Recognize the importance of determining the phenotypical characteristics of IBD and individualizing treatment accordingly.

Utilize serologic markers to identify patients at risk for aggressive disease.

Describe the normal gastrointestinal mucosa and gut microflora.

Recommend probiotics in conditions in which they have been shown to be effective.

Discuss the circumstances in which prebiotics and biotics appear to be beneficial.

Genetics, Serologic Markers, and 6-MP Metabolites
Maria C. Dubinksy, MD, Director, Pediatric Inflammatory Bowel Disease Program, Cedars-Sinai Medical Center, Los Angeles, CA

Introduction: terms “Crohn’s disease (CD)” and “ulcerative colitis (UC)” immaterial; immunology what matters; prognosis and treatment based on genetics and immunology

Pathophysiology of CD: defect in innate and adaptive immunity; innate immunity—first line of defense; guards against bacteria that leak across gut mucosa and trigger innate immune response (IR), ie, inability to tolerate gut flora, producing abnormal adaptive IR; adaptive IR—2 pathways include T-cell immunity and B-cell immunity; T-cell immunity targets tumor necrosis factor (TNF; cytokine released from lymphocytes); B-cell response—every time T-cell response occurs, B cells also produce antibodies against bacteria; serologic IR; clinical phenotype—necessary to determine; includes perianal CD, stricturing small bowel disease, pancolonic disease, upper tract disease, and internal fistulizing disease; each phenotype driven by different immune pathways; inappropriate to treat each patient in same manner; individualize therapy; must find markers (bacterial, genetic, or immune); IRs looked at thus far are microbial-driven antigens; serologic immune markers have significance beyond their presence or absence in patient with inflammatory bowel disease (IBD); presence means something scientifically and helps guide physician to immune pathway being experienced by patient; ≥32 immune pathways with markers

Serologic markers: in CD, anti-Saccharomyces cerevisiae antibody (ASCA) most commonly seen; outer membrane porin C (OmpC) found typically on wall of Escherichia coli; I2 marker for Pseudomonas fluorescence molecule (not commercially available; associated with interleukin [IL]-17 pathway)

New marker (CBir): found in CBir mouse; antigen in Clostridia family that causes colitis in mouse; in 50% of patients with CD, serum found positive for antibodies against same bacteria that caused colitis in mouse; differences present, based on age, as to which IR patients mount; correlation with age—children <8 yr of age, almost 95% of time, have colitis-like presentation; progresses to more small bowel phenotype as child gets older, probably because immunity changing as immune system matures; in pediatric age group, CBir most common antibody marker found (marker for colitis in children and small bowel disease in adults); ASCA more prominent and CBir less prominent as children become older; children who present older and ASCA-positive have more small bowel disease, while children <6 yr of age with CBir have more predominant colonic disease; IRs change over time, as genes change over time and with interaction with gut flora

ASCA in adults: association of markers with disease location reinforces importance of ASCA in adult cohort; ASCA factor in small bowel aggressive, fibrostenosing, internal penetrating disease and predictor of small bowel surgery; helps determine prognosis; double ASCA-positive (IgA and IgG) patient will have progressive disease and worst prognosis; in older studies, Nod2 associated with small bowel fibrostenosing disease; classic innate immune genes (eg, Nod2) associated with location of disease; location of gene defect helps determine site of disease

Perinuclear antineutrophil cytoplasmic antibodies (pANCA): definite colonic process; ≈25% of patients with CD pANCA-positive; more IRs patient has, means innate immunity ineffective and bacteria leaking across gut; presence of more antibodies means patient has higher risk for stricturing disease, internal penetrating disease, and nearly 10-fold increased risk of developing fistulizing CD, compared to patient with 0 or 1 antibody (2-fold increased risk); higher the number of markers above cutoff point, the worse the prognosis; not appropriate to place patient double ASCA-positive and CBir-positive on mesalamine (eg, Pentasa)

Other anticarbohydrate antibodies: similar to ASCA; include anti-laminaribioside antibodies (ALCA), anti-chitobioside antibodies (ACCA), and anti-mannobioside antibodies (AMCA)

Concept: more antibodies individual has, lower the level of innate immunity and higher the risk for progression of CD; study showed that ASCA alone predictive of earlier surgery in pediatric cohort; also, higher risk for second surgery and recurrence of complication in ASCA-positive patient who had surgery; IR marker of underlying pathogenesis of disease

