Paul A. Rufo

Fecal Lactoferrin is a sensitive and specific marker in identifying intestinal inflammation

OBJECTIVE:Lactoferrin is a glycoprotein expressed by activated neutrophils. The aim of this study was to determine the sensitivity and specificity of fecal lactoferrin concentrations for inflammatory bowel disease (IBD) or irritable bowel syndrome (IBS) versus healthy controls.METHODS:Fresh stool samples were collected from outpatients with ulcerative colitis (UC), Crohns disease (CD), or IBS. Clinical disease activity for IBD was assessed using a modified Harvey–Bradshaw Activity Index. Fecal lactoferrin concentrations were determined using a polyclonal antibody-based enzyme linked immunoassay. Mean fecal lactoferrin concentrations for each group and sensitivity and specificity of the assay were determined.RESULTS:One hundred-four CD patients, 80 UC patients, 31 IBS patients, and 56 healthy controls were recruited. The mean ± SE fecal lactoferrin concentration (μg/g fecal weight) was 440 ± 128 for CD patients, 1125 ± 498 for UC patients, 1.27 ± 0.29 for IBS patients, and 1.45 ± 0.4 for healthy controls. Fecal lactoferrin was 90% specific for identifying inflammation in patients with active IBD. Elevated fecal lactoferrin was 100% specific in ruling out IBS.CONCLUSIONS:Fecal lactoferrin is sensitive and specific for detecting inflammation in chronic IBD. This noninvasive test may prove useful in screening for inflammation in patients presenting with abdominal pain and diarrhea.

Fecal Lactoferrin Is a Sensitive and Specific Marker of Disease Activity in Children and Young Adults With Inflammatory Bowel Disease

Background and Aims: Fecal lactoferrin (FLA) is a neutrophil-derived surrogate marker of intestinal inflammation that is elevated in patients with inflammatory bowel disease. However, the correlation between FLA levels and serological markers of disease activity has not been previously reported, to our knowledge. In the present study we evaluated the ability of FLA levels to reflect disease activity in pediatric patients with inflammatory bowel disease. We further assessed the relationship between FLA levels and customary laboratory and clinical measures of inflammation. Patients and Methods: Fecal specimens were collected from 148 consecutive pediatric patients (79 with Crohn disease, 62 with ulcerative colitis, and 7 with irritable bowel syndrome) and 22 healthy control individuals. Lactoferrin was measured by enzyme-linked immunosorbent assay (IBD-SCAN, TECHLAB, Inc). Disease activity was assessed at the time of sample provision by laboratory measures (including erythrocyte sedimentation rate [ESR] and albumin) and previously validated disease activity indices (Pediatric Crohn Disease Activity Index, Kozarek, Harvey Bradshaw Activity Index). Results: Lactoferrin levels were significantly higher in patients with ulcerative colitis (1880 ± 565 μg/mL) (mean ± SE) or Crohn disease (1701 ± 382 μg/mL) than in healthy control individuals under 21 years of age (1.17 ± 0.47 μg/mL, P < 0.001). Lactoferrin levels correlated significantly with ESR, hematocrit, albumin, and platelet count (P < 0.001). Receiver operating characteristic curve analysis revealed that FLA levels were comparable to ESR in detecting patients with clinically active disease (P < 0.001). Patients who experienced a clinical flare within 2 months of specimen collection displayed higher lactoferrin levels (845 ± 452 μg/mL) than did those who remained in clinical remission (190 ± 90 μg/mL, P = 0.003). Conclusions: Data presented here demonstrate that FLA is a sensitive and specific biochemical marker of inflammation for use in the diagnosis and interval assessment of pediatric patients with IBD, and its level correlates well with both clinical disease activity indices and ESR. Elevated levels of FLA may also identify patients at greater risk for the development of subsequent clinical flares.

The antifungal antibiotic, clotrimazole, inhibits chloride secretion by human intestinal T84 cells via blockade of distinct basolateral K+ conductances. Demonstration of efficacy in intact rabbit colon and in an in vivo mouse model of cholera.

