Anti-infliximab antibodies and low infliximab levels correlate with drug discontinuation in pediatric inflammatory bowel disease.

BACKGROUND: Infliximab (IFX) use is limited by loss of response often due to the development of anti-IFX antibodies and low drug levels. METHODS: We performed a single center prospective observational cohort study of pediatric and young adult subjects with inflammatory bowel disease (IBD) on IFX with over 3 years of follow-up. Infliximab levels (IFXL) and antibodies to infliximab (ATI) were measured throughout the study. Subjects were followed until IFX was discontinued. RESULTS: We enrolled 219 subjects with IBD (184: Crohn's disease; 33: Ulcerative colitis; and 2 Indeterminant colitis; 84 female, median age 14.4 years, 37% on concomitant immunomodulator). Nine hundred and nineteen serum samples (mean 4.2 ± 2.1 per patient) were tested for IFXL and ATI. During the study, 31 (14%) subjects discontinued IFX. Sixty patients had ATI. Twenty-two of those 60 patients with ATI discontinued IFX; 14 of 31 patients who discontinued IFX had detectable ATI at study onset. The combination of ATI and IFXL < 5 µg/mL at study entry was associated with the highest risk of drug discontinuation (hazard ratios [HR] ATI 4.27 [p < 0.001] and IFXL < 5 µg/mL [HR]: 3.2 p = 0.001). Patients with IFXL 5-10 µg/mL had the lowest rate of discontinuation (6%). IFX dose escalation eliminated ATI in 21 of 60 subjects. CONCLUSIONS: ATI is a strong predictor of needing to stop IFX use and inversely correlates with IFXL. Detection of ATI during therapeutic drug monitoring postinduction but also periodically during maintenance therapy identifies individuals who may benefit from IFX dose escalation and/or the addition of an immunomodulator, as these interventions may reduce or eliminate ATI.

The Impact of Combination Therapy on Infliximab Levels and Antibodies in Children and Young Adults With Inflammatory Bowel Disease.

Goal: The aim of this study was to evaluate the effect of combination therapy with methotrexate or 6-mercaptopurine on infliximab levels (IFXL) and antibodies to infliximab (ATI). Background: Infliximab (IFX) is a highly effective therapy for inflammatory bowel disease (IBD). Unfortunately, 25%-50% of patients will lose response to IFX. Loss of response is correlated with low IFXL and ATI formation which accelerates drug clearance. Combination therapy is thought to decrease ATI formation. Methods: We performed a cross-sectional analysis of 223 pediatric and young adult patients with IBD on IFX. IFXL and ATI were measured and compared between subjects on current combination therapy, prior combination therapy, and IFX monotherapy. Results: Eighty-four (37.7%) patients were on combination therapy and 139 (62.3%) were on IFX monotherapy. Within the current monotherapy group, 112 (80.6%) had previously been on combination therapy, while 27 (19.4%) had never been on a concomitant immunomodulator. Patients currently on combination therapy had a higher IFXL (17.00 ± 1.33 µg/mL) than those currently on IFX monotherapy (13.18 ± 1.26 µg/mL), P < 0.01. IFXL was lowest in patients who had never been on combination therapy (11.53 ± 2.05 µg/mL) and highest in patients currently on combination therapy (17.00 ± 1.33 µg/mL). Patients currently on combination therapy had a lower rate of detectable ATI (9.5%) compared with those on monotherapy (20.0%) in multivariate analysis (odds ratio [OR]: 0.3; 95% confidence interval (CI), 0.1-0.7, P < 0.01). Conclusions: Current or prior combination therapy is associated with higher IFXL and lower rates of ATI formation.

ADAMTS13 Deficiency Worsens Colitis and Exogenous ADAMTS13 Administration Decreases Colitis Severity in Mice.

