Citations

Abstract Apoptosis induction by BH3 mimetics is a therapeutic strategy for human cancer. These mimetics exert single-agent activity in cells “primed” for cell death. Primed cells are dependent upon antiapoptotic Bcl-2 proteins for survival and are characterized by the ability of the BH3 mimetic to induce cytochrome c release from their isolated mitochondria. Our aim was to examine the single-agent activity of obatoclax, a BH3 mimetic in cholangiocarcinoma cell lines. In clonogenic assays, inhibition of colony formation was observed by obatoclax treatment. Despite single-agent activity by obatoclax, the mitochondria from these cells did not release cytochrome c after incubation with this BH3 mimetic. However, immunofluorescence and cell fractionation studies identified Bax activation and translocation to mitochondria after treatment with obatoclax. shRNA targeted knockdown of Bax doubled the IC50 for obatoclax but did not abrogate its cytotoxicity, whereas knockdown of Bak did not alter the IC50. In a cell-free system, obatoclax induced an activating conformational change of Bax, which was attenuated by a site-directed mutagenesis of a previously identified protein activation site. Finally, the drug also elicited a significant in vivo response in a rodent model of this disease. In conclusion, single-agent obatoclax treatment results in Bax activation, which contributes, in part, to cell death in cholangiocarcinoma cells. These data indicate that BH3 mimetics may also function as direct activators of Bax and induce cytotoxicity in cells not otherwise primed for cell death. Cancer Res; 70(5); 1960–9

AbstractThe effects of prostaglandin E2 (PGE2) given in controlled-release pellets or by daily sc injection for 21 days on mineral homeostasis and bone histomorphometry were compared in 7-week-old female rats. Sham operation and ovariectomy were performed at the beginning of the studies. In experiment 1, 7.5 mg PGE2 or drug-free, controlled-release pellets were implanted sc at the back of the neck on day 7. In experiment 2, 3 mg/kg body weight of PGE2 or vehicle was injected sc daily beginning on day 7. The animals were sacrificed on day 28 of the two experiments, and the tibiae were removed for histomorphometric analysis of the diaphysis and metaphysis. When administered by pellets in experiment 1, PGE2 lowered serum 1,25-dihydroxyvitamin D and did not influence weight gain, serum calcium, phosphorus, or magnesium, cross-sectional or medullary areas, periosteal bone formation and apposition rates, endosteal bone formation and apposition rates, or endosteal tetracycline-labeled perimeter. PGE2 lowered cancellous bone area and cancellous bone perimeter in both the sham-operated and ovariectomized rats. In contrast, when administered by sc injection in experiment 2, PGE2 reduced weight gain, increased serum magnesium, increased cortical area, and reduced medullary area without changing cross-sectional area, increased periosteal bone formation and apposition rates and endosteal bone and apposition rates, did not alter endosteal tetracycline-labeled perimeter, and increased cancellous bone area and cancellous bone perimeter in both sham-operated and ovariectomized animals. PGE2 produced local inflammation when given by pellets, and the serum concentration of 13,14-dihydro-15-ketoprostaglandin E2, the major metabolite of PGE2, increased when PGE2 was given by sc injection but not when administered by pellets. Thus, PGE2 given sc by controlled-release pellets (1) produces local inflammation and systemic bone loss without increasing PGE2 systemically and (2) provides a model for inflammation-induced loss of cancellous bone. The results also indicate that the pellet is not a valid means for the delivery of PGE2 to the general circulation.