The antifungal antibiotic clotrimazole (CLT) blocks directly and with high potency the Ca2+-activated K+ channels of human erythrocytes, erythroleukemia cells, and ferret vascular smooth muscle cells. We recently reported that CLT inhibits Cl- secretion in human intestinal T84 cells, likely by affecting K+ transport (Rufo, P.A., L. Jiang, S.J. Moe, C. Brugnara, S.L. Alper, and W.I. Lencer. 1996. J. Clin. Invest. 98:2066-2075). To determine if CLT had direct effects on K+ conductances in T84 cells, we selectively permeabilized apical membranes of confluent T84 cell monolayers using the ionophore amphotericin B. This technique permits direct measurement of basolateral K+ transport. We found that CLT and a stable des-imidazolyl derivative inhibited directly two pharmacologically distinct basolateral membrane K+conductances, but had no effect on apical membrane Cl- conductances. The effects of CLT on Cl- secretion were also examined in intact tissue. CLT inhibited forskolin-induced Cl- secretion in rabbit colonic mucosal sheets mounted in Ussing chambers by 91%. CLT also inhibited cholera toxin-induced intestinal Cl- secretion in intact mice by 94%. These data provide direct evidence that CLT blocks Cl- secretion in intestinal T84 cells by inhibition of basolateral K+ conductances, and show that CLT inhibits salt and water secretion from intact tissue in vitro and in vivo. The results further support the suggestion that CLT and its metabolites may show clinical efficacy in the treatment of secretory diarrheas of diverse etiologies.

Current therapy of inflammatory bowel disease in children.

Ulcerative colitis (UC) and Crohn disease (CD) are chronic intestinal inflammatory diseases that can present as bloody diarrhea, abdominal pain, and malnutrition. Collectively, these disorders are referred to as inflammatory bowel disease (IBD). All patients with IBD share a common pathophysiology. However, there are a number of developmental, psychosocial, and physiologic issues that are unique to the ≈20% of patients that present during childhood or adolescence. These include the possibility of disease-induced delays in linear growth or physical development, differences in drug dosing, and the changes in social and cognitive development that occur as children move from school-age years into adolescence and early adulthood. Gastroenterologists caring for these children must therefore develop an optimal regimen of pharmacologic therapies, nutritional management, psychologic support, and properly timed surgery (when necessary) that will maintain disease remission, minimize disease and drug-induced adverse effects, and optimize growth and development. This article reviews current approaches to the management of patients with UC and CD and highlights issues specific to the treatment of children with IBD.The principal medical therapies used to induce disease remission in patients with UC are aminosalicylates (for mild disease), corticosteroids (for moderate disease), and cyclosporine (ciclosporin) [for severe disease]. If a patient responds to the induction regimen, maintenance therapies that are used to prevent disease relapse include aminosalicylates, mercaptopurine, and azathioprine. Colectomy with creation of an ileal pouch anal anastomosis (J pouch) has become the standard of care for patients with severe or refractory colitis and results in an improved quality of life in most patients. Therefore, the risks associated with using increasingly potent immunosuppressant agents must be balanced in each case against a patient’s desire to retain their colon and avoid a temporary or potentially permanent ileostomy.Decisions about drug therapy in the management of patients with CD are more complex and depend on both the location (e.g. gastroduodenal vs small intestinal vs colonic), as well as the behavior of the disease (inflammatory/mucosal vs stricturing vs perforating) in a given patient. Induction therapies for CD typically include aminosalicylates and antibiotics (for mild mucosal disease), nutritional therapy (including elemental or polymeric formulas), corticosteroids (for moderate disease), and infliximab (for corticosteroid-resistant or fistulizing disease). Aminosalicylates, mercaptopurine, azathioprine, methotrexate, and infliximab can be used as maintenance therapies. Because surgical treatment of CD is not curative, it is typically reserved for those patients either with persistent symptoms and disease limited to a small section of the intestine (e.g. the terminal ileum and cecum) or for the management of complications of the disease including stricture or abdominal abscess. When surgery is necessary, maintenance medications administered postoperatively will postpone recurrence.Patients with UC and CD are at risk for the development of micronutrient deficiencies (including folate, iron, and vitamin D deficiencies) and require close nutritional monitoring. In addition, patients with UC and CD involving the colon are at increased risk of developing colon cancer, and should be enrolled into a colonoscopy surveillance program after 8–10 years of disease duration.

