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1.  Fatal outcome of human influenza A (H5N1) is associated with high viral load and hypercytokinemia 
Nature medicine  2006;12(10):1203-1207.
Avian influenza A (H5N1) viruses cause severe disease in humans1,2, but the basis for their virulence remains unclear. In vitro and animal studies indicate that high and disseminated viral replication is important for disease pathogenesis3-5. Laboratory experiments suggest that virus-induced cytokine dysregulation may contribute to disease severity6-9. To assess the relevance of these findings for human disease, we performed virological and immunological studies in 18 individuals with H5N1 and 8 individuals infected with human influenza virus subtypes. Influenza H5N1 infection in humans is characterized by high pharyngeal virus loads and frequent detection of viral RNA in rectum and blood. Viral RNA in blood was present only in fatal H5N1 cases and was associated with higher pharyngeal viral loads. We observed low peripheral blood T-lymphocyte counts and high chemokine and cytokine levels in H5N1-infected individuals, particularly in those who died, and these correlated with pharyngeal viral loads. Genetic characterization of H5N1 viruses revealed mutations in the viral polymerase complex associated with mammalian adaptation and virulence. Our observations indicate that high viral load, and the resulting intense inflammatory responses, are central to influenza H5N1 pathogenesis. The focus of clinical management should be on preventing this intense cytokine response, by early diagnosis and effective antiviral treatment.
PMCID: PMC4333202  PMID: 16964257
Nature medicine  2009;15(8):951-954.
Neutralizing antibodies are thought crucial to HIV vaccine protection but a major hurdle is the high antibody concentrations likely required as suggested by studies in animal models1. However, these studies typically apply a large virus inoculum to ensure infection in control animals in single challenge experiments. In contrast, most human infection via sexual encounter probably involves repeated exposures to much lower doses of virus2–4. Therefore, animal studies may have overestimated protective antibody levels in humans. To investigate the impact of virus challenge dose on antibody protection, we repeatedly exposed macaques intravaginally to low doses of a CCR5 coreceptor-using SHIV (an HIV/SIV chimera) in the presence of antibody at plasma concentrations leading to relatively modest neutralization titers of the order of 1:5 IC90 values in a PBMC assay. An effector function deficient variant of the neutralizing antibody was also included. The results show that a significantly greater number of challenges are required to infect animals treated with neutralizing antibody than control antibody-treated animals, and the notion that effector function may contribute to antibody protection is supported. Overall, the results imply that lower levels of antibody than considered hereto may provide benefit in the context of typical human exposure to HIV-1.
PMCID: PMC4334439  PMID: 19525965
3.  STAT3 signaling controls satellite cell expansion and skeletal muscle repair 
Nature medicine  2014;20(10):1182-1186.
The progression of disease- and age-dependent skeletal muscle wasting results in part from a decline in the number and function of satellite cells, the direct cellular contributors to muscle repair1–10. However, little is known about the molecular effectors underlying satellite cell impairment and depletion. Elevated levels of inflammatory cytokines, including interleukin-6 (IL-6), are associated with both age-related and muscle-wasting conditions11–13. The levels of STAT3, a downstream effector of IL-6, are also elevated with muscle wasting14,15, and STAT3 has been implicated in the regulation of self-renewal and stem cell fate in several tissues16–19. Here we show that IL-6–activated Stat3 signaling regulates satellite cell behavior, promoting myogenic lineage progression through myogenic differentiation 1 (Myod1) regulation. Conditional ablation of Stat3 in Pax7-expressing satellite cells resulted in their increased expansion during regeneration, but compromised myogenic differentiation prevented the contribution of these cells to regenerating myofibers. In contrast, transient Stat3 inhibition promoted satellite cell expansion and enhanced tissue repair in both aged and dystrophic muscle. The effects of STAT3 inhibition were conserved in human myoblasts. The results of this study indicate that pharmacological manipulation of STAT3 activity can be used to counteract the functional exhaustion of satellite cells, thereby maintaining the endogenous regenerative response and ameliorating muscle-wasting diseases.
PMCID: PMC4332844  PMID: 25194572
4.  Stem cell aging: mechanisms, regulators and therapeutic opportunities 
Nature medicine  2014;20(8):870-880.