OBJECTIVE: Despite the magnitude of the problem of catheter-associated bloodstream infections (CA-BSIs) in children, relatively little research has been performed to identify effective strategies to reduce these complications. In this study, we aimed to develop and evaluate effective catheter-care practices to reduce pediatric CA-BSIs. STUDY DESIGN AND METHODS: Our study was a multi-institutional, interrupted time-series design with historical control data and was conducted in 29 PICUs across the United States. Two central venous catheter–care practice bundles comprised our intervention: the insertion bundle of pediatric-tailored care elements derived from adult efforts and the maintenance bundle derived from the Centers for Disease Control and Prevention recommendations and expert pediatric clinician consensus. The bundles were deployed with quality-improvement teaching and methods to support their adoption by teams at the participating PICUs. The main outcome measures were the rate of CA-BSIs from January 2004 to September 2007 and compliance with each element of the insertion and maintenance bundles from October 2006 to September 2007. RESULTS: Average CA-BSI rates were reduced by 43% across 29 PICUs (5.4 vs 3.1 CA-BSIs per 1000 central-line-days; P < .0001). By September 2007, insertion-bundle compliance was 84% and maintenance-bundle compliance was 82%. Hierarchical regression modeling showed that the only significant predictor of an observed decrease in infection rates was the collective use of the insertion and maintenance bundles, as demonstrated by the relative rate (RR) and confidence intervals (CIs) (RR: 0.57 [95% CI: 0.45–0.74]; P < .0001). We used comparable modeling to assess the relative importance of the insertion versus maintenance bundles; the results showed that the only significant predictor of an infection-rate decrease was maintenance-bundle compliance (RR: 0.41 [95% CI: 0.20–0.85]; P = .017). CONCLUSIONS: In contrast with adult ICU care, maximizing insertion-bundle compliance alone cannot help PICUs to eliminate CA-BSIs. The main drivers for additional reductions in pediatric CA-BSI rates are issues that surround daily maintenance care for central lines, as defined in our maintenance bundle. Additional research is needed to define the optimal maintenance bundle that will facilitate elimination of CA-BSIs for children.

Rationale Carotid endarterectomy (CEA) and medical therapy were shown superior to medical therapy alone for symptomatic (ge;50%) and asymptomatic (≥60%) stenosis. Carotid angioplasty stenting (CAS) offers a less invasive alternative. Establishing safety, efficacy, and durability of CAS requires rigorous comparison with CEA in symptomatic and asymptomatic patients. Aims The objective is to compare the efficacy of CAS versus CEA in patients with symptomatic (≥50%) or asymptomatic (≥60%) extracranial carotid stenosis. Design The Carotid Revascularization Endarterectomy vs. Stenting Trial (CREST) is a prospective, randomized, parallel, two-arm, multi-center trial with blinded endpoint adjudication. Primary endpoints are analyzed using standard time-to-event statistical modeling with adjustment for major baseline covariates. Primary analysis is on an intent-to-treat basis. Study Outcomes The primary outcome is the occurrence of any stroke, myocardial infarction, or death during a 30-day peri-procedural period, and ipsilateral stroke during follow-up of up to four years. Secondary outcomes include restenosis and health-related quality of life.

Purpose We previously validated disease-free survival (DFS) as a surrogate for overall survival (OS) in fluorouracil-based adjuvant colon cancer clinical trials. New therapies have extended survival after recurrence from 1 to approximately 2 years. We examined the possible impact of this improvement on the DFS/OS association. Methods The Adjuvant Colon Cancer Endpoints (ACCENT) data set of 20,898 patients was analyzed. In an exploratory fashion, time from recurrence to death in patients experiencing recurrence was extended using several algorithms, and the association of DFS after 3 years of median follow-up and OS after varying lengths of follow-up (median of 5, 6, and 7 years) was assessed. Results Seven thousand four hundred two patients (35%) experienced recurrence. Median time from recurrence to death was 24 months in the hypothetical data sets. When times from recurrence to death were doubled, the association between treatment effects on DFS and 5-year OS was modest (R2 = 0.51 for both 2- and 3-year DFS) but remained strong for DFS and 6-year OS (R2 = 0.67 for both 2- and 3-year DFS) and 7-year OS (R2 = 0.70 for both 2- and 3-year DFS). The reduced DFS/OS association with extended survival after recurrence was greater in stage II than stage III patients. Multiple simulations provided consistent findings. Conclusion Extended survival after recurrence reduces the association between treatment effects on 3-year DFS and 5-year OS, particularly in stage II patients; longer follow-up strengthens the association. In modern adjuvant trials, 6 or 7 years may be required to demonstrate OS improvements, further supporting DFS as the preferred primary end point for future adjuvant colon cancer clinical trials.

