Naamah Zitomersky

Glypican-3 expression in Wilms tumor and hepatoblastoma.

Background Glypican-3 (GPC3) is a heparan sulfate proteoglycan. When it is disrupted, it causes the X-linked gigantism-overgrowth Simpson-Golabi-Behmel syndrome. Its involvement in growth control is consistent with recent reports that it can bind to growth factors, possibly including insulin-like growth factor 2. Further, it has been hypothesized that it may function as a tumor suppressor gene in breast and ovarian carcinomas and mesotheliomas. Patients and Methods RNA and protein were extracted from Wilms tumor and hepatoblastoma tissue samples and GPC3 levels were measured in these extracts by Northern blotting, reverse transcription polymerase chain reaction, and immunoblotting. Results In contrast to published results with carcinomas, high levels of GPC3 expression were found in Wilms tumor and hepatoblastoma. Low or undetectable expressions of this gene were found in normal tissue surrounding the tumor. Conclusions Increased expression of GPC3 in Wilms tumor and hepatoblastoma suggests a growth-promoting or neutral activity for this gene product rather than a growth-suppressive effect.

Thrombosis and inflammatory bowel disease: A call for improved awareness and prevention

&NA; 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. (Inflamm Bowel Dis 2011;)

Evidence of Extensive DNA Transfer between Bacteroidales Species within the Human Gut

ABSTRACT 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. 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.

Longitudinal Analysis of the Prevalence, Maintenance, and IgA Response to Species of the Order Bacteroidales in the Human Gut

ABSTRACT 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 × 108 to 8 × 1010 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.

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 Crohns 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.

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 Crohns 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 &mgr;g/mL, compared with 14% of patients with ATI <5 U/mL (P < 0.001). Ten (7%) patients (9 Crohns 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.

Thrombosis in Pediatric Patients With Inflammatory Bowel Disease; a Case for Risk Stratification, and Prophylactic Anticoagulation: P-161

ment for this study is ongoing. METHODS: English speaking youth ages 11-18 followed in one of two Pediatric IBD clinics in the Midwest, with a confirmed diagnosis of IBD, who had a parent/ guardian willing to participate, and who were prescribed an oral IBD maintenance medication were eligible to participate. Families completed a baseline assessment of adherence barriers (A1), and follow-up assessments at approximately 3 months (A2) and 5 months post recruitment (A3). The primary outcome was improvement in adherence to an oral maintenance medication, which was assessed via electronic monitoring. Following A1, families were randomized to either immediate treatment (i.e., 2 phone PSST sessions) or a wait list comparison group. Following A2, those in the immediate treatment group were re-randomized to either a maintenance condition or to receive two additional phone PSST intervention sessions, while those in the wait list group were automatically given treatment (i.e., 2 phone PSST intervention sessions). See Figure 1 for a diagram of the study procedure. RESULTS: To date, 76 participants (parent-child dyads or triads) have been consented and 38 have complete data available for the current analyses. Participating youth were predominately male (56%) and Caucasian (88%). Most participants had Crohn’s disease (71%). Eighteen of 38 youth (47%) had perfect adherence (i.e.,100%) during the baseline-monitoring interval and thus, had no improvement in adherence following the two intervention sessions. The remaining twenty youth had a mean pre-intervention adherence rate of 62% (SD 1⁄4 28%), which improved to 75% (SD 1⁄4 28%) following two phone intervention sessions. This finding was consistent with a medium effect size (d 1⁄4 0.59). Little additional benefit on adherence was seen among those receiving an additional 2 (4 total) PSST intervention sessions; however, this subsample was small (n 1⁄4 8). In addition, families reported high satisfaction with key intervention components. On an Intervention Satisfaction Scale ranging from 1 to 5, with higher scores reflecting higher satisfaction, mean youth satisfaction ratings ranged from 3.9 to 4.4. Mean mother satisfaction ratings ranged from 3.5 to 4.5. See Table 1 for satisfaction ratings by item. CONCLUSION(S): Results to date support phone-delivered PSST as a useful intervention for families of youth with IBD who have adherence problems. Additional analyses will explore differences in intervention efficacy as a function of youth age, SES, and disease severity. Analyses will also explore cost-savings of delivering the intervention via phone versus in person.