Citations

Abstract Transplantation of pluripotent stem cells has proven beneficial in heart failure, yet the proteomic landscape underlying repair remains largely uncharacterized. In a genetic model of dilated cardiomyopathy elicited by pressure overload in the KCNJ11 (potassium inwardly rectifying channel, subfamily J, member 11) null mutant, proteome-wide profiles were here resolved by means of a systems approach prior to and following disease manifestation in the absence or presence of embryonic stem cell treatment. Comparative two-dimensional gel electrophoresis revealed a unique cardiomyopathic proteome in the absence of therapy, remodeled in response to stem cell treatment. Specifically, linear ion trap quadrupole-Orbitrap mass spectrometry determined the identities of 93 and 109 differentially expressed proteins from treated and untreated cardiomyopathic hearts, respectively. Mapped protein–protein relationships and corresponding neighborhoods incorporated the stem cell-dependent subproteome into a nonstochastic network with divergent composition from the stem cell-independent counterpart. Stem cell intervention produced a distinct proteome signature across a spectrum of biological processes ranging from energetic metabolism, oxidoreductases, and stress-related chaperones to processes supporting protein synthesis/degradation, signaling, and transport regulation, cell structure and scaffolding. In the absence of treatment, bioinformatic interrogation of the disease-only proteome network prioritized adverse cardiac outcomes, ablated or ameliorated following stem cell transplantation. Functional and structural measurements validated improved myocardial contractile performance, reduced ventricular size and decreased cardiac damage in the treated cohort. Unbiased systems assessment unmasked “cardiovascular development” as a prioritized biological function in stem cell-reconstructed cardiomyopathic hearts. Thus, embryonic stem cell treatment transformed the cardiomyopathic proteome to demote disease-associated adverse effects and sustain a procardiogenic developmental response, supplying a regenerative substrate for heart failure repair.

The vasodilator signals regulating muscle blood flow during exercise are unclear. We tested the hypothesis that in young adults leg muscle vasodilation during steady-state exercise would be reduced independently by sequential pharmacological inhibition of nitric oxide synthase (NOS) and cyclooxygenase (COX) with NG-nitro-l-arginine methyl ester (l-NAME) and ketorolac, respectively. We tested a second hypothesis that NOS and COX inhibition would increase leg oxygen consumption (V̇o2) based on the reported inhibition of mitochondrial respiration by nitric oxide. In 13 young adults, we measured heart rate (ECG), blood pressure (femoral venous and arterial catheters), blood gases, and venous oxygen saturation (indwelling femoral venous oximeter) during prolonged (25 min) steady-state dynamic knee extension exercise (60 kick/min, 19 W). Leg blood flow (LBF) was determined by Doppler ultrasound of the femoral artery. Whole body V̇o2 was measured, and leg V̇o2 was calculated from blood gases and LBF. Resting intra-arterial infusions of acetylcholine (ACh) and nitroprusside (NTP) tested inhibitor efficacy. Leg vascular conductance (LVC) to ACh was reduced up to 53 ± 4% by l-NAME + ketorolac infusion, and the LVC responses to NTP were unaltered. Exercise increased LVC from 4 ± 1 to 33.1 ± 2 ml·min−1·mmHg−1 and tended to decrease after l-NAME infusion (31 ± 2 ml·min−1·mmHg−1, P = 0.09). With subsequent administration of ketorolac LVC decreased to 29.6 ± 2 ml·min−1·mmHg−1 ( P = 0.02; n = 9). While exercise continued, LVC returned to control values (33 ± 2 ml·min−1·mmHg−1) within 3 min, suggesting involvement of additional vasodilator mechanisms. In four additional subjects, LVC tended to decrease with l-NAME infusion alone ( P = 0.08) but did not demonstrate the transient recovery. Whole body and leg V̇o2 increased with exercise but were not altered by l-NAME or l-NAME + ketorolac. These data indicate a modest role for NOS- and COX-mediated vasodilation in the leg of exercising humans during prolonged steady-state exercise, which can be restored acutely. Furthermore, NOS and COX do not appear to influence muscle V̇o2 in untrained healthy young adults.

