Citations

Rationale: Although a cardioprotective function of target of rapamycin (TOR) signaling inhibition has been suggested by pharmacological studies using rapamycin, genetic evidences are still lacking. We explored adult zebrafish as a novel vertebrate model for dissecting signaling pathways in cardiomyopathy. Objective: We generated the second adult zebrafish cardiomyopathy model induced by doxorubicin. By genetically analyzing both the doxorubicin and our previous established anemia-induced cardiomyopathy models, we decipher the functions of TOR signaling in cardiomyopathies of different etiology. Methods and Results: Along the progression of both cardiomyopathy models, we detected dynamic TOR activity at different stages of pathogenesis as well as distinct effects of TOR signaling inhibition. Nevertheless, cardiac enlargement in both models can be effectively attenuated by inhibition of TOR signaling through short-term rapamycin treatment. To assess the long-term effects of TOR reduction, we used a zebrafish target of rapamycin ( ztor ) mutant identified from an insertional mutagenesis screen. We show that TOR haploinsufficiency in the ztor heterozygous fish improved cardiac function, prevented pathological remodeling events, and ultimately reduced mortality in both adult fish models of cardiomyopathy. Mechanistically, these cardioprotective effects are conveyed by the antihypertrophy, antiapoptosis, and proautophagy function of TOR signaling inhibition. Conclusions: Our results prove adult zebrafish as a conserved novel vertebrate model for human cardiomyopathies. Moreover, we provide the first genetic evidence to demonstrate a long-term cardioprotective effect of TOR signaling inhibition on at least 2 cardiomyopathies of distinct etiology, despite dynamic TOR activities during their pathogenesis.

PurposeLenalidomide is an oral immunomodulatory drug with multiple effects on the immune system and tumor cell microenvironment leading to inhibition of malignant cell growth. Based on encouraging reports of lenalidomide in relapsed and refractory chronic lymphocytic leukemia (CLL), we investigated the first-line use of single-agent lenalidomide in CLL.Patients and MethodsUsing a starting dose of lenalidomide 10 mg/d for 21 days of a 28-day cycle and weekly 5-mg dose escalations to a target of 25 mg, we encountered severe toxicities (tumor lysis, fatal sepsis) in the first two patients enrolled. The study was halted and the protocol amended to a more conservative regimen: starting dose of lenalidomide 2.5 mg with monthly escalations to a target dose of 10 mg, and extended tumor lysis prophylaxis and monitoring. Gene expression profiles from patient samples before and after 7 days of lenalidomide were performed.ResultsTwenty-five patients were enrolled on the amended protocol. No further tumor lysis events were reported. Tumor flare was common (88%) but mild. Grade 3 to 4 neutropenia occurred in 72% of patients, with only five episodes of febrile neutropenia. The overall response rate was 56% (no complete responses). Although rapid peripheral lymphocyte reductions were observed, rebound lymphocytoses during the week off-therapy were common. Lenalidomide-induced molecular changes enriched for cytoskeletal and immune-related genes were identified.ConclusionLenalidomide is clinically active as first-line CLL therapy and is well-tolerated if a conservative approach with slow dose escalation is used. A lenalidomide-induced molecular signature provides insights into its immunomodulatory mechanisms of action in CLL.

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.

Inactive cortisone is converted to active cortisol within the liver by 11β-hydroxysteroid dehydrogenase-1 (11β-HSD1), and impaired regulation of this process may be related to increased hepatic glucose production (HGP) in individuals with type 2 diabetes. The primary aim of this study was to investigate the effect of acute 11β-HSD1 inhibition on HGP and fat metabolism during insulin deficiency. Sixteen conscious, 42-h-fasted, lean, healthy dogs were studied. Somatostatin was infused to create insulin deficiency, and the animals were treated with a specific 11β-HSD1 inhibitor (compound 531) or placebo for 5 h. 11β-HSD1 inhibition completely suppressed hepatic cortisol production, and this attenuated the increase in HGP that occurred during insulin deficiency. PEPCK and glucose-6-phosphatase expression were decreased when 11β-HSD1 was inhibited, but gluconeogenic flux was unchanged, implying an effect on glycogenolysis. Since inhibition of hepatic cortisol production reduces HGP during insulin deficiency, 11β-HSD1 is a potential therapeutic target for the treatment of excess glucose production that occurs in diabetes.

Abstract Activating epidermal growth factor receptor (EGFR) mutations are common in many cancers including glioblastoma. However, clinical responses to EGFR inhibitors are infrequent and short-lived. We show that the Src family kinases (SFK) Fyn and Src are effectors of oncogenic EGFR signaling, enhancing invasion and tumor cell survival in vivo. Expression of a constitutively active EGFR mutant, EGFRvIII, resulted in activating phosphorylation and physical association with Src and Fyn, promoting tumor growth and motility. Gene silencing of Fyn and Src limited EGFR- and EGFRvIII-dependent tumor cell motility. The SFK inhibitor dasatinib inhibited invasion, promoted tumor regression, and induced apoptosis in vivo, significantly prolonging survival of an orthotopic glioblastoma model expressing endogenous EGFRvIII. Dasatinib enhanced the efficacy of an anti-EGFR monoclonal antibody (mAb 806) in vivo, further limiting tumor growth and extending survival. Examination of a large cohort of clinical samples showed frequent coactivation of EGFR and SFKs in glioblastoma patients. These results establish a mechanism linking EGFR signaling with Fyn and Src activation to promote tumor progression and invasion in vivo and provide rationale for combined anti-EGFR and anti-SFK targeted therapies. [Cancer Res 2009;69(17):6889–98]

