY have a lower placebo response. The ture is achieving its efficacy, particularly Phospholipids are readily taken up by choice of placebo, Eudragit-S 100 coated whether it is acting via reinforcement of mucus, so that the mucous content can cellulose, may not have been appropri- the mucus, as seems logical, or by other be substantially increased by their topi- ate. Cellulose would be delivered to the mechanisms, needs to be addressed. cal administration. In experimental ani- colonic lumen in an identical fashion to While there has been much previous mals, where there is no abnormality of that of the phospholipids and, since it is focus on the mucin glycoproteins, this mucus, topical PC protects the mucosa not fermented by colonic bacteria, it work shows that more attention needs from luminal insults in the form of might be considered benign and inert. to be drawn to the less glamorous lipid acetic acid and trinitrobenzenesulphonic However, cellulose might potentially be component, which is not simply playing acid, which usually induce colitis.41 42 It abrasive to the mucosa, this being a an inert structural role in the mucus gel might be anticipated that the protective postulated mechanism by which non- layer but rather is a dynamic component and lubricant function of mucus would fermentable fibre stimulates epithelial of a complex barrier system. The puta- be considerably enhanced by topical proliferation in fibre starved atrophic tive efficacy of PC might be enhanced by application of PC in a situation such as colon in otherwise healthy rats.44 The better distribution using spreading ulcerative colitis where PC content is placebo therefore might have worsened agents. Perhaps liposomes of PC could reduced.13 Such a simple concept has the outcome. On the other hand, there is be used to introduce other therapies recently been evaluated43 and the results no evidence that non-fermented fibre is such as anti-inflammatory drugs or even of a randomised controlled trial are detrimental to the course of ulcerative antisense RNA that will assist with reported in this issue of Gut (see page colitis. Whatever the case, it is still epithelial healing—two for the price of 966). Delivery to the large bowel lumen reasonable to say that three months of one! There is also the potential to was achieved by coating of PC enriched therapy with PC rich phospholipids introduce phospholipids and fatty acid phospholipids with Eudragit-S 100. delivered to the colonic lumen were species, such as arachidonic and butyric Release of phospholipids from this pH convincingly efficacious in inducing acids that may have cytoprotective dependent coating would be expected to remission in patients with chronically properties. Welcome phospholipids to predominantly occur in the terminal active ulcerative colitis. the cutting edge of ulcerative colitis! ileum and proximal colon, a situation mimicking the putative main source of What about the mechanism of action? Gut 2005;54:900–903. phospholipids in the colonic mucus. It would be precarious to assign this doi: 10.1136/gut.2004.058453 http://gut.bmj.com/ Details of the origin (for example, from efficacy to the improved hydrophobicity soy or egg), and the type of phospho- and barrier function of the mucus ...... lipids and fatty acid species used in this without confirmatory data. The hydro- Authors’ affiliations study were not described. Nevertheless, phobicity of the mucosal layer can be P R Gibson, J G Muir, Monash University Department of Medicine and Department of PC was successfully incorporated into quantified from the ‘‘contact angle’’ Gastroenterology, Box Hill Hospital, Box Hill, rectal mucus confirming that the deliv- after a drop of saline is placed on the mucosal surface.27 Whether this simple Victoria, Australia ery system works. on October 1, 2021 by guest. Protected copyright. test can be carried out on biopsy speci- In a population of 60 patients with mens or even at colonoscopy is uncer- Correspondence to: Professor P Gibson, chronically active disease, the efficacy of tain, but such an assessment in patients Department of Gastroenterology, Box Hill the PC was astoundingly good over a Hospital, Box Hill, Victoria 3128, Australia; with ulcerative colitis seems worth- three month treatment period. [email protected] while. Alternatively, effects on mucus Compared with a response of 10% in