We recently showed that the Wiskott–Aldrich syndrome protein (WASP) family member, WASH, localizes to endosomal subdomains and regulates endocytic vesicle scission in an Arp2/3-dependent manner. Mechanisms regulating WASH activity are unknown. Here we show that WASH functions in cells within a 500 kDa core complex containing Strumpellin, FAM21, KIAA1033 (SWIP), and CCDC53. Although recombinant WASH is constitutively active toward the Arp2/3 complex, the reconstituted core assembly is inhibited, suggesting that it functions in cells to regulate actin dynamics through WASH. FAM21 interacts directly with CAPZ and inhibits its actin-capping activity. Four of the five core components show distant (approximately 15% amino acid sequence identify) but significant structural homology to components of a complex that negatively regulates the WASP family member, WAVE. Moreover, biochemical and electron microscopic analyses show that the WASH and WAVE complexes are structurally similar. Thus, these two distantly related WASP family members are controlled by analogous structurally related mechanisms. Strumpellin is mutated in the human disease hereditary spastic paraplegia, and its link to WASH suggests that misregulation of actin dynamics on endosomes may play a role in this disorder.

Abstract Genomic perturbations that challenge normal signaling at the pluripotent stage may trigger unforeseen ontogenic aberrancies. Anticipatory systems biology identification of transcriptome landscapes that underlie latent phenotypes would offer molecular diagnosis before the onset of symptoms. The purpose of this study was to assess the impact of calreticulin-deficient embryonic stem cell transcriptomes on molecular functions and physiological systems. Bioinformatic surveillance of calreticulin-null stem cells, a monogenic insult model, diagnosed a disruption in transcriptome dynamics, which re-prioritized essential cellular functions. Calreticulin-calibrated signaling axes were uncovered, and network-wide cartography of undifferentiated stem cell transcripts suggested cardiac manifestations. Calreticulin-deficient stem cell-derived cardiac cells verified disorganized sarcomerogenesis, mitochondrial paucity, and cytoarchitectural aberrations to validate calreticulin-dependent network forecasts. Furthermore, magnetic resonance imaging and histopathology detected a ventricular septal defect, revealing organogenic manifestation of calreticulin deletion. Thus, bioinformatic deciphering of a primordial calreticulin-deficient transcriptome decoded at the pluripotent stem cell stage a reconfigured multifunctional molecular registry to anticipate predifferentiation susceptibility toward abnormal cardiophenotype.

Abstract Innate immune effector mechanisms triggered by oncolytic viruses may contribute to the clearance of both infected and uninfected tumor cells in immunocompetent murine hosts. Here, we developed an in vitro tumor cell/bone marrow coculture assay and used it to dissect innate immune sensor and effector responses to intratumoral vesicular stomatitis virus (VSV). We found that the type III IFN interleukin-28 (IL-28) was induced by viral activation of innate immune-sensing cells, acting as a key mediator of VSV-mediated virotherapy of B16ova melanomas. Using tumor variants which differentially express the IL-28 receptor, we showed that IL-28 induced by VSV within the tumor microenvironment sensitizes tumor cells to natural killer cell recognition and activation. These results revealed new insights into the immunovirological mechanisms associated with oncolytic virotherapy in immune-competent hosts. Moreover, they defined a new class of tumor-associated mutation, such as acquired loss of responsiveness to IL-28 signaling, which confers insensitivity to oncolytic virotherapy through a mechanism independent of viral replication in vitro. Lastly, the findings suggested new strategies to manipulate immune signals that may enhance viral replication, along with antitumor immune activation, and improve the efficacy of oncolytic virotherapies. Cancer Res; 70(11); 4539–49. ©2010 AACR.

The study looks at the high cost of new treatments for chronic lymphocytic leukemia and calls for the consideration of a standardized and transparent method to determine the price of new drugs based on comparative effectiveness.

