Structural insights into humanization of anti-tissue factor antibody 10H10.

Murine antibody 10H10 raised against human tissue factor is unique in that it blocks the signaling pathway, and thus inhibits angiogenesis and tumor growth without interfering with coagulation. As a potential therapeutic, the antibody was humanized in a two-step procedure. Antigen-binding loops were grafted onto selected human frameworks and the resulting chimeric antibody was subjected to affinity maturation by using phage display libraries. The results of humanization were analyzed from the structural perspective through comparison of the structure of a humanized variant with the parental mouse antibody. This analysis revealed several hot spots in the framework region that appear to affect antigen binding, and therefore should be considered in human germline selection. In addition, some positions in the Vernier zone, e.g., residue 71 in the heavy chain, that are traditionally thought to be crucial appear to tolerate amino acid substitutions without any effect on binding. Several humanized variants were produced using both short and long forms of complementarity-determining region (CDR) H2 following the difference in the Kabat and Martin definitions. Comparison of such pairs indicated consistently higher thermostability of the variants with short CDR H2. Analysis of the binding data in relation to the structures singled out the ImMunoGeneTics information system® germline IGHV1-2*01 as dubious owing to two potentially destabilizing mutations as compared to the other alleles of the same germline and to other human germlines.

Emergency Department Visits Related to Inflammatory Bowel Disease: Results From Nationwide Emergency Department Sample.

We analyzed a national US database to study the presentation of children with inflammatory bowel disease (IBD) to the emergency department (ED). Our results indicate that from 2006 to 2010, there was a significant increase in the number of ED visits related to children with IBD accompanied by a contemporaneous decline in the rate of hospitalization that followed these ED visits. Earlier published results have highlighted an increased overall rate of hospitalizations in the United States related to children with IBD. In this context, our results support the evidence for an increased prevalence of pediatric IBD in the United States in recent years.

Targeting the ion channel Kv1.3 with scorpion venom peptides engineered for potency, selectivity, and half-life.

Ion channels are an attractive class of drug targets, but progress in developing inhibitors for therapeutic use has been limited largely due to challenges in identifying subtype selective small molecules. Animal venoms provide an alternative source of ion channel modulators, and the venoms of several species, such as scorpions, spiders and snails, are known to be rich sources of ion channel modulating peptides. Importantly, these peptides often bind to hyper-variable extracellular loops, creating the potential for subtype selectivity rarely achieved with small molecules. We have engineered scorpion venom peptides and incorporated them in fusion proteins to generate highly potent and selective Kv1.3 inhibitors with long in vivo half-lives. Kv1.3 has been reported to play a role in human T cell activation, and therefore, these Kv1.3 inhibitor fusion proteins may have potential for the treatment of autoimmune diseases. Our results support an emerging approach to generating subtype selective therapeutic ion channel inhibitors.

Trends in hospitalizations of children with inflammatory bowel disease within the United States from 2000 to 2009.

BACKGROUND: The incidence and prevalence of pediatric inflammatory bowel disease (IBD) seems to be increasing in North America and Europe. Our objective was to evaluate hospitalization rates in children with IBD in the United States during the decade 2000 to 2009. METHODS: We analyzed cases with a discharge diagnosis of Crohn disease (CD) and ulcerative colitis (UC) within the Healthcare Cost and Utilization Project Kids' Inpatient Database, Agency for Healthcare Research and Quality. RESULTS: We identified 61,779 pediatric discharges with a diagnosis of IBD (CD, 39,451 cases; UC, 22,328 cases). The number of hospitalized children with IBD increased from 11,928 to 19,568 (incidence, 43.5-71.5 cases per 10,000 discharges per year; P < 0.001). For CD, the number increased from 7757 to 12,441 (incidence, 28.3-45.0; P < 0.001) and for UC, 4171 to 7127 (15.2-26.0; P < 0.001). Overall, there was a significant increasing trend for pediatric hospitalizations with IBD, CD, and UC (P < 0.001). In addition, there was an increase in IBD-related complications and comorbid disease burden (P < 0.01). CONCLUSION: There was a significant increase in the number and incidence of hospitalized children with IBD in the United States from 2000 to 2009.