Clonal disorders of large granular lymphocytes (LGL) result in leukemia due to the expansion of a discrete subset of either CD3 + T cells or natural killer (NK) cells. It has been hypothesized that a viral antigen acts as the initial stimulus causing the expansion of these cells. The possible involvement of human T cell lymphotropic virus types 1 and 2 (HTLV-1 and HTLV-2) in this disease has been studied but no conclusive evidence has linked either virus with LGL leukemia. In this study, we examined whether HTLV-3 or HTLV-4, two newly identified HTLV groups discovered in Central Africa in primate hunters, is involved in LGL leukemia. We developed two specific real-time PCR quantitative assays that are highly sensitive, capable of detecting 10 copies of HTLV-3 or HTLV-4 pol sequences in a background of 1 μ g of DNA from human peripheral blood lymphocytes (PBL). We tested PBL DNA samples from 40 LGL leukemia patients in the United States and found that all samples were negative for HTLV-3 or HTLV-4 infection. These results suggest that HTLV-3 and HTLV-4 are not the causative agent of LGL leukemia.

Abstract Microsatellite instability (MSI) occurs in 10% to 20% of colorectal cancers (CRC) and has been attributed to both MLH1 promoter hypermethylation and germline mutation in the mismatch repair (MMR) genes. We present results from a large population- and clinic-based study of MLH1 methylation, immunohistochemistry, and MMR germline mutations that enabled us to (a) estimate the prevalence of MMR germline mutations and MLH1 methylation among MSI-H cases and help us understand if all MSI-H CRC is explained by these mechanisms and (b) estimate the associations between MLH1 methylation and sex, age, and tumor location within the colon. MLH1 methylation was measured in 1,061 population-based and 172 clinic-based cases of CRC. Overall, we observed MLH1 methylation in 60% of population-based MSI-H cases and in 13% of clinic-based MSI-H cases. Within the population-based cases with MMR mutation screening and conclusive immunohistochemistry results, we identified a molecular event in MMR in 91% of MSI-H cases: 54% had MLH1 methylation, 14% had a germline mutation in a MMR gene, and 23% had immunohistochemistry evidence for loss of a MMR protein. We observed a striking age difference, with the prevalence of a MMR germline mutation more than 4-fold lower and the prevalence of MLH1 methylation more than 4-fold higher in cases diagnosed after the age of 50 years than in cases diagnosed before that age. We also determined that female sex is an independent predictor of MLH1 methylation within the MSI-H subgroup. These results reinforce the importance of distinguishing between the underlying causes of MSI in studies of etiology and prognosis. (Cancer Epidemiol Biomarkers Prev 2008;17(11):3208–15)

Dynamin is a 100-kDa microtubule-activated GTPase originally isolated from mammalian brain that has been proposed to be crucial in the early steps of endocytosis. Previous studies on the primary structure, biochemical properties, and functional role of dynamin indicated that it was neuron-specific. However, using an antibody against a synthetic peptide representing an enzymatic region of rat brain dynamin (D100), we identified a 100-kDa protein doublet in rat liver, suggesting that dynamin exists as different isoforms that are distinct from the brain counterpart. We then initiated a search for distinctive dynamin isoforms with antibodies and cDNA probes. A 500-bp PCR-generated cDNA probe corresponding to the enzymatic region of the rat brain dynamin-encoding gene was used to isolate six overlapping clones from a rat liver cDNA library that together span the complete coding sequence of another dynamin gene, "Dyn2." Sequence analyses reveal that dynamin 2 (Dyn2) is 75% identical to brain dynamin at the DNA level and is 79% identical at the protein level. By Northern blot analysis and isoform-specific PCR, Dyn2 was found ubiquitously in adult rat tissues as two transcripts of 3.5 kb and 4 kb; the highest levels were found in testis. These results indicate that dynamin proteins are encoded by at least two genes expressed differentially in mammalian tissues and that the expression of Dyn2, and not of brain dynamin, accounts for the ubiquitous distribution of dynamin in rat tissues.

KLF11 (TIEG2) is a pancreas-enriched transcription factor that has elicited significant attention because of its role as negative regulator of exocrine cell growth in vitro and in vivo . However, its functional role in the endocrine pancreas remains to be established. Here, we report, for the first time, to our knowledge, the characterization of KLF11 as a glucose-inducible regulator of the insulin gene. A combination of random oligonucleotide binding, EMSA, luciferase reporter, and chromatin immunoprecipitation assays shows that KLF11 binds to the insulin promoter and regulates its activity in beta cells. Genetic analysis of the KLF11 gene revealed two rare variants (Ala347Ser and Thr220Met) that segregate with diabetes in families with early-onset type 2 diabetes, and significantly impair its transcriptional activity. In addition, analysis of 1,696 type 2 diabetes mellitus and 1,776 normoglycemic subjects show a frequent polymorphic Gln62Arg variant that significantly associates with type 2 diabetes mellitus in North European populations (OR = 1.29, P = 0.00033). Moreover, this variant alters the corepressor mSin3A-binding activity of KLF11, impairs the activation of the insulin promoter and shows lower levels of insulin expression in pancreatic beta cells. In addition, subjects carrying the Gln62Arg allele show decreased plasma insulin after an oral glucose challenge. Interestingly, all three nonsynonymous KLF11 variants show increased repression of the catalase 1 promoter, suggesting a role in free radical clearance that may render beta cells more sensitive to oxidative stress. Thus, both functional and genetic analyses reveal that KLF11 plays a role in the regulation of pancreatic beta cell physiology, and its variants may contribute to the development of diabetes.