Aging tissues experience a progressive decline in homeostatic and regenerative capacities, which has been attributed to degenerative changes in tissue-specific stem cells, stem cell niches and systemic cues that regulate stem cell activity. Understanding the molecular pathways involved in this age-dependent deterioration of stem cell function will be critical for developing new therapies for diseases of aging that target the specific causes of age-related functional decline. Here we explore key molecular pathways that are commonly perturbed as tissues and stem cells age and degenerate. We further consider experimental evidence both supporting and refuting the notion that modulation of these pathways per se can reverse aging phenotypes. Finally, we ask whether stem cell aging establishes an epigenetic ‘memory’ that is indelibly written or one that can be reset.
PMCID: PMC4160113  PMID: 25100532
5.  The ribonuclease activity of SAMHD1 is required for HIV-1 restriction 
Nature medicine  2014;20(8):936-941.
The HIV-1 restriction factor SAMHD11,2 is proposed to inhibit HIV-1 replication by depleting the intracellular dNTP pool3-5. However, the phosphorylation of SAMHD1 regulates its ability to restrict HIV-1 without decreasing cellular dNTP levels6-8, which is not consistent with a role for SAMHD1 dNTPase activity in HIV-1 restriction. Here, we show that SAMHD1 possesses RNase activity and that the RNase but not the dNTPase function is essential for HIV-1 restriction. By enzymatically characterizing Aicardi-Goutières syndrome (AGS)-associated SAMHD1 mutations and mutations in the allosteric dGTP-binding site of SAMHD1, we identify SAMHD1 mutants that are RNase-positive but dNTPase-negative (SAMHD1D137N) or RNase-negative but dNTPase-positive (SAMHD1Q548A). The allosteric mutant SAMHD1D137N is able to restrict HIV-1 infection, whereas the AGS mutant SAMHD1Q548A is defective for HIV-1 restriction. SAMHD1 associates with HIV-1 RNA and degrades it during the early phases of infection. SAMHD1 silencing in macrophages and CD4+ T cells from healthy donors increases HIV-1 RNA stability, rendering the cells permissive for HIV-1 infection. Furthermore, the phosphorylation of SAMHD1 at T592 negatively regulates its RNase activity in vivo and impedes HIV-1 restriction. Our results reveal that the RNase activity of SAMHD1 is responsible for preventing HIV-1 infection by directly degrading the HIV-1 RNA.
PMCID: PMC4318684  PMID: 25038827
SAMHD1; ribonuclease; HIV-1 restriction factor; HIV-1 genomic RNA; Aicardi-Goutières syndrome
6.  Regulation of the hepatitis C virus RNA replicase by endogenous lipid peroxidation 
Nature medicine  2014;20(8):927-935.
Although oxidative tissue injury often accompanies viral infection, there is little understanding of how it influences virus replication. We show that multiple hepatitis C virus (HCV) genotypes are exquisitely sensitive to oxidative membrane damage, a property distinguishing them from other pathogenic RNA viruses. Lipid peroxidation, regulated in part through sphingosine kinase 2, severely restricts HCV replication in Huh-7 cells and primary human hepatoblasts. Endogenous oxidative membrane damage lowers the 50% effective concentration of direct-acting antivirals, suggesting critical regulation of the conformation of the NS3/4A protease and NS5B polymerase, membrane-bound HCV replicase components. Resistance to lipid peroxidation maps genetically to trans-membrane and membrane-proximal residues within these proteins, and is essential for robust replication in cell culture, as exemplified by the atypical JFH1 strain. Thus, the typical, wild-type HCV replicase is uniquely regulated by lipid peroxidation, providing a novel mechanism for attenuating replication in stressed tissue and possibly facilitating long-term viral persistence.
PMCID: PMC4126843  PMID: 25064127
7.  Asfotase-α improves bone growth, mineralization and strength in mouse models of neurofibromatosis type-1 
Nature medicine  2014;20(8):904-910.