Analysis of the structural and molecular basis of voltage-sensitive sodium channel inhibition by the spider toxin huwentoxin-IV (µ-TRTX-Hh2a).

Voltage-gated sodium channels (VGSCs) are essential to the normal function of the vertebrate nervous system. Aberrant function of VGSCs underlies a variety of disorders, including epilepsy, arrhythmia, and pain. A large number of animal toxins target these ion channels and may have significant therapeutic potential. Most of these toxins, however, have not been characterized in detail. Here, by combining patch clamp electrophysiology and radioligand binding studies with peptide mutagenesis, NMR structure determination, and molecular modeling, we have revealed key molecular determinants of the interaction between the tarantula toxin huwentoxin-IV and two VGSC isoforms, Nav1.7 and Nav1.2. Nine huwentoxin-IV residues (F6A, P11A, D14A, L22A, S25A, W30A, K32A, Y33A, and I35A) were important for block of Nav1.7 and Nav1.2. Importantly, molecular dynamics simulations and NMR studies indicated that folding was normal for several key mutants, suggesting that these amino acids probably make specific interactions with sodium channel residues. Additionally, we identified several amino acids (F6A, K18A, R26A, and K27A) that are involved in isoform-specific VGSC interactions. Our structural and functional data were used to model the docking of huwentoxin-IV into the domain II voltage sensor of Nav1.7. The model predicts that a hydrophobic patch composed of Trp-30 and Phe-6, along with the basic Lys-32 residue, docks into a groove formed by the Nav1.7 S1-S2 and S3-S4 loops. These results provide new insight into the structural and molecular basis of sodium channel block by huwentoxin-IV and may provide a basis for the rational design of toxin-based peptides with improved VGSC potency and/or selectivity.

Diarrhea in solid-organ transplant recipients: a review of the evidence.

OBJECTIVE: To provide a comprehensive review of the literature as it relates to diarrhea in solid organ transplant (SOT) recipients. In this article, we review the epidemiology, pathogenesis, clinical manifestations, diagnosis and management of diarrhea in SOT recipients and discuss recent advances and challenges. METHODS: Two investigators conducted independent literature searches using PubMed, Web of Science, and Scopus until January 1st, 2013. All databases were searched using a combination of the terms diarrhea, solid organ transplant, SOT, transplant associated diarrhea, and transplant recipients. Articles that discussed diarrhea in SOT recipients were reviewed and relevant cross-references also read and evaluated for inclusion. Selection bias could be a possible limitation of the approach used in selecting or finding articles for this article. FINDINGS: Post-transplant diarrhea is a common and distressing occurrence in patients, which can have significant deleterious effects on the clinical course and well-being of the organ recipient. A majority of cases are due to infectious and drug-related etiologies. However, various other etiologies including inflammatory bowel disease must be considered in the differential diagnosis. A step-wise, informed approach to post-transplant diarrhea will help the clinician achieve the best diagnostic yield. The use of diagnostic endoscopy should be preceded by exclusion of an infectious or drug-related cause of diarrhea. Empiric management with antidiarrheal agents, probiotics, and lactose-free diets may have a role in managing patients for whom no cause can be determined even after an extensive investigation. CONCLUSIONS: Physicians should be familiar with the common etiologies that result in post-transplant diarrhea. A directed approach to diagnosis and treatment will not only help to resolve the diarrhea but also prevent potentially life-threatening consequences including loss of the graft as well. Prospective studies are required to determine the etiology of post-transplant diarrhea in different clinical and geographic settings.

Esophageal impedance monitoring for gastroesophageal reflux.