BACKGROUND: Inflammatory bowel disease (IBD) affects 1.6 million people in the United States. IBD is associated with an increased risk of thrombosis, which rises with disease activity. The pathogenesis of IBD and its increased thrombotic risk is not completely understood. Ultra large von Willebrand factor (ULVWF) multimers are secreted from activated endothelium, leading to recruitment of platelets and leukocytes. A disintegrin and metalloproteinase with thrombospondin type I repeats motif 13 (ADAMTS13) cleaves highly adhesive ULVWF into smaller, less bioactive, multimers, releasing them into circulation. Mice deficient in ADAMTS13 (ADAMTS13-/-) have heightened inflammatory and thrombotic responses. OBJECTIVES: We hypothesized that upon colitis induction, ADAMTS13-/- mice would have more severe symptoms compared with wild-type (WT) mice, and rhADAMTS13 administration to mice with colitis would improve their condition. RESULTS: Dextran sodium sulfate-induced colitis was worse in ADAMTS13-/- mice than WT. ADAMTS13-/- showed increased weight loss, worse anemia, and increased clinical and histologic colitis severity, compared with WT mice. ADAMTS13-/- mice had increased VWF release, with accumulation at inflamed colonic sites. Also, the majority of mice showed one or more submucosal colonic thrombi. ADAMTS13 deficiency worsened colitis and propagated intestinal inflammation, most likely through increased platelet-leukocyte recruitment by VWF. Treatment of WT mice with rhA-DAMTS13 decreased colitis severity without worsening anemia. Additionally, several immune-mediated chronic murine colitis models, and inflamed colon tissue specimens from IBD patients, showed increased VWF release at inflamed sites, suggesting a generalizability of our findings. CONCLUSION: Measuring VWF/ADAMTS13 levels could have clinical utility. When applicable, the administration of ADAMTS13, in addition to primary treatment, may improve outcomes for IBD patients.

13-year-old girl with recurrent, episodic, persistent vomiting: out of the pot and into the fire.

Cyclic vomiting syndrome (CVS) is a well-established cause of recurrent vomiting in the pediatric population. Severe vomiting with chronic cannabis use, known as cannabinoid hyperemesis syndrome, has recently been more widely recognized as an etiology of persistent episodic vomiting. In turn, patients presenting with frequent episodes of CVS are now increasingly being screened for cannabinoid use. Because patients with persistent vomiting are also frequently prescribed a proton pump inhibitor (PPI) for their gastrointestinal symptoms, it is important to be aware of the potential for a PPI to cause an interaction that can lead to false-positive urine cannabinoid screening. We describe a case of a false-positive urine cannabinoid screen in a patient with CVS who received a dose of intravenous pantoprazole. The primary reference regarding drug screen interference from PPIs can be found in the pantoprazole package insert that refers to pre-Food and Drug Administration approval data. Although multiple sources on the Internet report the possibility of positive cannabinoid screens from pantoprazole, there are no known published reports of the phenomenon in the medical literature.

Antibodies to infliximab are associated with lower infliximab levels and increased likelihood of surgery in pediatric IBD.

BACKGROUND: Adult studies suggest antibodies to infliximab (ATI) correlate with loss of response in inflammatory bowel disease but pediatric data are limited. METHODS: We conducted a cross-sectional study of trough infliximab levels and ATI in 134 pediatric and young adult patients receiving infliximab. At the time serum was obtained demographics, disease phenotype, duration of infliximab therapy, use of combination therapy (methotrexate or 6-mercaptopurine with infliximab), and surgery were recorded. RESULTS: Assays were performed on 134 subjects currently receiving infliximab (85 male; mean age, 17.3 ± 4.3 years; 114 Crohn's disease and 20 ulcerative colitis). Infliximab use ranged from 12 days to 12 years: median 2.0 (interquartile range [1.1-4.3]) years. Twenty-seven of 134 (20%) patients had ATI ≥5 U/mL. Of patients with ATI ≥5 U/mL, 59% had infliximab levels <5 µg/mL, compared with 14% of patients with ATI <5 U/mL (P < 0.001). Ten (7%) patients (9 Crohn's disease, 1 ulcerative colitis) underwent bowel resections after beginning infliximab infusions. Sixty percent who underwent surgery had ATI ≥12 U/mL; in contrast, only 8% of patients who did not undergo surgery had ATI ≥12 U/mL (P = 0.01). At the time of serum sampling, 50 (37%) patients were receiving combination therapy, compared with 84 (63%) on infliximab alone. Combination therapy at the time of serum sampling did not correlate with either increase infliximab levels or lower ATI compared with infliximab monotherapy. However, prior immunomodulator use was associated with lower antibody levels (P = 0.007). CONCLUSIONS: ATI correlates with reduction in infliximab level and a higher risk of surgery in patients with inflammatory bowel disease.