Mineralization of the skeleton depends on the balance between levels of pyrophosphate (PPi), an inhibitor of hydroxyapatite formation, and phosphate generated from PPi breakdown by alkaline phosphatase (ALP). We report here that ablation of Nf1, encoding the RAS/GTPase–activating protein neurofibromin, in bone–forming cells leads to supraphysiologic PPi accumulation, caused by a chronic ERK–dependent increase in genes promoting PPi synthesis and extracellular transport, namely Enpp1 and Ank. It also prevents BMP2–induced osteoprogenitor differentiation and, consequently, expression of ALP and PPi breakdown, further contributing to PPi accumulation. The short stature, impaired bone mineralization and strength in mice lacking Nf1 in osteochondroprogenitors or osteoblasts could be corrected by enzyme therapy aimed at reducing PPi concentration. These results establish neurofibromin as an essential regulator of bone mineralization, suggest that altered PPi homeostasis contributes to the skeletal dysplasiae associated with neurofibromatosis type-1 (NF1), and that some of the NF1 skeletal conditions might be preventable pharmacologically.
PMCID: PMC4126855  PMID: 24997609
bone mineralization; neurofibromin; Neurofibromatosis type I; osteoblast; mesenchymal stem cell; pyrophosphate; Ank; Enpp1/PC1
8.  Intracellular Calcium Regulates Nonsense-Mediated mRNA Decay 
Nature medicine  2014;20(8):961-966.
The nonsense-mediated mRNA decay (NMD) pathway selectively eliminates aberrant transcripts containing premature translation termination codons (PTCs) and regulates the levels of a number of physiological mRNAs. NMD modulates the clinical outcome of a variety of human diseases, including cancer and many genetic disorders, and may represent an important target for therapeutic intervention. Here we have developed a novel multicolored, bioluminescence-based reporter system that can specifically and effectively assay NMD in live human cells. Using this reporter system, we conducted a robust high-throughput small-molecule screen in human cells and, unpredictably, identified a group of cardiac glycosides including ouabain and digoxin as potent inhibitors of NMD. Cardiac glycoside-mediated effects on NMD are dependent on binding and inhibiting the Na+/K+-ATPase on the plasma membrane and subsequent elevation of intracellular calcium levels. Induction of calcium release from endoplasmic reticulum also leads to inhibition of NMD. Thus, this study reveals intracellular calcium as a key regulator of NMD and has important implications for exploiting NMD in the treatment of disease.
PMCID: PMC4126864  PMID: 25064126
11.  An epithelial circadian clock controls pulmonary inflammation and glucocorticoid action 
Nature medicine  2014;20(8):919-926.
The circadian system is as an important regulator of immune function. Human inflammatory lung diseases frequently show time-of-day variation in symptom severity and lung function, but the mechanisms and cell types that are underlying these effects remain unclear. We show that pulmonary antibacterial responses are modulated by a circadian clock within epithelial club (Clara) cells. These drive circadian neutrophil recruitment to the lung via the chemokine CXCL5. Genetic ablation of the clock gene Bmal1 (also called Arntl or MOP3) in bronchiolar cells disrupts rhythmic Cxcl5 expression, resulting in exaggerated inflammatory responses to lipopolysaccharide and bacterial infection. Adrenalectomy blocks rhythmic inflammatory responses and the circadian regulation of CXCL5, suggesting a key role for the adrenal axis in driving CXCL5 expression and pulmonary neutrophil recruitment. Glucocorticoid receptor occupancy at the Cxcl5 locus shows circadian oscillations, but this is disrupted in mice with bronchiole-specific ablation of Bmal1, leading to enhanced CXCL5 expression despite normal corticosteroid secretion. In clock-gene disrupted mice the synthetic glucocorticoid dexamethasone loses anti-inflammatory efficacy. We now define a regulatory mechanism that links the circadian clock and glucocorticoid hormones to control both time-of-day variation and also the magnitude of pulmonary inflammation and responses to bacterial infection.
PMCID: PMC4268501  PMID: 25064128
12.  The Ryanodine Receptor Store Sensing Gate Controls Ca2+ Waves and Ca2+ Triggered Arrhythmias 
Nature medicine  2014;20(2):184-192.