might only be a minor player in the Conflict of interest: None declared. the placebo arm, 90% of the phospho- efficacy. Replenishment of the epithelial lipid treated group responded and 53% pool of PC, with its potential positive were in remission after three months of REFERENCES effects on improving epithelial health, therapy. No clinically significant side 1 Gibson PR. Ulcerative colitis—an epithelial limiting epithelial destruction, and sup- disease? Ballieres Clin Gastroenterol effects were noted. Is this too good to be pressing its involvement with inflam- 1997;1:17–33. true? 2 Pallone F, Monteleone G, Monteleone I, et al. The matory mechanisms, as discussed There were problems with the study immune system in inflammatory bowel disease. In: above, might also be an important Satsangi L, Sutherland LR, eds. Inflammatory design. Despite being described as a mechanism of action. bowel diseases. Edinburgh: Churchill Livingstone, single centre study, end points were 2003:85–93. scored in multiple centres for practical 3 Pavli P, Gibson PR. Pathogenic factors in inflammatory bowel disease. II. Crohn’s disease. reasons. However, as blinding and ran- FUTURE DIRECTIONS Dig Dis 1992;10:72–86. domisation seemed appropriate, hetero- The findings that treatment with PC rich 4 Roediger WEW. The colonic epithelium in geneity of assessment is unlikely to be a phospholipids permits healing of other- ulcerative colitis—an energy deficiency disease? significant factor in skewing the results. wise difficult to treat ulcerative colitis Lancet 1980;2:712–15. 5 Ramakrishna BS, Roberts-Thompson JC, There were also no data on the time opens a new approach to the treatment Pannall PR, et al. Impaired sulphation of phenol course of efficacy, although a comment of ulcerative colitis. There is the impor- by the colonic mucosa in quiescent and active in the discussion indicated a slow onset tant need to confirm the results using a ulcerative colitis. Gut 1991;32:46–9. 6 Gibson PR, Rosella O. Interleukin-8 secretion by of effect over weeks. The dearth of more appropriate placebo (perhaps free colonic crypt cells in vitro: a response to injury that adverse events in a three month study unsaturated fatty acid such as linoleic is suppressed by butyrate and enhanced in

www.gutjnl.com COMMENTARIES 903

inflammatory bowel disease. Gut 18 Tsai HH, Dwarakanath AD, Hart CA, et al. mucosa: arachidonic acid-rich phosphatidyl/ 1995;37:536–43. Increased faecal mucin sulphatase activity in plasmenyl-ethanolamine in the mucosa in 7 Gibson PR, Rosella O. Abnormalities of the ulcerative colitis: a potential target for treatment. inflammatory bowel disease. J Gastroenterol urokinase system in colonic crypt cells from Gut 1995;36:570–6. 1999;34:46–53. patients with ulcerative colitis. Inflamm Bowel Dis 19 Hills BA. Surface-acting phospholipid: a 33 Minami T, Shinomura Y, Miyagawa J, et al. Gut: first published as 10.1136/gut.2004.055889 on 10 June 2005. Downloaded from 1996;2:105–14. Pandora’s box of clinical applications. Part II. Immunohistochemical localization of group II 8 Gibson P, Rosella R, Nov R, et al. Colonic Barrier and lubricating properties. Int Med J phospholipase A2 in colonic mucosa of patients epithelium is diffusely abnormal in ulcerative 2002;32:242–51. with inflammatory bowel disease. colitis and colorectal carcinoma. Gut 20 Lichtenberger LM. The hydrophobic barrier Am J Gastroenterol 1997;92:289–92. 1995;36:857–63. properties of gastrointestinal mucus. Annu Rev 34 Brown JF, Chang Q, Soper BD, et al. Protein 9 Pullan RD, Thomas GA, Rhodes M, et al. Physiol 1995;57:565–83. kinase C mediates experimental colitis in the rat. Thickness of adherent mucus gel on colonic 21 Clamp J. The role of mucus in human intestinal Am J Physiol 1999;276:G583–90. mucosa in humans and its relevance to colitis. Gut defence. In: Losowsky MS, Heatley RV, eds. Gut 35 Nishizuka Y. Intracellular signaling by hydrolysis 1994;35:353–9. defences in clinical practice. Edinburgh: Churchill of phospholipids and activation of protein kinase 10 Rhodes JM, Black RR, Gallimore R, et al. Livingstone, 1986:83–94. C. Science 1992;258:607–14. Histochemical demonstration of desialation and 22 Lake AM, Bloch KJ, Neutra MR, et al. Intestinal 36 Sakanoue Y, Hatada T, Horai T, et al. Protein desulphation of normal and inflammatory bowel goblet cell mucus release. II. In vivo stimulation by kinase C activity of colonic mucosa in disease rectal mucus by faecal extracts. Gut antigen in the immunized rat. J Immunol ulcerative colitis. Scand J Gastroenterol 1985;26:1312–18. 