Citations

Purpose We conducted a pediatric phase I trial of the vascular endothelial growth factor (VEGF)–neutralizing antibody bevacizumab (BV). Primary aims included estimating the maximum-tolerated dose (MTD) and determining the dose-limiting toxicities (DLTs), pharmacokinetics, and biologic effects of BV in children with cancer. Patients and Methods BV (5, 10, 15 mg/kg) was administered intravenously every 2 weeks in 28-day courses to children with refractory solid tumors. Results Twenty-one patients enrolled, 20 (median age, 13 years) were eligible, and 18 completed one course and were fully assessable for toxicity. A total of 67 courses were administered (median, three courses per patient; range, one to 16 courses). Treatment was well tolerated with no DLTs observed. Non-DLTs included infusional reaction, rash, mucositis, proteinuria, and lymphopenia. Increases in systolic and diastolic blood pressure not meeting Common Terminology Criteria for Adverse Events (CTCAEv3) pediatric-specific criteria for hypertension were observed. There was no hemorrhage or thrombosis. Growth perturbation was not detected in a limited sample over the first course. The serum exposure to BV as measured by area under the concentration-time curve (AUC) seemed to increase in proportion to dose. The median clearance of BV was 4.1 mL/d/kg (range, 3.1 to 15.5 mL/d/kg), and the median half-life was 11.8 days (range, 4.4 to 14.6 days). No objective responses were observed. Exploratory analyses on circulating endothelial mobilization and viability are consistent with the available adult data. Conclusion BV is well tolerated in children. Phase II pediatric studies of BV in combination with chemotherapy in dosing schedules similar to adults are planned.

ABSTRACT We previously showed that the cysteines flanking the internal fusion peptide of the avian sarcoma/leukosis virus subtype A (ASLV-A) Env (EnvA) are important for infectivity and cell-cell fusion. Here we define the stage of fusion at which the cysteines are required. The flanking cysteines are dispensable for receptor-triggered membrane association but are required for the lipid mixing step of fusion, which, interestingly, displays a high pH onset and a biphasic profile. Second-site mutations that partially restore infection partially restore lipid mixing. These findings indicate that the cysteines flanking the internal fusion peptide of EnvA (and perhaps by analogy Ebola virus glycoprotein) are important for the foldback stage of the conformational changes that lead to membrane merger.

A-kinase anchoring proteins (AKAPs) recruit signaling molecules and present them to downstream targets to achieve efficient spatial and temporal control of their phosphorylation state. In the heart, sympathetic nervous system (SNS) regulation of cardiac action potential duration (APD), mediated by β-adrenergic receptor (βAR) activation, requires assembly of AKAP9 (Yotiao) with the I Ks potassium channel α subunit (KCNQ1). KCNQ1 mutations that disrupt this complex cause type 1 long-QT syndrome (LQT1), one of the potentially lethal heritable arrhythmia syndromes. Here, we report identification of ( i ) regions on Yotiao critical to its binding to KCNQ1 and ( ii ) a single putative LQTS-causing mutation (S1570L) in AKAP9 (Yotiao) localized to the KCNQ1 binding domain in 1/50 (2%) subjects with a clinically robust phenotype for LQTS but absent in 1,320 reference alleles. The inherited S1570L mutation reduces the interaction between KCNQ1 and Yotiao, reduces the cAMP-induced phosphorylation of the channel, eliminates the functional response of the I Ks channel to cAMP, and prolongs the action potential in a computational model of the ventricular cardiocyte. These reconstituted cellular consequences of the inherited S1570L-Yotiao mutation are consistent with delayed repolarization of the ventricular action potential observed in the affected siblings. Thus, we have demonstrated a link between genetic perturbations in AKAP and human disease in general and AKAP9 and LQTS in particular.

The role of B-cell receptor (BCR)–mediated survival signals in diffuse large B-cell lymphoma (DLBCL) remains undefined. Ligand-induced BCR signaling induces receptor oligomerization, Igα/β immunoreceptor tyrosine-based activation motif (ITAM) phosphorylation, and activation of the spleen tyrosine kinase (SYK), which initiates downstream events and amplifies the initial BCR signal. BCRs also transmit low-level tonic survival signals in the absence of receptor engagement. Herein, we assess the role of SYK-dependent tonic BCR survival signals in DLBCL cell lines and primary tumors and evaluate the efficacy of an ATP-competitive inhibitor of SYK, R406, in vitro. R406 induced apoptosis of the majority of examined DLBCL cell lines. In R406-sensitive DLBCL cell lines, R406 specifically inhibited both tonic- and ligand-induced BCR signaling (autophosphorylation of SYK525/526 and SYK-dependent phosphorylation of the B-cell linker protein [BLNK]). The majority of examined primary DLBCLs also exhibited tonic- and ligand-induced BCR signaling; in these primary tumors, BCR signaling was also inhibited by R406. Of note, BCR-dependent and R406-sensitive DLBCL cell lines were independently identified as “BCR-type” tumors by transcriptional profiling. Therefore, SYK-dependent tonic BCR signaling is an important and potentially targetable survival pathway in some, but not all, DLBCLs. In addition, R406-sensitive DLBCLs can be identified by their transcriptional profiles.