Abstract Anti-Smith (Sm) D autoantibodies are specific for systemic lupus erythematosus. In this investigation, the influence of HLA-D genes on immune responses to SmD was investigated. Mice with HLA-DR3, HLA-DR4, HLA-DQ0601, HLA-DQ0604, or HLA-DQ8 transgenes were immunized with recombinant SmD1, and their Ab responses were analyzed. Analysis by ELISA showed that all strains responded well to SmD. However, when synthetic SmD peptides were used as substrate, DR3 mice had the highest Ab response followed by DQ8, DQ0604, DQ0601, and DR4. A similar trend was observed in Western blot analysis using WEHI 7.1 cell lysate as the substrate, with the exception that DR4 mice did not generate detectable amounts of Abs. Only sera from DR3 and DQ0604 mice immunoprecipitated A-ribonucleoprotein (RNP), SmB, and SmD. Intermolecular epitope spreading to A-RNP and SmB was evident in DR3 and DQ0604 mice, as sera depleted of anti-SmD Abs were reactive with these proteins. DR3 mice also generated an immune response to C-RNP. Anti-nuclear Abs were detected in the majority of the DR3 mice, whereas moderate reactivities were seen in DQ0604 and DQ8 mice. Interestingly, only DR3 mice mounted an anti-dsDNA Ab response. Approximately half of the anti-dsDNA Abs were cross-reactive with SmD. Ab responses correlated with the strength of the T cell responses. Thus, HLA-DR3 appears to be the dominant HLA-D gene that determines the magnitude and quality of the anti-SmD immune response. In addition, our findings provide insights into the origin of the anti-dsDNA Abs often detected in patients with systemic lupus erythematosus.

Abstract Purpose: Carfilzomib (formerly PR-171) is a novel proteasome inhibitor of the epoxyketone class that is selective and structurally distinct from bortezomib. Proteasome inhibition by carfilzomib is mechanistically irreversible. Consequently, proteasome inhibition is more sustained with carfilzomib than with bortezomib. Experimental Design: In a phase 1 trial evaluating the safety and efficacy of carfilzomib in relapsed or refractory hematologic malignancies, eight dose groups of three to six patients received 5 consecutive days of carfilzomib i.v. push at doses of 1.2, 2.4, 4, 6, 8.4, 11, 15, and 20 mg/m2 within 14-day cycles. Results: Twenty-nine patients enrolled that were relapsed or refractory after at least two prior therapies. Nonhematologic toxicities included fatigue, nausea, and diarrhea in more than one third of patientsmostly grade 1 or 2 in severity. At 20 mg/m2, grade 3 febrile neutropenia and grade 4 thrombocytopenia were reported, establishing 15 mg/m2 as the maximum tolerated dose. No grade 3 or 4 peripheral neuropathies were reported. Antitumor activity was observed at doses 11 mg/m2: one unconfirmed complete response (mantle cell), one partial response (multiple myeloma), and two minimal responses (multiple myeloma and Waldenstrm's macroglobulinemia). Conclusion: This is the first clinical use of carfilzomib that shows tolerability and clinical activity in multiple hematologic malignancies using consecutive-day dosing. (Clin Cancer Res 2009;15(22):708591)

Abstract Eosinophils are multifunctional leukocytes implicated in the pathogenesis of asthma and in immunity to certain organisms. Associations between exposure to an environmental fungus, such as Alternaria, and asthma have been recognized clinically. Protease-activated receptors (PARs) are G protein-coupled receptors that are cleaved and activated by serine proteases, but their roles in innate immunity remain unknown. We previously found that human eosinophils respond vigorously to Alternaria organisms and to the secretory product(s) of Alternaria with eosinophils releasing their proinflammatory mediators. In this study, we investigated the roles of protease(s) produced by Alternaria and of PARs expressed on eosinophils in their immune responses against fungal organisms. We found that Alternaria alternata produces aspartate protease(s) and that human peripheral blood eosinophils degranulate in response to the cell-free extract of A. alternata. Eosinophils showed an increased intracellular calcium concentration in response to Alternaria that was desensitized by peptide and protease ligands for PAR-2 and inhibited by a PAR-2 antagonistic peptide. Alternaria-derived aspartate protease(s) cleaved PAR-2 to expose neo-ligands; these neo-ligands activated eosinophil degranulation in the absence of proteases. Finally, treatment of Alternaria extract with aspartate protease inhibitors, which are conventionally used for HIV-1 and other microbes, attenuated the eosinophils’ responses to Alternaria. Thus, fungal aspartate protease and eosinophil PAR-2 appear critical for the eosinophils’ innate immune response to certain fungi, suggesting a novel mechanism for pathologic inflammation in asthma and for host-pathogen interaction.