Spontaneous Ca2+ release from intracellular stores is important for various physiological and pathological processes. In cardiac muscle cells, spontaneous store overload-induced Ca2+ release (SOICR) can result in Ca2+ waves, a major cause of ventricular tachyarrhythmias (VTs) and sudden death. The molecular mechanism underlying SOICR has been a mystery for decades. Here, we show that a point mutation E4872A in the helix bundle crossing (the proposed gate) of the cardiac ryanodine receptor (RyR2) completely abolishes luminal, but not cytosolic, RyR2 Ca2+ activation. Introducing metal-binding histidines at this site converts RyR2 into a luminal Ni2+ gated channel. Mouse hearts harboring an RyR2 mutation at this site (E4872Q+/−) are resistant to store overload-induced Ca2+ waves and completely protected against Ca2+-triggered VTs. These data show that the RyR2 gate directly senses store Ca2+, explaining RyR2 store Ca2+ regulation, Ca2+ wave initiation, and Ca2+-triggered arrhythmias. This novel store-sensing gate structure is conserved in all RyRs and inositol 1,4,5-trisphosphate receptors.
PMCID: PMC4269524  PMID: 24441828
13.  The Ashwell-Morell receptor regulates hepatic thrombopoietin production via JAK2-STAT3 signaling 
Nature medicine  2014;21(1):47-54.
The hepatic Ashwell-Morell receptor (AMR) can bind and remove desialylated platelets. We demonstrate that platelets become desialylated as they circulate and age in blood. Binding of desialylated platelets to the AMR induces hepatic thrombopoietin (TPO) gene transcription and translation, thereby regulating platelet production. The highly conserved endocytic AMR signals through Janus kinase 2 (JAK2) and the acute phase response signal transducer and activator of transcription 3 (STAT3) in vivo and in vitro. Recognition of this novel physiological feedback mechanism illuminates the pathophysiology of platelet diseases, such as Essential Thrombocythemia and Immune Thrombocytopenia, and contributes to our understanding of the mechanisms of thrombocytopenia observed with JAK1/2 inhibition.
PMCID: PMC4303234  PMID: 25485912
14.  Interleukin-6 enhances insulin secretion by increasing glucagon-like peptide-1 secretion from L cells and alpha cells 
Nature medicine  2011;17(11):1481-1489.
Exercise, obesity and type 2 diabetes are associated with elevated plasma concentrations of interleukin-6 (IL-6). Glucagon-like peptide-1 (GLP-1) is a hormone that induces insulin secretion. Here we show that administration of IL-6 or elevated IL-6 concentrations in response to exercise stimulate GLP-1 secretion from intestinal L cells and pancreatic alpha cells, improving insulin secretion and glycemia. IL-6 increased GLP-1 production from alpha cells through increased proglucagon (which is encoded by GCG) and prohormone convertase 1/3 expression. In models of type 2 diabetes, the beneficial effects of IL-6 were maintained, and IL-6 neutralization resulted in further elevation of glycemia and reduced pancreatic GLP-1. Hence, IL-6 mediates crosstalk between insulin-sensitive tissues, intestinal L cells and pancreatic islets to adapt to changes in insulin demand. This previously unidentified endocrine loop implicates IL-6 in the regulation of insulin secretion and suggests that drugs modulating this loop may be useful in type 2 diabetes.
PMCID: PMC4286294  PMID: 22037645
15.  Increased gene copy number of the vesicle SNARE VAMP7 disrupts human male urogenital development through altered estrogen action 
Nature medicine  2014;20(7):715-724.
Vesicle transport is intimately connected with key nuclear functions and transcriptional regulation. Here, children born with congenital genitourinary tract masculinization disorders were analyzed by array-Comparative Genomic Hybridization, which revealed the presence of de novo copy number gains on Xq28 encompassing the VAMP7 gene encoding a vesicle-trafficking protein. Humanized VAMP7 BAC transgenic mice displayed cryptorchidism, urethral defects, and hypospadias. Mutant mice exhibited reduced penile length, focal spermatogenic anomalies, diminished sperm motility, and subfertility. VAMP7 colocalized with estrogen receptor alpha (ESR1) in the presence of ligand. Elevated levels of VAMP7 markedly intensified ESR1 transcriptional activity by increasing ESR1 protein cellular content upon ligand stimulation and up-regulated the expression of estrogen-responsive genes including ATF3, CYR61, and CTGF, all of which are implicated in human hypospadias. Hence, increased gene dosage of the SNARE protein, VAMP7, enhances estrogen receptor action in male genitourinary tissues, affects the virilization of the reproductive tract, and results in genitourinary birth defects in humans.