1979;122:834–7. 1992;27:275–80. 37 Glatz JA, Muir JG, Murray AW. Direct evidence 11 Raouf AH, Tsai HH, Parker N, et al. Sulphation of 23 Brandtzaeg P. Molecular and cellular aspects of for phorbol ester-stimulated accumulation of colonic and rectal mucin in inflammatory bowel the secretory immunoglobulin system. APMIS 1995;103:1–19. diacylglycerol derived from phosphatidylcholine. disease: reduced sulphation of rectal mucus in 24 Weinberg, ed. Human lactoferrin: a novel Carcinogenesis 1987;8:1943–5. ulcerative colitis. Clin Sci 1992;83:623–6. therapeutic with broad spectrum potential. 38 Schultsz C, Van Den Berg FM, Ten Kate FW, et al. 12 Morita H, Kettlewell MG, Jewell DP, et al. J Pharm Pharmacol 2001;53:1303–10. The intestinal mucus layer from patients with Glycosylation and sulphation of colonic mucus 25 Sands BE, Podolsky DK. The trefoil peptide family. inflammatory bowel disease harbors high glycoproteins in patients with ulcerative colitis and Annu Rev Physiol 1996;58:253–73. numbers of bacteria compared with controls. in healthy subjects. Gut 1993;34:926–32. 26 Prokazova NV, Zvezdina ND, Korotaeva AA. Gastroenterology 1999;117:1089–97. 13 Ehehalt R, Wagenblast J, Erben G, et al. Effect of lysophosphatidylcholine on 39 Kleessen B, Kroesen AJ, Buhr HJ, et al. Mucosal Phosphatidylcholine and lysophosphatidylcholine transmembrane signal transduction. Biochemistry and invading bacteria in patients with in intestinal mucus of ulcerative colitis patients. A 1998;63:31–7. inflammatory bowel disease compared with quantitative approach by nanoelectrospray 27 Hills BA. Surface-acting phospholipid: a controls. Scand J Gastroenterol tandem mass spectrometry. Scand J Gastroenterol Pandora’s box of clinical applications. Part I. The 2002;37:1034–41. 2004;39:737–42. lung and air spaces. Int Med J 2002;32:170–8. 40 Gjone E. Plasma phospholipids in ulcerative 14 Howarth GS, Xian CJ, Read LC. Insulin-like 28 Wright MM, Howe AG, Zaremberg V. Cell colitis. A quantitative thin layer chromatographic growth factor-I partially attenuates colonic membranes and apoptosis: role of cardiolipin, study. Scand J Clin Lab Invest 1966;18:263–7. damage in rats with experimental colitis induced phosphatidylcholine, and anticancer lipid 41 Fabia R, Ar’Rajab A, Willen R, et al. Effects of by oral dextran sulphate sodium. analogues. Biochem Cell Biol 2004;82:18–28. phosphatidylcholine and phosphatidylinositol on Scand J Gastroenterol 1998;33:180–90. 29 Iwamoto M, Koji T, Makiyama K, et al. Apoptosis acetic-acid-induced colitis in the rat. Digestion 15 FitzGerald AJ, Pu M, Marchbank T, et al. of crypt epithelial cells in ulcerative colitis. J Pathol 1992;53:35–44. Synergistic effects of systemic trefoil factor family 1996;180:152–9. 42 Mourelle M, Guarner F, Malagelada JR. 1 (TFF1) peptide and epidermal growth factor in a 30 Kruidenier L, Kuiper I, Lamers CB, et al. Intestinal Polyunsaturated phosphatidylcholine prevents rat model of colitis. Peptides 2004;25:793–801. oxidative damage in inflammatory bowel disease: stricture formation in a rat model of colitis. 16 Scheppach W. Treatment of distal ulcerative semi-quantification, localization, and association Gastroenterology 1996;110:1093–7. colitis with short-chain fatty acid enemas. A with mucosal antioxidants. J Pathol 43 Stremmel W, Merle U, Zahn A, et al. Retarded placebo-controlled trial. German-Austrian SCFA 2003;201:28–36. release phsophatidylcholine benefits patients with Study Group. Dig Dis Sci 1996;41:2254–9. 31 Minami T, Tojo H, Shinomura Y, et al. Increased chronic active ulcerative colitis. Gut 17 Vandenbroucke K, Hans W, Van Huysse J, et al. group II phospholipase A2 in colonic mucosa of 2005;54:966–71. Active delivery of trefoil factors by genetically patients with Crohn’s disease and ulcerative 44 Mariadason JM, Catto-Smith A, Gibson PR. modified Lactococcus lactis prevents and heals colitis. Gut 1994;35:1593–9. Modulation of distal colonic epithelial barrier http://gut.bmj.com/ acute colitis in mice. Gastroenterology 32 Morita H, Nakanishi K, Dohi T, et al. function by dietary fibre in normal rats. Gut 2004;127:502–13. Phospholipid turnover in the inflamed intestinal 1999;44:394–9.