PAD4-deficiency does not affect bacteremia in polymicrobial sepsis and ameliorates endotoxemic shock.

Neutrophil extracellular traps (NETs), consisting of nuclear DNA with histones and microbicidal proteins, are expelled from activated neutrophils during sepsis. NETs were shown to trap microbes, but they also fuel cardiovascular, thrombotic, and autoimmune disease. The role of NETs in sepsis, particularly the balance between their antimicrobial and cytotoxic actions, remains unclear. Neutrophils from peptidylarginine deiminase 4-(PAD4(-/-)) deficient mice, which lack the enzyme allowing for chromatin decondensation and NET formation, were evaluated. We found that neutrophil functions involved in bacterial killing, other than NETosis, remained intact. Therefore, we hypothesized that prevention of NET formation might not have devastating consequences in sepsis. To test this, we subjected the PAD4(-/-) mice to mild and severe polymicrobial sepsis produced by cecal ligation and puncture. Surprisingly, under septic conditions, PAD4(-/-) mice did not fare worse than wild-type mice and had comparable survival. In the presence of antibiotics, PAD4-deficiency resulted in slightly accelerated mortality but bacteremia was unaffected. PAD4(-/-) mice were partially protected from lipopolysaccharide-induced shock, suggesting that PAD4/NETs may contribute to the toxic inflammatory and procoagulant host response to endotoxin. We propose that preventing NET formation by PAD4 inhibition in inflammatory or thrombotic diseases is not likely to increase host vulnerability to bacterial infections.

Evidence of extensive DNA transfer between bacteroidales species within the human gut.

UNLABELLED: The genome sequences of intestinal Bacteroidales strains reveal evidence of extensive horizontal gene transfer. In vitro studies of Bacteroides and other bacteria have addressed mechanisms of conjugative transfer and some phenotypic outcomes of these DNA acquisitions in the recipient, such as the acquisition of antibiotic resistance. However, few studies have addressed the horizontal transfer of genetic elements between bacterial species coresident in natural microbial communities, especially microbial ecosystems of humans. Here, we examine the genomes of Bacteroidales species from two human adults to identify genetic elements that were likely transferred among these Bacteroidales while they were coresident in the intestine. Using seven coresident Bacteroidales species from one individual and eight from another, we identified five large chromosomal regions, each present in a minimum of three of the coresident strains at near 100% DNA identity. These five regions are not found in any other sequenced Bacteroidetes genome at this level of identity and are likely all integrative conjugative elements (ICEs). Such highly similar and unique regions occur in only 0.4% of phylogenetically representative mock communities, providing strong evidence that these five regions were transferred between coresident strains in these subjects. In addition to the requisite proteins necessary for transfer, these elements encode proteins predicted to increase fitness, including orphan DNA methylases that may alter gene expression, fimbriae synthesis proteins that may facilitate attachment and the utilization of new substrates, putative secreted antimicrobial molecules, and a predicted type VI secretion system (T6SS), which may confer a competitive ecological advantage to these strains in their complex microbial ecosystem. IMPORTANCE: By analyzing Bacteroidales strains coresident in the gut microbiota of two human adults, we provide strong evidence for extensive interspecies and interfamily transfer of integrative conjugative elements within the intestinal microbiota of individual humans. In the recipient strain, we show that the conjugative elements themselves can be modified by the transposition of insertion sequences and retroelements from the recipient's genome, with subsequent transfer of these modified elements to other members of the microbiota. These data suggest that the genomes of our gut bacteria are substantially modified by other, coresident members of the ecosystem, resulting in highly personalized Bacteroidales strains likely unique to that individual. The genetic content of these ICEs suggests that their transfer from successful adapted members of an ecosystem confers beneficial properties to the recipient, increasing its fitness and allowing it to better compete within its particular personalized gut microbial ecosystem.