PMCID: PMC4283218  PMID: 24880616
16.  Oncogenic transformation of diverse gastrointestinal tissues in primary organoid culture 
Nature medicine  2014;20(7):769-777.
The application of primary organoid cultures containing epithelial and mesenchymal elements to cancer modeling holds promise for combining the accurate multilineage differentiation and physiology of in vivo systems with the facile in vitro manipulation of transformed cell lines. Here, a single air-liquid interface culture method was used without modification to engineer oncogenic mutations into primary epithelial/mesenchymal organoids from mouse colon, stomach and pancreas. Pancreatic and gastric organoids exhibited dysplasia upon KrasG12D expression and/or p53 loss, and readily generated adenocarcinoma upon in vivo transplantation. In contrast, primary colon organoids required combinatorial Apc, p53, KrasG12D and Smad4 mutations for progressive transformation to invasive adenocarcinoma-like histology in vitro and tumorigenicity in vivo, recapitulating multi-hit models of colorectal cancer (CRC), and versus more promiscuous transformation of small intestinal organoids. Colon organoid culture functionally validated the microRNA miR-483 as a dominant driver oncogene at the Insulin-like growth factor-2 (IGF2) 11p15.5 CRC amplicon, inducing dysplasia in vitro and tumorigenicity in vivo. These studies demonstrate the general utility of a highly tractable primary organoid system for cancer modeling and driver oncogene validation in diverse gastrointestinal tissues.
PMCID: PMC4087144  PMID: 24859528
17.  miR-1202: A Primate Specific and Brain Enriched miRNA Involved in Major Depression and Antidepressant Treatment 
Nature medicine  2014;20(7):764-768.
Major depressive disorder (MDD), is a prevalent mood disorder that associates with differential prefrontal brain expression patterns1. Treatment of MDD includes a variety of biopsychosocial approaches, but in medical practice, antidepressant drugs are the most common treatment for depressive episodes, and not surprisingly, they are among the most prescribed medications in North America2,3. While they are clearly effective, particularly for moderate to severe depressive episodes, there is important variability in how individuals respond to antidepressant treatment. Failure to respond has important individual, economic and social consequences for patients and their families4. Several lines of evidence demonstrate that genes are regulated through the activity of microRNAs (miRNAs), which act as fine–tuners and on–off switches in gene expression patterns5–7. Here we report on complementary studies using postmortem human brain samples, cellular assays and samples from clinical trials of depressed patients, and show that miR-1202, a miRNA specific to primates and enriched in the human brain, is differentially expressed in depressed individuals. Additionally, miR-1202 regulates the expression of the Metabotropic Glutamate Receptor 4 (GRM4) gene and predicts antidepressant response at baseline. These results suggest that miR-1202 is associated with the pathophysiology of depression and is a potential target for novel antidepressant treatments.
PMCID: PMC4087015  PMID: 24908571 CAMSID: cams4401
18.  Altered translation of GATA1 in Diamond-Blackfan anemia 
Nature medicine  2014;20(7):748-753.
Ribosomal protein haploinsufficiency occurs in diverse human diseases including Diamond-Blackfan anemia (DBA),1,2 congenital asplenia,3 and T-cell leukemia.4 Yet how mutations in such ubiquitously expressed proteins result in cell-type and tissue specific defects remains a mystery.5 Here, we show that GATA1 mutations that reduce full-length protein levels of this critical hematopoietic transcription factor can cause DBA in rare instances. We show that ribosomal protein haploinsufficiency, the more common cause of DBA, can similarly reduce translation of GATA1 mRNA - a phenomenon that appears to result from this mRNA having a higher threshold for initiation of translation. In primary hematopoietic cells from patients with RPS19 mutations, a transcriptional signature of GATA1 target genes is globally and specifically reduced, confirming that the activity, but not the mRNA level, of GATA1 is reduced in DBA patients with ribosomal protein mutations. The defective hematopoiesis observed in DBA patients with ribosomal protein haploinsufficiency can be at least partially overcome by increasing GATA1 protein levels. Our results provide a paradigm by which selective defects in translation due to mutations in ubiquitous ribosomal proteins can result in human disease.