The antifungal antibiotic, clotrimazole, inhibits Cl- secretion by polarized monolayers of human colonic epithelial cells.

Clotrimazole (CLT) prevents dehydration of the human HbSS red cell through inhibition of Ca++-dependent (Gardos) K+ channels in vitro (1993. J. Clin Invest. 92:520-526.) and in patients (1996. J. Clin Invest. 97:1227-1234.). Basolateral membrane K+ channels of intestinal crypt epithelial cells also participate in secretagogue-stimulated Cl- secretion. We examined the ability of CLT to block intestinal Cl- secretion by inhibition of K+ transport. Cl- secretion was measured as short-circuit current (Isc) across monolayers of T84 cells. CLT reversibly inhibited Cl- secretory responses to both cAMP- and Ca2+-dependent agonists with IC50 values of approximately 5 microM. Onset of inhibition was more rapid when CLT was applied to the basolateral cell surface. Apical Cl- channel and basolateral NaK2Cl cotransporter activities were unaffected by CLT treatment as assessed by isotopic flux measurement. In contrast, CLT strongly inhibited basolateral 86Rb efflux. These data provide evidence that CLT reversibly inhibits Cl- secretion elicited by cAMP-, cGMP-, or Ca2+-dependent agonists in T84 cells. CLT acts distal to the generation of cAMP and Ca2+ signals, and appears to inhibit basolateral K+ channels directly. CLT and related drugs may serve as novel antidiarrheal agents in humans and animals.

Proteolytic Activation of Cholera Toxin and Escherichia coli Labile Toxin by Entry into Host Epithelial Cells SIGNAL TRANSDUCTION BY A PROTEASE-RESISTANT TOXIN VARIANT

Cholera and Escherichia coliheat-labile toxins (CT and LT) require proteolysis of a peptide loop connecting two major domains of their enzymatic A subunits for maximal activity (termed “nicking”). To test whether host intestinal epithelial cells may supply the necessary protease, recombinant rCT and rLT and a protease-resistant mutant CTR192H were prepared. Toxin action was assessed as a Cl− secretory response (Isc) elicited from monolayers of polarized human epithelial T84 cells. When applied to apical cell surfaces, wild type toxins elicited a brisk increase in Isc (80 μA/cm2). Isc was reduced 2-fold, however, when toxins were applied to basolateral membranes. Pretreatment of wild type toxins with trypsin in vitro restored the “basolateral” secretory responses to “apical” levels. Toxin entry into T84 cells via apical but not basolateral membranes led to nicking of the A subunit by a serine-type protease. T84 cells, however, did not nick CTR192H, and the secretory response elicited by CTR192H remained attenuated even when applied to apical membranes. Thus, T84 cells express a serine-type protease(s) fully sufficient for activating the A subunits of CT and LT. The protease, however, is only accessible for activation when the toxin enters the cell via the apical membrane.

Increased incidence of urinary matrix metalloproteinases as predictors of disease in pediatric patients with inflammatory bowel disease

Background: Matrix metalloproteinases (MMPs) are a family of metal‐dependent enzymes responsible for the degradation and remodeling of extracellular matrix and basement membrane proteins that occurs during both normal physiologic activity and disease. It has been suggested that MMPs may also play a role in the pathogenesis of inflammatory bowel disease (IBD) by mediating mucosal breakdown in response to an enhanced inflammatory cascade. We previously demonstrated that elevated urinary MMP levels are independent predictors of disease status in cancer patients. Here we demonstrate that elevated urinary MMP levels may be biomarkers of disease activity in patients with IBD. Methods: We analyzed 95 urine samples prospectively collected from 55 children and young adults with known or suspected IBD who presented for evaluation to the Gastrointestinal Procedure Unit at Childrens Hospital Boston. Urinary MMPs were analyzed in patients by zymography and compared to 40 age‐ and sex‐matched controls. Results: Urinary MMP levels were significantly elevated (P < 0.0001) in patients with IBD, as well as in each subgroup (Crohns disease or ulcerative colitis), relative to controls. Multiple logistic regression revealed that urinary MMP‐2 and MMP‐9 NGAL levels were independent predictors of Crohns disease and ulcerative colitis (P < 0.0001). Conclusions: These data are the first to demonstrate that urinary MMPs may represent novel noninvasive biomarkers for use in the evaluation of patients with IBD.