Characterization of adherent bacteroidales from intestinal biopsies of children and young adults with inflammatory bowel disease.

There is extensive evidence implicating the intestinal microbiota in inflammatory bowel disease [IBD], but no microbial agent has been identified as a sole causative agent. Bacteroidales are numerically dominant intestinal organisms that associate with the mucosal surface and have properties that both positively and negatively affect the host. To determine precise numbers and species of Bacteroidales adherent to the mucosal surface in IBD patients, we performed a comprehensive culture based analysis of intestinal biopsies from pediatric Crohn's disease [CD], ulcerative colitis [UC], and control subjects. We obtained biopsies from 94 patients and used multiplex PCR or 16S rDNA sequencing of Bacteroidales isolates for species identification. Eighteen different Bacteroidales species were identified in the study group, with up to ten different species per biopsy, a number higher than demonstrated using 16S rRNA gene sequencing methods. Species diversity was decreased in IBD compared to controls and with increasingly inflamed tissue. There were significant differences in predominant Bacteroidales species between biopsies from the three groups and from inflamed and uninflamed sites. Parabacteroides distasonis significantly decreased in inflamed tissue. All 373 Bacteroidales isolates collected in this study grew with mucin as the only utilizable carbon source suggesting this is a non-pathogenic feature of this bacterial order. Bacteroides fragilis isolates with the enterotoxin gene [bft], previously associated with flares of colitis, were not found more often at inflamed colonic sites or within IBD subjects. B. fragilis isolates with the ability to synthesize the immunomodulatory polysaccharide A [PSA], previously shown to be protective in murine models of colitis, were not detected more often from healthy versus inflamed tissue.

Risk factors, morbidity, and treatment of thrombosis in children and young adults with active inflammatory bowel disease.

OBJECTIVES: Pediatric inpatients with inflammatory bowel disease (IBD) are rarely considered for thromboprophylaxis because of concerns about safety and underappreciation of thrombotic risk. We characterized thromboembolism (TE) in children and young adults with inflammatory bowel disease (IBD) at a single tertiary care hospital. METHODS: We performed a retrospective review of an inpatient billing database for all IBD admissions with colonic involvement and an anticoagulation database for thrombotic complications from 2006 to 2011. RESULTS: Of 532 patients admitted with IBD with colonic involvement, 10 (1.9%) had TE (9 venous, 1 arterial), 2 of whom had recurrent thrombosis. Many of the events resulted in considerable morbidity, including 4 cerebrovascular events and 2 pulmonary emboli. Established risk factors in IBD colitis inpatients with TE included: indwelling catheter (4/10), first-degree family member with TE (2/10), hereditary thrombophilia (3/10), smoking (1/10), oral contraceptive (1/5 females), and thalidomide (1/10). Additionally, most (8/10) patients had acquired thrombophilia, mostly elevation of factor VIII and anticardiolipin antibodies. Patients with IBD and TE received therapeutic anticoagulation without significantly increased bleeding. Thrombus resolution was documented in 7 cases, persistence in 2 cases and recurrence in 2 cases. CONCLUSIONS: Pediatric inpatients hospitalized with IBD with colonic involvement have increased risk of TE, including complications of pulmonary embolism, recurrence, persistence, and indefinite long-term anticoagulation. Therapeutic anticoagulation in patients with IBD with active colitis appears safe. We identified both inherited thrombophilias and acquired risk factors in patients with IBD and TE. We presently use risk stratification and recommend prophylactic anticoagulation in high-risk patients.

Insulin-like growth factor 1 receptor as a therapeutic target in ewing sarcoma: lack of consistent upregulation or recurrent mutation and a review of the clinical trial literature.