It is well-known that dynamin 2 (Dyn2) participates in clathrin- and caveolae-mediated endocytosis; however, the role of Dyn2 in coat-independent endocytic processes remains controversial. Here we demonstrate a role for specific spliced variants of Dyn2 in the micropinocytosis of fluid in epithelial cells, independent of coat-mediated endocytic pathways. A general inhibition of Dyn2 was first performed using either microinjection of anti-dynamin antibodies or Dyn2-siRNA treatment. Both of these methods resulted in reduced uptake of transferrin, a marker for clathrin-mediated endocytosis, and, under unstimulated conditions, reduced the uptake of the fluid-phase markers dextran and horseradish peroxidase (HRP). By contrast, cells treated similarly but stimulated with serum or EGF internalized substantial amounts of dextran or HRP, indicating that Dyn2 is not required for stimulated fluid uptake via macropinocytosis. We next tested whether a specific spliced variant might selectively affect fluid-phase endocytosis. Mutation of specific Dyn2 spliced variants resulted in a differential attenuation of transferrin and dextran internalization. Furthermore, the reduction in fluid uptake in Dyn2-siRNA-treated cells was only rescued upon re-expression of select spliced variants. These findings suggest that Dyn2 function is required for the coat-independent internalization of fluid through endocytic pathways distinct from macropinocytosis and, in addition, implicate different Dyn2 spliced variants in specific endocytic functions.

Background Inhibition of leukocyte adhesion can reduce myocardial infarct size in animals. This study was designed to define the safety and efficacy of a recombinant, humanized, monoclonal antibody to the CD18 subunit of the β2 integrin adhesion receptors (rhuMAb CD18), in reducing infarct size in patients treated with a thrombolytic agent. Methods and Results The Limitation of Myocardial Infarction following Thrombolysis in Acute Myocardial Infarction Study (LIMIT AMI) was a randomized, double-blind, placebo-controlled, multicenter study conducted in 60 centers in the United States and Canada. A total of 394 subjects who presented within 12 hours of symptom onset with ECG findings (ST-segment elevation) consistent with AMI were treated with recombinant tissue plasminogen activator and were also given an intravenous bolus of 0.5 or 2.0 mg/kg rhuMAb CD18 or placebo. Coronary angiography was performed at 90 minutes, 12-lead ECGs were obtained at baseline, 90, and 180 minutes, and resting sestamibi scans were performed at ≥120 hours. Adjunctive angioplasty and use of glycoprotein IIb/IIIa antiplatelet agents at the time of angiography were discretionary. There were no treatment effects on coronary blood flow, infarct size, or the rate of ECG ST-segment elevation resolution, despite the expected induction of peripheral leukocytosis. A slight trend toward an increase in bacterial infections was observed with rhuMAb CD18 ( P =0.33). Conclusions RhuMAb CD18 was well tolerated but not effective in modifying cardiac end points.

Background— Renal insufficiency is associated with increased morbidity and mortality after cardiopulmonary bypass cardiac surgery. B-type natriuretic peptide is a cardiac hormone that enhances glomerular filtration rate and inhibits aldosterone. Cystatin has been shown to be a better endogenous marker of renal function than creatinine. Methods and Results— We performed a double-blinded placebo-controlled proof of concept pilot study in patients (n=40) with renal insufficiency preoperatively (defined as an estimated creatinine clearance of <60 mL/min determined by the Cockroft-Gault formula), undergoing cardiopulmonary bypass cardiac surgery. Patients were randomized to placebo (n=20) or IV low dose nesiritide (n=20; 0.005 μg/Kg/min) for 24 hours started after the induction of anesthesia and before cardiopulmonary bypass. Patients in the nesiritide group had an increase of plasma B-type natriuretic peptide and its second messenger cGMP with a decrease in plasma cystatin levels at the end of the 24-hour infusion. These changes were not observed in the placebo group. There was a significant activation of aldosterone in the placebo group at the end of the 24-hour infusion, but not in the nesiritide group. At 48 and 72 hours, there was a decrease in estimated creatinine clearance and an increase in plasma cystatin as compared with end of the 24-hour infusion in the placebo group. In contrast, renal function was preserved in the nesiritide group with no significant change in estimated creatinine clearance and a trend for plasma cystatin to increase as compared with end of the 24-hour infusion. Conclusion— This proof of concept pilot study supports the conclusion that perioperative administration of low dose nesiritide is biologically active and decreases plasma cystatin in patients with renal insufficiency undergoing cardiopulmonary bypass cardiac surgery. Further studies are warranted to determine whether these physiological observations can be translated into improved clinical outcomes.

Hip simulators are regularly used by researchers to assess total hip arthroplasty (THA) implants, range of motion, stability, and alignment of acetabular cup and stem. Previous papers have described three types of simulators: three-dimensional protractors, biaxial rocking motion (BRM) protractors, and single-axis prosthetic range-of-motion (PROM) devices. We have developed a new hip simulator in which the ROM device is completely automated in three independent axes (elevation, internal/external rotation, and plane of elevation). Coupled with the simulator, we used a FaroArm Gold Series coordinate measuring machine (CMM) to accurately align the implant components. The results show that the methodology and alignment setup are accurate and repeatable. With this simulator and digitizer, we are able to study the characteristics of numerous THA implants at various orientations of the pelvis, acetabular cup, stem, and femur.