Impact of therapy: use of biologic agent within 30 days highest predictor of surgery (sickest patients); study found that Nod2 not particularly associated; should look at pathway (not gene in isolation); concluded that Nod2 innate immune defect; in patients homozygous to Nod2 gene, more serologic IRs (poor innate immunity)

pANCA-positive patient: colitis-like phenotype; in pANCA-positive patients with CD, CBir present in 44%; in pANCA-positive patients with UC, CBir almost negligible; CBir differentiates between pANCA-positive patient with CD vs pure UC patient; patient with colitis must be seen by colorectal surgeon to have colon removed; high preoperative levels of pANCA predict development of chronic pouchitis; CBir positivity not huge factor in development of pouchitis; if pANCA-positive and CBir-positive, risk for pouchitis nearly doubles; of patients with high pANCA levels (>100 EU/mL) and who are CBir-positive, 50% develop chronic pouchitis; other factors—family history of CD number one predictor of developing CD after colectomy; other factor ASCA IgA positivity; if both ASCA and family history positive, >50% probability of developing CD after colectomy

Management: individualize drug therapy (patients immunologically heterogeneous); immunologically, some predictors of nonresponse present; ASCA good predictor of response to infliximab or any biologic therapy; infliximab—shown to have negative influence on outcome; poor outcome for patients who have undergone 2-stage procedure; also infectious complications; pANCA had no greater rate of response, compared to placebo, in patients with UC-like CD; pANCA positivity and ASCA negativity negative predictor of early response to biologics; pANCA probably not TNF-driven response; thiopurine methyltransferase (TPMT) enzyme—determines whether patient metabolizer of thiopurine (eg, 6-mercaptopurine [MP], azathioprine); azathioprine becomes 6-MP in body, which breaks down to 6-MMP and 6-thioguanine nucleotide (TGN) to determine whether patient ideal responder; data based on using 6-TGN as predictor of response confirmed by meta-analysis; overall, close to 3-fold odds ratio of being responder if able to achieve TGN level >235 pmol/8 x 108 red blood cells (RBCs); those patients who obtain therapeutic levels of 6-TGN have better ability to respond; clinically, patient with low 6-MMP and low 6-TG underdosed; ideally, 6-TG 235 to 450 pmol/8 x 108 RBCs and 6-MMP <5700 pmol/8 x 108 RBCs; if patient has high levels of both, decrease dose (risk for liver toxicity and leukopenia); problem patient is preferential MMP metabolizer with low 6-TG (unable to achieve therapeutic level)

Prebiotics, Probiotics, and Biotics in IBD
David G. Binion, MD, Visiting Professor of Medicine, University of Pittsburgh School of Medicine, Co-Director, Inflammatory Bowel Disease Center, and Director, Translational Inflammatory Bowel Disease Research, Division of Gastroenterology, Hepatology, and Nutrition, UPMC Presbyterian Hospital, Pittsburgh, PA

Gut microflora, immune homeostasis, and chronic intestinal inflammation: only 10% of cells in body human; remaining 90% bacteria in gastrointestinal (GI) tract (trillions); 10-fold higher number of organisms in GI tract, compared to human cells (weight ≈2 kg); ≈500 species; 2 to 4 million bacterial genes, but only ≈20% culturable and identified; normal enteric flora necessary for health; each person has own microflora

Normal GI mucosa: crypt architecture comprised of single layer of epithelial cells separating gut lumen from inside of body; massive surface area; intestines largest immune organ in body (existing below epithelium); immune cells in gut protect and allow for tolerance to food antigens; physiologic level of inflammation exists in GI tract; in CD, inflammation chronic and destructive, with massive influx of immune cells and breakdown of blood vessels and hemorrhage

Gut microflora: concerted effort in past to locate autoimmune target during inflammation; probable immune target in IBD; concept of impaired tolerance to own enteric flora focus of current thinking on IBD; stomach acid kills off most bacteria; at upper portion of GI tract, low levels of bacteria present; trillions of bacteria in distal ileum; right lower quadrant of abdomen has most bacteria; study found that highest populations of bacteria in GI tract found in ileoanal pouch and terminal ileum in patient who has undergone terminal ileal resection; normal bacteria more than capable of inducing inflammatory response in GI tract; study—looked at mouse pups delivered by cesarean section and placed in germ-free environment; without bacteria, animals unable to fully develop architecture of bowel; putting back regular bacteria after being germ-free for few days caused “explosion” of inflammation throughout GI tract; immune system controlled over next 2 wk; gut microflora in humans has changed through time; in primitive man, more Bifidobacterium and fewer anaerobes; way of living in Westernized developed society probably changed flora and has impact on various disease processes; study—22 control participants underwent immune assay to look at immune reactivity; found random scattering of immune reactivity; in 31 patients with CD, immune reactivity found against antigens (eg, E coli, Bacteroides, Klebsiella, yeast); different for each patient, so CD not one entity immunologically and genetically; in celiac disease, antigen is gluten