Dual pH-multichannel intraluminal impedance (pH-MII) is a sensitive tool for evaluating overall gastroesophageal reflux disease, and particularly for permitting detection of nonacid reflux events. pH-MII technology is especially useful in the postprandial period or at other times when gastric contents are nonacidic.pH-MII was recently recognized by the North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition and the European Society for Pediatric Gastroenterology, Hepatology, and Nutrition as being superior to pH monitoring alone for evaluation of the temporal relation between symptoms and gastroesophageal reflux. In children, pH-MII is useful to correlate symptoms with reflux (particularly nonacid reflux), to quantify reflux during tube feedings and the postprandial period, and to assess efficacy of antireflux therapy. This clinical review is simply an evidence-based overview addressing the indications, limitations, and recommended protocol for the clinical use of pH-MII in children.

'Normal' vital signs belie occult hypoperfusion in geriatric trauma patients.

Base deficit (BD) and lactic acid (LA) are accepted markers of hypoperfusion and predictors of outcome in the trauma patient and we aim to assess the value of these markers in the triage of the elderly with "normal" vital signs. Patients older than age 65 who presented between 1997 and 2004 but who did not have isolated head injuries were included. Three patient groups were established: normal, occult hypoperfusion (OH), and shock. Outcome measures included mortality, hospital length of stay, intensive care unit length of stay, and discharge disposition. One hundred six patients were included in the analysis and had similar Injury Severity Scores. Mean systolic blood pressure was similar in the normal and OH groups. Forty-two per cent of patients had abnormal BD or LA in the emergency room indicating OH. These patients were more likely to have a longer intensive care unit length of stay (8.6 days vs. 3 days; P = 0.01) and were also more likely to be discharged to a nursing facility (P = 0.03). The trend was toward increased mortality in the OH group. OH is a common finding in elderly trauma patients. Outcomes in these patients are different and more like those presenting in shock.

Evaluation of gastroesophageal reflux in pediatric patients with asthma using impedance-pH monitoring.

OBJECTIVES: To determine the proportion of acid and nonacid reflux events in children with asthma suspected to have gastroesophageal reflux (GER) using combined impedance-pH monitoring, and to determine the symptom index (SI) for nonacid and acid reflux events. STUDY DESIGN: This was a prospective study of children with asthma (age 5 months to 6 years) referred for evaluation of GER. Exclusion criteria were congenital anomalies, cerebral palsy, mental retardation, and cardiac disease. The children underwent a 20-hour multichannel intraluminal impedance (MII)-pH study. RESULTS: A total of 24 children (17 male; mean age, 33 months) were enrolled from March 2004 to February 2005. MII-pH detected 1184 reflux events, versus 419 reflux events by pH alone; 51% (605 events) were nonacid. The proportion of nonacid reflux events decreased with time elapsed from last meal (P < .0001 by Pearson's chi2 test). A total of 555 symptoms were recorded, including 331 cases of cough, 243 of which (73.4%) were not associated with a reflux event. The SI for MII-pH was significantly different than that for the pH probe (37% vs 0%; P = .008). CONCLUSIONS: Acid and nonacid reflux occurs with equal frequency in children with asthma. Most symptoms occur in the absence of a reflux event.

Evaluation of infantile acid and nonacid gastroesophageal reflux using combined pH monitoring and impedance measurement.

OBJECTIVE: Characterize the proportion of acid and nonacid esophageal reflux events in young infants with suspected gastroesophageal reflux (GER) using combined pH-multichannel intraluminal impedance (MII) monitoring. Determine the symptom index correlation with nonacid reflux and acid reflux events. STUDY DESIGN: Prospective study of children, aged 2 weeks to 1 year, referred to The Children's Hospital of Denver Gastroenterology Clinic for evaluation of GER. Exclusion criteria were congenital anomalies or syndromes, cerebral palsy, mental retardation, and pulmonary or cardiac disease. The children were admitted to The Children's Hospital General Clinical Research Center for a 20 hour pH-MII study. Acid suppression was either never used or discontinued 2 weeks before testing. RESULTS: Thirty-four infants were enrolled from February 2004 to February 2005. Ages ranged from 2 months to 11 months, median = 7 (20 females/14 males). One thousand eight hundred ninety reflux events were detected by MII, and 588 reflux events were detected by pH probe alone. The percent of reflux that was acid was 47% (888 events) versus 53% of (1,002 events) nonacid reflux events. The proportion of nonacid reflux decreased with age (P < 0.0001 by Pearson chi test) and with increasing time elapsed from last meal. There were 958 total symptoms evaluated. The most frequently reported symptom was fussiness/pain, which correlated with nonacid reflux events 24.6% and acid reflux 25.2%. The proximal height of a reflux was predictive for symptoms of fussiness/pain, arching, and burping. CONCLUSION: MII detects more reflux events than pH monitoring alone. The proportion of nonacid reflux to acid reflux events in infants is more similar to adults than previously reported. Combined pH-MII esophageal monitoring identifies more reflux events and improves clinical correlation with symptoms.