Health supervision in the management of children and adolescents with IBD: NASPGHAN recommendations

Ulcerative colitis (UC) and Crohn disease (CD), collectively referred to as inflammatory bowel disease (IBD), are chronic inflammatory disorders that can affect the gastrointestinal tract of children and adults. Like other autoimmune processes, the cause(s) of these disorders remain unknown but likely involves some interplay between genetic vulnerability and environmental factors. Children, in particular with UC or CD, can present to their primary care providers with similar symptoms, including abdominal pain, diarrhea, weight loss, and bloody stool. Although UC and CD are more predominant in adults, epidemiologic studies have demonstrated that a significant percentage of these patients were diagnosed during childhood. The chronic nature of the inflammatory process observed in these children and the waxing and waning nature of their clinical symptoms can be especially disruptive to their physical, social, and academic development. As such, physicians caring for children must consider these diseases when evaluating patients with compatible symptoms. Recent research efforts have made available a variety of more specific and effective pharmacologic agents and improved endoscopic and radiologic assessment tools to assist clinicians in the diagnosis and interval assessment of their patients with IBD; however, as the level of complexity of these interventions has increased, so too has the need for practitioners to become familiar with a wider array of treatments and the risks and benefits of particular diagnostic testing. Nonetheless, in most cases, and especially when frequent visits to subspecialty referral centers are not geographically feasible, primary care providers can be active participants in the management of their pediatric patients with IBD. The goal of this article is to educate and assist pediatricians and adult gastroenterology physicians caring for children with IBD, and in doing so, help to develop more collaborative care plans between primary care and subspecialty providers.

Severe colitis in children.

*Department of Pediatrics, Medical College of Wisconsin, Milwaukee, Wisconsin; †Department of Pediatrics, Cedars-Sinai Medical center, Los Angeles, California; ‡Department of Pediatrics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania; §Department of Pediatrics, University of Cincinnati, Cincinnati Children’s Hospital Medical center, Cincinnati, Ohio; ¶Combined Program in Gastroenterology and Nutrition, Children’s Hospital, Boston, Massachusetts; #Department of Pediatrics, Cleveland Clinic Foundation, Cleveland, Ohio; kDivision of Gastoenterology, Hepatology and Nutrition, Hospital for Sick Children, Toronto, Canada; and **Department of Pediatrics, Connecticut Children’s Medical Center, Hartford, CT University of Connecticut School of Medicine, Farmington, Connecticut

Adalimumab for the treatment of Crohn-like colitis and enteritis in glycogen storage disease type Ib

SummaryGlycogen storage disease (GSD) type Ib is a congenital disorder of glycogen metabolism that is associated with neutropenia, neutrophil dysfunction, and an inflammatory bowel disease that mimics a Crohn phenotype. Gastrointestinal inflammation in GSD Ib has been successfully treated with 5-aminosalicylic acid and granulocyte colony-stimulating factor (G-CSF). However, therapeutic options for patients not responding to traditional therapies have been limited owing to untoward effects of glucocorticoids and immunomodulators in this metabolic disorder. Adalimumab is a monoclonal antibody targeting tumour necrosis factor-α that has shown promise for the treatment of patients with Crohn disease. Due to the limited options for treating GSD-associated inflammatory bowel disease, use of adalimumab was attempted in a case unresponsive to aminosalicylate, G-CSF, and antibiotic therapy. Significant clinical and histological improvement was observed in our patient, and the medication was well tolerated.