Probiotics: definition—microbial preparations which contain live and/or dead bacteria, including their components and products, which produce therapeutic benefit beyond their nutritional value; 119 active trials, mostly in Canada and Western Europe; long history in veterinary medicine (probiotics in animal feed prevent disease); human studies still in infancy; available brands include, eg, Florajen, VSL#3 (best data; more expensive; comprised of 8 strains of lyophilized bacteria); Lactobacillus GG—patented in 1987; in Finland, found no increase in Lactobacillus bacteremia in treated people; should not be given to patients in intensive care unit with damaged gut; study in Finland showed benefit in atopy and hay fever; another study looked at pregnant mothers with atopy and hay fever given Lactobacillus GG; found that their children had significantly lower rates of atopy and hay fever, extending out 4 yr; also beneficial in antibiotic-associated diarrhea and vaginal infections; minimal hepatic encephalopathy (MHE)—seen in 60% of patients with cirrhosis; difficult to diagnose; causes severe problems in processing information (eg, driving car); only therapy lactulose; speaker attempted trial with probiotics, but shut down by Food and Drug Administration (FDA); eating 2 yogurts daily proven beneficial (MHE resolved)

Probiotics in IBD: not enough data; probably multiple mechanisms, including antimicrobial activity, better barrier function (improving health of epithelium), and immunomodulatory effect; no doubt that probiotics effective in animal models of IBD; Cochrane analysis—showed no evidence to suggest benefit in CD (probably because studies small and limited, with insufficient data to make recommendations); studies on UC—utilized E coli Nissle strain; randomized controlled trials show clear benefit in UC patients, compared to 5-aminosalicylic acid (ASA) compounds; also benefit in UC patients intolerant of 5-ASA compounds; failed trial of VSL#3 with rifaximin (50% effective) compared to mesalamine; real success seen in patients with surgically created ileoanal pouch reconstructions; in patients antibiotic-dependent with chronic refractory pouchitis, 85% of those who received VSL#3 remitted; VSL#3 effective in preventing first bout of pouchitis

Prebiotics: definition—substrates for support of bacteria; nondigestible food ingredients that beneficially affect host by selectively stimulating growth and/or activity of one of limited number of bacteria in colon, improving host health; includes lactulose, fructo-oligosaccharides, inulin, and fiber; nutritional substrates important for support of healthy enteric flora; in diversion colitis—necessary to ferment fiber from diet to create short-chain fatty acids utilized for energy in left colon; lack of short-chain fatty acids causes problems in colon; in CD—before biologic agents available, very ill patient advised to stop eating and provided total parenteral nutrition (TPN); hypothesized that “resting” gut to heal; actually reducing bacterial flora; Clostridium difficile infection—when patient not eating, C difficile has growth advantage; healthy bacteria killed, further disturbing flora and increasing growth of anaerobes (worsening patient’s condition); food prebiotic for normal enteric flora; in presence of diverted segments of bowel, pouch itself affected by C difficile; found that inserting fiber into pouch acts as prebiotic (in patient with C difficile enteritis); C difficile in small bowel extremely dangerous; in study utilizing fructo-oligosaccharide (Prebio 1), responders had increased bifidobacteria while nonresponders did not

Biotics: definition—live organisms ingested and maintained in GI tract which produce local or systemic therapeutic benefit distinct from effect of vaccination; best example helminths; IBD most common in North America, Western Europe, and Australia; clear that wealthier areas of world affected by IBD; hygiene hypothesis postulates that effective public health strategies and vaccination eradicated infections, leading to increase of inflammatory diseases; found that where helminth infections occur, IBD absent; helminths—powerful stimulators of cytokine response (IL-10 and IL-4) that suppress cytokines implicated in IBD; Trichuris suis utilized due to self-limited colonization, inability to multiply in host, absence of direct transmission, and eggs stable and easy to produce from pigs; study—≈90 participants with CD and UC treated; found high rates of remission and response, with no side effects; appears safe and effective; shut down by FDA

Summary: improved understanding of molecular and cellular mechanisms underlying IBD will lead to improved therapy; relationship between enteric flora and mucosal immune system will become focus of mechanism-based therapy; solid evidence for probiotic therapy exists in treatment of pouchitis, antibiotic-associated diarrhea, and in C difficile prophylaxis; further studies required before solid recommendations possible on use of prebiotic, probiotic, and biotic therapy for treatment of IBD