Hepatitis C virus has recently been directed towards ...... understanding the mechanisms under-

lying the development of these meta- on October 1, 2021 by guest. Protected copyright. bolic manifestations in HCV infected Insulin resistance and steatosis in patients and their implications in the progression of liver disease. Several hepatitis C virus infection important questions have been exam- ined: are these metabolic disorders in A Zekry, J G McHutchison, A M Diehl HCV infected patients a result of viral or host factors and, if viral, how do viral ...... proteins interfere with lipid and insulin metabolism? What is the primary event The relationship between hepatitis C virus (HCV), steatosis, and in these patients (steatosis or IR) and insulin resistance is genotype specific, and steatosis and insulin what are the implications of steatosis resistance are closely linked to the progression of liver disease in and IR in the pathogenesis of liver HCV infected patients disease? Finally, how can we exploit our current knowledge for developing effective therapeutic strategies for HCV ince the identification of hepatitis infection than other chronic inflamma- infected patients? C virus (HCV) in the late 1980s, tory liver disease.1 A proportion of HCV In this issue of Gut, Fartoux and Schronic HCV infection has emerged infected patients with steatosis also colleagues2 utilise the homeostasis as a complex multifaceted disease with exhibit several of the clinical features model assessment for IR (HOMA IR) manifestations extending beyond the seen in non-alcoholic steatohepatitis index to study the association between liver. As such, hepatic steatosis, insulin (NASH), questioning the significance steatosis and IR in patients with chronic resistance (IR), and type II diabetes of these metabolic disorders in the HCV infection (see page 1003). In order have been observed to occur more pathogenesis of HCV related liver dis- to examine this association, the authors frequently in association with HCV ease. Hence, considerable HCV research excluded patients with alternate factors

www.gutjnl.com 904 COMMENTARIES

Table 1 Clinical evidence for a steatogenic effect for genotype 3 increased in patients with NASH and chronic HCV infection.22 23 Moreover, in l Hepatic steatosis is present in the majority of patients with genotype 3 infection each of these diseases, serum leptin l Hepatic steatosis correlates with the level of HCV RNA in serum and the liver level has been reported to independently Gut: first published as 10.1136/gut.2004.055889 on 10 June 2005. Downloaded from l In patients infected with genotype 3 (but not genotype 1), successful viral clearance with antiviral correlate with hepatic steatosis.22 24 therapy results in the disappearance of steatosis, only to recur on viral relapse l IR has been observed to be less common among genotype 3 infected patients, even in those with While it is recognised that the effect of extensive hepatic steatosis. leptin on insulin sensitivity is variable, increased expression of leptin in hepa- HCV, hepatitis C virus; IR, insulin resistance. tocytes has been shown to attenuate several insulin induced activities, including tyrosine phosphorylation of known to be associated with the devel- INSULIN RESISTANCE AND IRS-1.25 opment of steatosis (such as alcohol STEATOSIS IN HCV INFECTED Adiponectin is abundantly synthe- intake .20 g/day or diabetes). Their PATIENTS sised and secreted by adipose tissue. findings confirm earlier observations3–5 Steatosis in patients with genotype 1 Serum levels of adiponectin correlate that differing and genotype specific infection is increasingly recognised as a with systemic insulin sensitivity. More mechanisms characterise the develop- component of the metabolic syndrome, recently, low levels of serum adiponec- ment of hepatic steatosis in HCV a condition invariably associated with tin have been shown to correlate with infected patients. Accordingly, steatosis IR. In support, host factors such as age, the presence of steatosis in patients with in genotype 3 infected patients is pri- body mass index (BMI), and central chronic HCV infection. The latter obser- marily viral mediated (cytopathic) obesity, have been shown to correlate vation suggests a role for adiponectin in whereas host factors, principally those with the development of steatosis in the development of IR and steatosis in associated with IR and its clinical genotype 1 infected patients (but not these patients.26 attributes, are responsible for the devel- genotype 3).18 Fartoux and colleagues2 In contrast with the effect of fat on opment of steatosis in the genotype 1 have further demonstrated that HOMA IR, insulin itself controls the regulation infected patient. IR was the only risk factor indepen- of a host of genes involved in lipid dently associated with the development metabolism.27 The ability of insulin to of steatosis in genotype 1 infected activate lipogenesis is partly mediated DIRECT STEATOGENIC EFFECT OF patients (p = 0.001). Moreover, the by inducing the transcription of the HCV degree of steatosis was significantly sterol regulatory element binding pro- Evidence