PMCID: PMC4087046  PMID: 24952648
19.  Chronic variable stress activates hematopoietic stem cells 
Nature medicine  2014;20(7):754-758.
Exposure to psychosocial stress is a risk factor for many diseases, including atherosclerosis1,2. While incompletely understood, interaction between the psyche and the immune system provides one potential mechanism linking stress and disease inception and progression. Known crosstalk between the brain and immune system includes the hypothalamic–pituitary–adrenal axis, which centrally drives glucocorticoid production in the adrenal cortex, and the sympathetic–adrenal–medullary axis, which controls stress–induced catecholamine release in support of the fight–or–flight reflex3,4. It remains unknown however if chronic stress changes hematopoietic stem cell activity. Here we show that stress increases proliferation of these most primitive progenitors, giving rise to higher levels of disease–promoting inflammatory leukocytes. We found that chronic stress induced monocytosis and neutrophilia in humans. While investigating the source of leukocytosis in mice, we discovered that stress activates upstream hematopoietic stem cells. Sympathetic nerve fibers release surplus noradrenaline, which uses the β3 adrenergic receptor to signal bone marrow niche cells to decrease CXCL12 levels. Consequently, elevated hematopoietic stem cell proliferation increases output of neutrophils and inflammatory monocytes. When atherosclerosis–prone ApoE−/− mice encounter chronic stress, accelerated hematopoiesis promotes plaque features associated with vulnerable lesions that cause myocardial infarction and stroke in humans.
PMCID: PMC4087061  PMID: 24952646
20.  Noninvasive two-photon fluorescence microscopy imaging of mouse retina and RPE through the pupil of the eye 
Nature medicine  2014;20(7):785-789.
Two-photon excitation microscopy (TPM) can image retinal molecular processes in vivo. Intrinsically fluorescent retinyl esters in sub-cellular structures called retinosomes are an integral part of the visual chromophore regeneration pathway. Fluorescent condensation products of all–trans–retinal accumulate in the eye with age and are also associated with age-related macular degeneration (AMD). Here we report repetitive, dynamic imaging of these compounds in live mice, through the pupil of the eye. Leveraging advanced adaptive optics we developed a data acquisition algorithm that permitted the identification of retinosomes and condensation products in the retinal pigment epithelium (RPE) by their characteristic localization, spectral properties, and absence in genetically modified or drug-treated mice. This imaging approach has the potential to detect early molecular changes in retinoid metabolism that trigger light and AMD-induced retinal defects and to assess the effectiveness of treatments for these conditions.
PMCID: PMC4087080  PMID: 24952647
21.  Epigenetic targeting of Hedgehog pathway transcriptional output through BET bromodomain inhibition 
Nature medicine  2014;20(7):732-740.
Hedgehog signaling drives oncogenesis in several cancers and strategies targeting this pathway have been developed, most notably through inhibition of Smoothened. However, resistance to Smoothened inhibitors occurs via genetic changes of Smoothened or other downstream Hedgehog components. Here, we overcome these resistance mechanisms by modulating GLI transcription via inhibition of BET bromodomain proteins. We show the BET bromodomain protein, BRD4, regulates GLI transcription downstream of SMO and SUFU and chromatin immunoprecipitation studies reveal BRD4 directly occupies GLI1 and GLI2 promoters, with a substantial decrease in engagement of these sites upon treatment with JQ1, a small molecule inhibitor targeting BRD4. Globally, genes associated with medulloblastoma-specific GLI1 binding sites are downregulated in response to JQ1 treatment, supporting direct regulation of GLI activity by BRD4. Notably, patient- and GEMM-derived Hedgehog-driven tumors (basal cell carcinoma, medulloblastoma and atypical teratoid/rhabdoid tumor) respond to JQ1 even when harboring genetic lesions rendering them resistant to Smoothened antagonists.
PMCID: PMC4108909  PMID: 24973920
Hedgehog; SHH; medulloblastoma; basal cell carcinoma; ATRT; BRD4; BET bromodomain; GLI1; GLI2; epigenetics; JQ1
22.  Successful Supercooled Liver Storage for 4 Days 
Nature medicine  2014;20(7):790-793.