Background and Purpose— Telemedicine techniques can be used to address the rural–metropolitan disparity in acute stroke care. The Stroke Team Remote Evaluation Using a Digital Observation Camera (STRokE DOC) trial reported more accurate decision making for telemedicine consultations compared with telephone-only and that the California-based research network facilitated a high rate of thrombolysis use, improved data collection, low risk of complications, low technical complications, and favorable assessment times. The main objective of the STRokE DOC Arizona TIME (The Initial Mayo Clinic Experience) trial was to determine the feasibility of establishing, de novo, a single-hub, multirural spoke hospital telestroke research network across a large geographical area in Arizona by replicating the STRokE DOC protocol. Methods— Methods included prospective, single-hub, 2-spoke, randomized, blinded, controlled trial of a 2-way, site-independent, audiovisual telemedicine system designed for remote examination of adult patients with acute stroke versus telephone consultation to assess eligibility for treatment with intravenous thrombolysis. The primary outcome measure was whether the decision to give thrombolysis was correct. Secondary outcomes were rate of thrombolytic use, 90-day functional outcomes, incidence of intracerebral hemorrhages, and technical observations. Results— From December 2007 to October 2008, 54 patients were assessed, 27 of whom were randomized to each arm. Mean National Institutes of Health Stroke Scale score at presentation was 7.3 (SD 6.2) points. No consultations were aborted; however, technical problems (74%) were prevalent in the telemedicine arm. Overall, the correct treatment decision was established in 87% of the consultations. Both modalities, telephone (89% correct) and telemedicine (85% correct), performed well. Intravenous thrombolytic treatment was used in 30% of the telemedicine and telephone consultations. Good functional outcomes at 90 days were not significantly different. There were no statistically significant differences in mortality (4% in telemedicine and 11% in telephone) or rates of intracerebral hemorrhage (4% in telemedicine and 0% in telephone). Conclusions— It is feasible to extend the original STRokE DOC trial protocol to a new state and establish an operational single-hub, multispoke rural hospital telestroke research network in Arizona. The trial was not designed to have sufficient power to detect a difference between the 2 consultative modes: telemedicine and telephone-only. Whether by telemedicine or telephone consultative modalities, there were appropriate treatment decisions, high rates of thrombolysis use, improved data collection, low rates of intracerebral hemorrhage, and equally favorable time requirements. The learning curve was steep for the hub and spoke personnel of the new telestroke network, as reflected by frequent technical problems. Overall, the results support the effectiveness of highly organized and structured stroke telemedicine networks for extending expert stroke care into rural remote communities lacking sufficient neurological expertise.

Abstract Background: There is strong and consistent evidence that a genetic component contributes to the etiology of chronic lymphocytic leukemia (CLL). A recent genome-wide association study of CLL identified seven genetic variants that increased the risk of CLL within a European population. Methods: We evaluated the association of these variants, or variants in linkage disequilibrium with these variants, with CLL risk in an independent sample of 438 CLL cases and 328 controls. Results: Of these seven single nucleotide polymorphisms (SNP), six had P trend < 0.05 and had estimated odds ratios (OR) that were strikingly comparable to those of the previous study. Associations were seen for rs9378805 [OR, 1.47; 95% confidence intervals (CI), 1.19-1.80; P trend = 0.0003] near IRF4 and rs735665 near GRAMD1B (OR, 1.47; 95% CI, 1.14-1.89; P trend = 0.003). However, no associations (P > 0.05) were found for rs11083846, nor were any found for any SNP in linkage disequilibrium with rs11083846. Conclusions: Our results confirm the previous findings and further support the role of a genetic basis in the etiology of CLL; however, more research is needed to elucidate the causal SNP(s) and the potential manner in which these SNPs or linked SNPs function in CLL pathogenesis. Cancer Epidemiol Biomarkers Prev; 19(4); 1098–102. ©2010 AACR.