for a HCV genotype 3 specific predictive of HOMA IR (p = 0.004). tein 1c (SREBP-1c), a key regulator of steatogenic effect has come from several There are several uncertainties fatty acid synthesis in the liver. The clinical observations (table 1).36–8 regarding the cascade of events leading effect of insulin on SREBP-1c transcrip- Furthermore, this direct steatogenic to the development of IR and steatosis tion is even more apparent in the 28 effect of HCV is highly reproducible in in HCV infected patients with features presence of profound IR. In this transgenic mice and cell culture models of the metabolic syndrome. As such, setting, obese animals with IR have of hepatitis C, in which overexpression whether IR or steatosis is the primary or increased levels of SREBP-1c resulting of viral protein (such as core and NS5A) secondary event in these patients is in increased rates of fatty acid synthesis http://gut.bmj.com/ has been shown to induce accumula- unclear. and the development of hepatic steato- tion of intracytoplasmic triglyceride rich sis.29 30 Subsequent normalisation of 9 Overall, there are sufficient clinical droplets. and experimental data indicating that SREBP-1 expression in these animals 28 There are several identified mechan- excess fat can perpetuate IR: large dramatically ameliorates steatosis. isms whereby HCV may alter lipid epidemiological studies reveal that the metabolism. Firstly, HCV core protein risk of IR rises as body fat content HCV AND INSULIN RESISTANCE has been shown to directly inhibit the (determined by BMI) increases, from Several studies (including that of on October 1, 2021 by guest. Protected copyright. function of microsomal triglyceride the very lean to the very obese.19 Fartoux and colleagues2) evaluating IR transfer protein, a major regulator of Experimental evidence also indicates in patients with chronic HCV infection hepatic assembly, and secretion of nas- that intracellular accumulation of fatty have found that the development of IR cent triglyceride rich very low density acid metabolites (either through can occur early in the course of the lipoproteins (VLDL). The latter effect increased delivery or decreased metabo- disease. This effect appears to be inde- impairs the ability of hepatocytes to lism) in the liver or muscle directly pendent of body weight, stage of liver 10 11 assemble and secret VLDL. Secondly, promotes serine phosphorylation (in disease, and presence or absence of HCV core protein has been observed to contrast with tyrosine phosphorylation) overt diabetes.31–33 There are other induce mitochondrial injury resulting in of insulin receptor substrate 1 (IRS-1). clinical observations supporting a ‘‘fat oxidative stress. Oxidative stress per- This effect leads to impaired glucose independent’’ mechanism in the devel- turbs lipid peroxidation, thereby con- transport activity and other events opment of IR in HCV infected patients. tributing to the development of downstream of insulin receptor signal- Firstly, Fartoux and colleagues2 and steatosis.12–14 Finally, microarray studies ling.20 In this respect, measures to others32 have observed that patients have illustrated the ability of HCV (in reduce circulating free fatty acid and infected with HCV genotype 3 have particular genotype 3) to induce tran- liver triglyceride content have been more extensive hepatic steatosis but a scription of several genes involved in shown to restore insulin signalling and lower incidence of IR.32 This observation lipid metabolism in the liver.15 16 Among reverse both hepatic and muscle IR supports a genotype specific mechanism these is stearoyl coenzyme A desaturase induced by high fat diets.17 21 underlying IR in HCV infected 4 (SCD4), a rate limiting enzyme in the Another common link in the deve- patients.32 Secondly, two studies synthesis of monounsaturated fats.15 lopment of IR and steatosis is the demonstrated that clearance of HCV Reduced expression of SCD4 in livers adipocyte secreted proteins leptin and with antiviral therapy resulted in of ob/ob mice has been shown to adiponectin. Leptin is a protein product restoration of insulin sensitivity and significantly ameliorate hepatic steato- of the adipocyte obese (ob) gene. Serum euglycaemia.34 35 Finally, the extent of sis.17 leptin levels have been observed to be IR has been shown to correlate with the

www.gutjnl.com COMMENTARIES 905

observed an association between stea- HCV tosis and severity of fibrosis, irrespective ? of HCV genotype. While insulin levels