The realization of long–term human organ preservation will have groundbreaking effects on the current practice of transplantation. Herein we present a novel technique based on sub–zero non–freezing tissue preservation and extracorporeal machine perfusion that allows transplantation of rat livers preserved for up to 4 days, thereby tripling the viable preservation duration.
PMCID: PMC4141719  PMID: 24973919
23.  Mfge8 promotes obesity by mediating the uptake of dietary fats and serum fatty acids 
Nature medicine  2014;20(2):175-183.
Fatty acids are integral mediators of energy storage, membrane formation and cell signaling. The pathways that orchestrate uptake of fatty acids remain incompletely understood. Expression of the integrin ligand Mfge8 is increased in human obesity and in mice on a high-fat diet, but its role in obesity is unknown. We show here that Mfge8 promotes the absorption of dietary triglycerides and the cellular uptake of fatty acid and that Mfge8-deficient (Mfge8−/−) mice are protected from diet-induced obesity, steatohepatitis and insulin resistance. Mechanistically, we found that Mfge8 coordinates fatty acid uptake through αvβ3 integrin– and αvβ5 integrin–dependent phosphorylation of Akt by phosphatidylinositide-3 kinase and mTOR complex 2, leading to translocation of Cd36 and Fatp1 from cytoplasmic vesicles to the cell surface. Collectively, our results imply a role for Mfge8 in regulating the absorption and storage of dietary fats, as well as in the development of obesity and its complications.
PMCID: PMC4273653  PMID: 24441829
24.  A next-generation dual-recombinase system for time and host specific targeting of pancreatic cancer 
Nature medicine  2014;20(11):1340-1347.
Genetically engineered mouse models (GEMMs) have dramatically improved our understanding of tumor evolution and therapeutic resistance. However, sequential genetic manipulation of gene expression and targeting of the host is almost impossible using conventional Cre-loxP–based models. We have developed an inducible dual-recombinase system by combining flippase-FRT (Flp-FRT) and Cre-loxP recombination technologies to improve GEMMs of pancreatic cancer. This enables investigation of multistep carcinogenesis, genetic manipulation of tumor subpopulations (such as cancer stem cells), selective targeting of the tumor microenvironment and genetic validation of therapeutic targets in autochthonous tumors on a genome-wide scale. As a proof of concept, we performed tumor cell–autonomous and nonautonomous targeting, recapitulated hallmarks of human multistep carcinogenesis, validated genetic therapy by 3-phosphoinositide-dependent protein kinase inactivation as well as cancer cell depletion and show that mast cells in the tumor microenvironment, which had been thought to be key oncogenic players, are dispensable for tumor formation.
PMCID: PMC4270133  PMID: 25326799
25.  Randomized dose-finding clinical trial of oncolytic immunotherapeutic vaccinia JX-594 in liver cancer 
Nature medicine  2013;19(3):329-336.
Oncolytic viruses and active immunotherapeutics have complementary mechanisms of action (MOA) that are both self amplifying in tumors, yet the impact of dose on subject outcome is unclear. JX-594 (Pexa-Vec) is an oncolytic and immunotherapeutic vaccinia virus. To determine the optimal JX-594 dose in subjects with advanced hepatocellular carcinoma (HCC), we conducted a randomized phase 2 dose-finding trial (n = 30). Radiologists infused low-or high-dose JX-594 into liver tumors (days 1, 15 and 29); infusions resulted in acute detectable intravascular JX-594 genomes. Objective intrahepatic Modified Response Evaluation Criteria in Solid Tumors (mRECIST) (15%) and Choi (62%) response rates and intrahepatic disease control (50%) were equivalent in injected and distant noninjected tumors at both doses. JX-594 replication and granulocyte-macrophage colony-stimulating factor (GM-CSF) expression preceded the induction of anticancer immunity. In contrast to tumor response rate and immune endpoints, subject survival duration was significantly related to dose (median survival of 14.1 months compared to 6.7 months on the high and low dose, respectively; hazard ratio 0.39; P = 0.020). JX-594 demonstrated oncolytic and immunotherapy MOA, tumor responses and dose-related survival in individuals with HCC.
PMCID: PMC4268543  PMID: 23396206

Results 1-25 (780)