Portopulmonary hypertension in pediatric patients.

OBJECTIVES: To investigate the clinical presentation, manifestations, and response to therapy of portopulmonary hypertension (PPHTN) in pediatric patients. STUDY DESIGN: This study was a retrospective chart review describing the evaluation and course of 7 patients with PPHTN. RESULTS: Causes of portal hypertension (HTN) included biliary atresia (3 cases), cavernous transformation of the portal vein (2 cases), and primary sclerosing cholangitis and cryptogenic cirrhosis (1 case each). The median interval from the diagnosis of portal HTN to PPHTN was 12.1 years. Four patients presented with a new heart murmur, 4 presented with syncope, and 3 presented with dyspnea. Although electrocardiograms (ECGs) and chest x-rays were normal in 3 and 2 patients, respectively, echocardiograms diagnosed pulmonary HTN in all 7 patients. Five patients had cardiac catheterizations; the average mean pulmonary artery pressure was 65 +/- 20 mm Hg. Response to therapy was variable, and 4 patients died. Postmortem lung tissue examination revealed plexiform lesions and pulmonary arteriopathy. CONCLUSIONS: Because symptoms are subtle and may be overlooked, pediatric patients with portal HTN who develop a new heart murmur, dyspnea, syncope, or who are being evaluated for liver transplantation require evaluation for PPHTN. ECG and chest x-ray are insensitive screens for PPHTN. An echocardiogram and cardiology evaluation is essential for the diagnosis.

Frequency of ASCA seropositivity in children with cystic fibrosis.

OBJECTIVE: To determine the frequency of anti-Saccharomyces cerevisiae antibodies (ASCA) seropositivity in pediatric patients with cystic fibrosis (CF) and correlate ASCA with clinical features. METHODS: Prospective study of children with CF aged 2 to 21 years enrolled from The Children's Hospital Cystic Fibrosis Center. Exclusion criteria included Crohn disease, immunodeficiency or immunoglobulin A deficiency. The frequency of ASCA (ASCA immunoglobulin A and immunoglobulin G) was measured by an enzyme-linked immunosorbent assay kit provided by Inova, Inc. (San Diego, CA). The CF Foundation database was queried for clinical data on ASCA seropositive patients. RESULTS: Seventeen (20.7%) of 82 patients were seropositive for ASCA. Of these, eight had immunoglobulin A antibodies, six had immunoglobulin G antibodies and three had both. ASCA seropositivity in CF patients was significantly greater than the general population (20.7% versus 4%, P < 0.0001), using an exact binomial test on a single proportion. The 95% confidence limits around our observed proportion were 12.6% to 31.1%. Of 17 ASCA positive patients, 1 (5.8%) had a history of meconium ileus, 14 (82.4%) had DeltaF508 gene mutation, 9 (52.9%) had positive sputum cultures for fungal organisms, and 17 (100%) had an ideal body weight % >or=85%. CONCLUSION: Patients with CF have a higher frequency of ASCA seropositivity than the general population. When evaluating CF patients for Crohn disease, ASCA should be used with caution. The reasons for higher ASCA seropositivity in CF patients are unknown, but may include exposure to fungal organisms via intestinal or pulmonary sources.