were predictive of fibrosis in their Gut: first published as 10.1136/gut.2004.055889 on 10 June 2005. Downloaded from ↑ TNF-α ↑ SOC3 ↓ STAT-3 univariate analysis, subsequent multi- variate analysis confirmed steatosis, but not insulin levels, to be independently Decreased tyrosine Reduced activity of Altered expression associated with fibrosis (p = 0.02). kinase activity of IRS-1 PI3K and Akt of PEPCK gene Thus two keys questions arise: is fat per se fibrogenic and what is the role of insulin in the fibrotic process? Leptin Steatosis is associated with increased production of reactive oxygen species which initiate lipid peroxidation, result- 42 Insulin resistance ↓ Adiponectin ing in hepatic stellate cell activation. However, in NASH, disease progression is recognised as being slower than that observed in patients with HCV infection Figure 1 Molecules that are likely to be involved in mediating insulin resistance (IR) in hepatitis C virus (HCV) infection. Tumour necrosis factor a (TNF-a) is increased in the serum and liver of HCV and steatosis. Thus it is likely that the infected patients. TNF induces IR by decreasing the tyrosine kinase activity of insulin receptor coexistence of HCV and steatosis aggra- substrate 1 (IRS-1).39 Treatment of HCV core transgenic mice with anti-TNF-a has been shown to vates and accelerates the injury induced restore insulin sensitivity. High levels of suppressor of cytokine signalling 3 (SOC3) have been by each alone. In this setting, hepatic detected in association with IR in HCV infection. This effect was associated with reduced insulin 37 inflammation induced by the host induced phosphorylation of the p85 subunit of phosphatidylinositol 3-kinase (PI3K) and Akt. response, together with the increased SOC3 (and possibly HCV) can attenuate the activity of signal transducer and activator of transcription 3 (STAT-3). Mice lacking STAT-3 specifically in the liver have been reported to exhibit production of several proinflammatory IR and, when fed a high fat diet, glucose intolerance. The latter was associated with increased and profibrotic cytokines, provide the expression of the phosphoenolpyruvate carboxykinase (PEPCK) gene.38 The PEPCK gene itself is a substrate for the ‘‘second hit’’ in the target for leptin (also increased in HCV infection) mediated regulation of gluconeogenesis in the steatotic liver. Also, the ability of the liver. Finally, reduced expression of adiponectin is associated with obesity and IR. Low serum levels 26 virus itself to induce oxidative stress and of adiponectin have been shown to correlate with steatosis in HCV infected patients. promote lipid peroxidation may further aggravate the pathogenic process grade of portal inflammation in HCV activator of transcription 3 (STAT-3) induced by steatosis. Consider also that infected patients.32 Collectively, these mediated inhibition of this region.38 fat may render HCV infected liver more data suggest that either HCV per se or The net effect in these animals was vulnerable to injury. Livers with steato- the inflammatory response incited by development of systemic IR and hepatic sis are more sensitive to TNF-a mediated HCV in the liver is central to the steatosis. Conversely, inhibiting expres- inflammation and liver injury.43 development of IR. This first hypothesis sion of SOCs protein in obese animals Moreover, in HCV livers with steatosis, has been recently verified in HCV core normalised levels of SREBP-1 and apoptosis activity has been noted to be http://gut.bmj.com/ transgenic mice, observed to develop IR improved insulin sensitivity and hepatic increased compared with infected livers resistance as early as one month of age, steatosis. Consistent with these data, without steatosis.44 in the absence of either overt liver injury hepatic STAT-3 signalling has recently While Fartoux and colleagues2 could or excessive body weight gain. been shown to be essential for normal not find a direct association between IR The precise mechanisms whereby glucose homeostasis and insulin sensi- and fibrosis, others have.32 33 45 The HCV induces IR remain elusive but tivity.39 These observations are relevant authors do concede that insulin plays a recent progress has shed light on several as HCV is recognised as influencing the significant role in the development of on October 1, 2021 by guest. Protected copyright. critical pathways. Impairment of IRS-1 activity of STAT-3. It is therefore intri- fibrosis via a mechanism involving and IRS-2 expression has been observed guing to speculate that STAT-3 may be steatosis. In this regard, steatosis pro- in the livers of both HCV infected one of the key molecules involved in motes cellular IR which, in turn, induces patients as well as in HCV core trans- HCV mediated IR (fig 1). compensatory hyperinsulinaemia. genic mice.36 37 Specifically, HCV core Hyperinsulinaemia has been shown to protein has been shown to inhibit ROLE OF CYTOKINES directly stimulate hepatic stellate cell insulin induced phosphorylation of the Tumour necrosis factor a (TNF-a) levels proliferation and increase expression of p85 subunit of phosphatidylinositol are elevated in the liver and serum of connective tissue growth factor, a key 46 3-kinase (PI3K) and Akt, which are patients with chronic HCV infection. factor in the progression of fibrosis. downstream components of IRS in the TNF-a plays an important role in the Collectively, the data implicate both liver.37 Interestingly, HCV has been development of IR through interfering steatosis and IR in liver disease progres- reported to mediate dysfunction of the with insulin signalling.40 In IR HCV core sion in HCV infected patients. insulin signalling pathways by upregu- transgenic mice, treatment with anti- lating the expression of suppressor of TNF-a restored insulin sensitivity, thus