The insulin-like growth factor 1 receptor (IGF-1R) has been considered an important therapeutic target in Ewing sarcoma (ES), generating a need to identify the subset of patients most likely to respond to IGF-1R inhibitors. We assessed IGF-1R expression in ES cell lines and patient tumors to understand the variable clinical responses to anti-IGF-1R therapy. Using ligand-binding displacement, we measured between 13,000 and 40,000 receptors per cell in ES cell lines. We used ELISA to quantify IGF-1R in patient tumors, which expressed 4.8% ± 3.7 to 20.0% ± 0.2 of the levels in a positive control cell line overexpressing IGF-1R. Flow cytometry showed markedly reduced IGF-1R expression in ES cell lines compared to a standard positive control cell line. The IGF1R gene was sequenced in 47 ES tumor samples and 8 ES cell lines; only one tumor sample showed a nonsynonymous mutation, R1353H, in a region with low functional impact. Finally, we assessed IGF-1R pathway activity in the ES stem cell (ESSC) population, to characterize its potential for resistance to anti-IGF-1R therapy, using Luminex technology. We found no significant differences in IGF-1R pathway activity between ESSCs and the total cell population. Overall, our findings suggest that IGF-1R as a therapeutic target in this sarcoma may require reevaluation.

Longitudinal analysis of the prevalence, maintenance, and IgA response to species of the order Bacteroidales in the human gut.

Bacteroidales species are the most abundant Gram-negative bacteria of the human intestinal microbiota. These bacteria evolved to synthesize numerous capsular polysaccharides (PS) that are subject to phase variation. In Bacteroides fragilis, PS synthesis is regulated so that only one of the eight PS biosynthesis loci is transcribed at a time in each bacterium. To determine if the bacteria evolved this unusual property to evade a host IgA response, we directly studied the human fecal ecosystem. We performed a longitudinal analysis of the abundant Bacteroidales species from 15 healthy adults at four intervals over a year. For this study, we used bacterial culture to perform analyses not accurate with DNA-based methods, including quantification of total viable Bacteroidales bacteria, strain maintenance, and IgA responses. Abundant Bacteroidales isolates were identified to the species level using multiplex PCR and 16S rRNA gene sequencing. Arbitrarily primed PCR was used for strain typing. IgA responses to endogenous strains carried over the year were analyzed, and the orientations of the invertible PS locus promoters from the ecosystem were quantified. Subjects consistently harbored from 5 × 10(8) to 8 × 10(10) Bacteroidales bacteria/g of feces. Within the cohort, 20 different Bacteroidales species were detected at high concentrations. Bacteroides uniformis was the most prevalent; however, abundant Bacteroidales species varied between subjects. Strains could be maintained over the year within the ecosystem at high density. IgA responses were often not induced and did not correlate with the elimination of a strain or major changes in the orientations of the capsular PS locus promoters.

Thrombosis and inflammatory bowel disease: a call for improved awareness and prevention.

Thrombotic complications in patients with inflammatory bowel disease (IBD) are common and require improved awareness and prevention. In this review the interface between IBD and thrombosis is discussed, with emphasis on risk assessment and data to aid clinical decision making. Thromboembolic complications are 3-fold more likely in IBD patients than controls and the relative risk exceeds 15 during disease flares. Improved assessment of thrombosis risk for an individual patient includes thorough personal and family history and awareness of prothrombotic medications and lifestyle choices. Patients with the highest risk of thrombosis are those with active colonic disease, personal or strong family history of thrombosis, and those with significant acquired risk factors. Combined risk factors or hospitalization should prompt mechanical thromboprophylaxis. Indications for prophylactic anticoagulation are not defined currently by clinical studies, especially in pediatric patients, although some groups now advocate prophylactic anticoagulation for all hospitalized IBD patients and even some outpatients with disease flares. Thrombosis management requires a multidisciplinary therapeutic approach to balance anticoagulation and bleeding risk. While bleeding may occur with anticoagulation in IBD, data and experience indicate that therapeutic heparin is safe and bleeding manifestations can be managed supportively in most patients. Until prospective trials of prophylactic anticoagulation are published, management of thrombotic risk and prophylaxis in IBD will remain a clinical challenge.