CONCLUSIONS cytokine signalling 3 (SOC3).37 This supporting the notion that TNF-a is a Our knowledge of the mechanisms and observation is quite important in light major factor for the development of IR significance of steatosis and IR in 31 of recent data demonstrating direct in HCV infection. patients with chronic HCV infection involvement of SOC1 and SOC3 in has advanced. The work by Fartoux regulating expression of SREBP-1c in ROLE OF STEATOSIS AND and colleagues2 adds to the growing the livers of obese animals.38 In these INSULIN RESISTANCE IN evidence that the relationship between animal models, overexpression of SOC1 PROGRESSION OF HCV LIVER HCV, steatosis, and IR is genotype and SOC3 proteins has been shown to DISEASE specific and that steatosis and IR are enhance SREBP-1c promoter activity In agreement with previous closely linked to the progression of liver by attenuating signal transducer and reports,34741 Fartoux and colleagues2 disease in HCV infected patients. As

www.gutjnl.com 906 COMMENTARIES such, we need to clarify the molecular steatosis in transgenic mice. J Gen Virol 29 Yahagi N, Shimano H, Hasty AH, et al. Absence pathways involved in mediating these 1997;78:1527–31. of sterol regulatory element-binding protein-1 10 Perlemuter G, Sabile A, Letteron P, et al. Hepatitis (SREBP-1) ameliorates fatty livers but not obesity inter-relationships. In light of our cur- C virus core protein inhibits microsomal or insulin resistance in Lep(ob)/Lep(ob) mice. rent knowledge of the implications of triglyceride transfer protein activity and very low J Biol Chem 2002;277:19353–7. Gut: first published as 10.1136/gut.2004.055889 on 10 June 2005. Downloaded from steatosis and IR in liver disease, the density lipoprotein secretion: a model of viral- 30 Horton JD, Shimomura I, Ikemoto S, et al. related steatosis. FASEB J 2002;16:185–94. Overexpression of sterol regulatory element- importance of lifestyle changes (such as 11 Serfaty L, Andreani T, Giral P, et al. Hepatitis C binding protein-1a in mouse adipose tissue weight loss) need to be an emphasised virus induced hypobetalipoproteinemia: a produces adipocyte hypertrophy, increased fatty in treating HCV infected patients. possible mechanism for steatosis in chronic acid secretion, and fatty liver. J Biol Chem hepatitis C. J Hepatol 2001;34:428–34. 2003;278:36652–60. Similarly, new therapeutic approaches 12 Okuda M, Li K, Beard MR, et al. Mitochondrial 31 Shintani Y, Fujie H, Miyoshi H, et al. Hepatitis C exploiting the interaction between HCV injury, oxidative stress, and antioxidant gene virus infection and diabetes: direct involvement of and lipid metabolism are eagerly expression are induced by hepatitis C virus core the virus in the development of insulin resistance. Gastroenterology 2004;126:840–8. awaited. protein. Gastroenterology 2002;122:366–75. 13 Moriya K, Fujie H, Shintani Y, et al. The core 32 Hui JM, Sud A, Farrell GC, et al. Insulin resistance protein of hepatitis C virus induces hepatocellular is associated with chronic hepatitis C virus ACKNOWLEDGEMENTS carcinoma in transgenic mice. Nat Med infection and fibrosis progression[corrected]. 1998;4:1065–7. Gastroenterology 2003;125:1695–704. This work was supported by a grant from 33 Petit JM, Bour JB, Galland-Jos C, et al. Risk NIH, 2RO1 DK053792. 14 Lerat H, Honda M, Beard MR, et al. Steatosis and liver cancer in transgenic mice expressing the factors for diabetes mellitus and early insulin Gut 2005;54:903–906. structural and nonstructural proteins of hepatitis C resistance in chronic hepatitis C. J Hepatol 2001;35:279–83. doi: 10.1136/gut.2004.059873 virus. Gastroenterology 2002;122:352–65. 15 Bigger CB, Guerra B, Brasky KM, et al. 34 Tanaka H, Shiota G, Kawasaki H. Changes in Intrahepatic Gene Expression during Chronic glucose tolerance after interferon-alpha therapy ...... Hepatitis C Virus Infection in Chimpanzees. J Virol in patients with chronic hepatitis C. J Med Authors’ affiliations 2004;78:13779–92. 1997;28:335–46. 16 Shackel NA, McGuinness PH, Abbott CA, et al. 35 Konrad T, Zeuzem S, Vicini P, et al. Evaluation of A Zekry, J G McHutchison, A M Diehl, factors controlling glucose tolerance in patients Division of Gastroenterology and Duke Insights into the pathobiology of hepatitis C virus- associated cirrhosis: analysis of intrahepatic with HCV infection before and after 4 months Clinical Research Institute, Duke University differential gene expression. Am J Pathol therapy with interferon-alpha. Eur J Clin Invest Medical Centre, Durham, North Carolina, 2002;160:641–54. 2000;30:111–21. 36 Aytug S, Reich D, Sapiro LE, et al. Impaired IRS- USA 17 Sekiya M, Yahagi N, Matsuzaka T, et al. 1/PI3-kinase signaling in patients with HCV: a Polyunsaturated fatty acids ameliorate hepatic mechanism for increased prevalence of type 2 steatosis in obese mice by SREBP-1 suppression. diabetes. Hepatology 2003;38:1384–92. Correspondence to: Dr A M Diehl, Hepatology 2003;38:1529–39. 37 Kawaguchi T, Yoshida T, Harada M, et al. Gastroenterology Division, Duke University, 18 Adinolfi LE, Utili R, Ruggiero G. Body composition Hepatitis C virus down-regulates insulin receptor Snyderman Building, GSRB-1, Suite 1073, Box and hepatic steatosis as precursors of fibrosis in substrates 1 and 2 through up-regulation of 3256, 595 LaSalle St, Durham, NC 27710, chronic hepatitis C patients. Hepatology suppressor of cytokine signaling 3. Am J Pathol USA; [email protected] 1999;30:1530–1. 2004;165:1499–508. 19 Colditz GA, Willett WC, Rotnitzky A, et al. Conflict of interest: None declared. 38 Ueki K, Kondo T, Kahn CR. Suppressor of Weight gain as a risk factor for clinical diabetes cytokine signaling 1 (SOCS-1) and SOCS-3 mellitus in women. Ann Intern Med cause insulin resistance through inhibition of 1995;122:481–6. REFERENCES tyrosine phosphorylation of insulin receptor 20 Shulman GI. Cellular mechanisms of insulin substrate proteins by discrete mechanisms. Mol 1 Goodman ZD, Ishak KG. Histopathology of resistance. J Clin Invest 2000;106:171–6. Cell Biol 2004;24:5434–46. hepatitis C virus infection. Sem Liver Dis 21 An J, Muoio DM, Shiota M, et al. Hepatic 39 Inoue H, Ogawa W, Ozaki M, et al. Role of 1995;15:70–81. expression of malonyl-CoA decarboxylase STAT-3 in regulation of hepatic gluconeogenic

2 Fartoux L, Poujol-Robert A, Gue´chot J, et al. reverses muscle, liver and whole-animal insulin genes and carbohydrate metabolism in vivo. Nat http://gut.bmj.com/ Insulin resistance is a cause of steatosis and resistance. Nat Med 2004;10:268–74. Med 2004;10:168–74. fibrosis progression in chronic hepatitis C. Gut 22 Chitturi S, Farrell G, Frost L, et al. Serum leptin in 40 Hotamisligil GS, Murray DL, Choy LN, et al. 2005;54:1003–8. NASH correlates with hepatic steatosis but not Tumor necrosis factor alpha inhibits signaling 3 Kumar D, Farrell GC, Fung C, et al. Hepatitis C fibrosis: a manifestation of lipotoxicity? from the insulin receptor. Proc Natl Acad Sci U S A virus genotype 3 is cytopathic to hepatocytes: Hepatology 2002;36:403–9. 1994;91:4854–8. Reversal of hepatic steatosis after sustained 23 Patel K, Muir A, McHutchison JG, et al. A link 41 Hourigan LF, Macdonald GA, Purdie D, et al. therapeutic response. Hepatology between leptin and steatosis in chronic hepatitis Fibrosis in chronic hepatitis C correlates 2002;36:1266–72. C? Time to weigh up the fats. Am J Gastroenterol significantly with body mass index and steatosis. 4 Adinolfi LE, Gambardella M, Andreana A, et al. 2003;98:952–5. Hepatology 1999;29:1215–9. Steatosis accelerates the progression of liver 24 Romero-Gomez M, Castellano-Megias VM, 42 Chavin KD, Yang S, Lin HZ, et al. Obesity induces on October 1, 2021 by guest. Protected copyright. damage of chronic hepatitis C patients and Grande L, et al. Serum leptin levels correlate with expression of uncoupling protein-2 in hepatocytes correlates with specific HCV genotype and hepatic steatosis in chronic hepatitis C. and promotes liver ATP depletion. J Biol Chem visceral obesity. Hepatology 2001;33:1358–64. Am J Gastroenterol 2003;98:1135–41. 1999;274:5692–700. 5 Rubbia-Brandt L, Quadri R, Abid K, et al. 25 Cohen B, Novick D, Rubinstein M. Modulation of 43 Yang SQ, Lin HZ, Lane MD, et al. Obesity Hepatocyte steatosis is a cytopathic effect of insulin activities by leptin. Science increases sensitivity to endotoxin liver injury: hepatitis C virus genotype 3. J Hepatol 1996;274:1185–8. implications for the pathogenesis of 2000;33:106–15. 26 Michel PJ, Anne M, Valerie J, et al. Decreased steatohepatitis. Proc Natl Acad Sci U S A 6 Poynard T, Ratziu V, McHutchison J, et al. Effect plasma adiponectin concentrations are closely 1997;94:2557–62. of treatment with peginterferon or interferon alfa- related to steatosis in HCV-infected patients. J Clin 44 Walsh MJ, Vanags DM, Clouston AD, et al. 2b and ribavirin on steatosis in patients infected Endocrinol Metab, 2005;not on p/m*. Steatosis and liver cell apoptosis in chronic with hepatitis C. Hepatology 2003;38:75–85. 27 Shimomura I, Matsuda M, Hammer RE, et al. hepatitis C: a mechanism for increased liver 7 Patton HM, Patel K, Behling C, et al. The impact of Decreased IRS-2 and increased SREBP-1c lead to injury. Hepatology 2004;39:1230–8. steatosis on disease progression and early and mixed insulin resistance and sensitivity in livers of 45 McCaughan GW, George J. Fibrosis progression sustained treatment response in chronic hepatitis lipodystrophic and ob/ob mice. Mol Cell in chronic hepatitis C virus infection. Gut C patients. J Hepatol 2004;40:484–90. 2000;6:77–86. 2004;53:318–21. 8 Hui JM, Kench J, Farrell GC, et al. Genotype- 28 Shimomura I, Shimano H, Korn BS, et al. Nuclear 46 Paradis V, Perlemuter G, Bonvoust F, et al. High specific mechanisms for hepatic steatosis in sterol regulatory element-binding proteins glucose and hyperinsulinemia stimulate chronic hepatitis C infection. J Gastroenterol activate genes responsible for the entire program connective tissue growth factor expression: a Hepatol 2002;17:873–81. of unsaturated fatty acid biosynthesis in potential mechanism involved in progression to 9 Moriya K, Yotsuyanagi H, Shintani Y, et al. transgenic mouse liver. J Biol Chem fibrosis in nonalcoholic steatohepatitis. Hepatitis C virus core protein induces hepatic 1998;273:35299–306. Hepatology 2001;34:738–44.

www.gutjnl.com