Targeted T cell immunotherapies using engineered T lymphocytes expressing tumor-directed chimeric antigen receptors (CARs) are designed to benefit patients with cancer. Although incorporation of costimulatory endodomains within these CARs increases the proliferation of CAR-redirected T lymphocytes, it has proven difficult to draw definitive conclusions about the specific effects of costimulatory endodomains on the expansion, persistence, and antitumor effectiveness of CAR-redirected T cells in human subjects, owing to the lack of side-by-side comparisons with T cells bearing only a single signaling domain. We therefore designed a study that allowed us to directly measure the consequences of adding a costimulatory endodomain to CAR-redirected T cells. Patients with B cell lymphomas were simultaneously infused with 2 autologous T cell products expressing CARs with the same specificity for the CD19 antigen, present on most B cell malignancies. One CAR encoded both the costimulatory CD28 and the ζ-endodomains, while the other encoded only the ζ-endodomain. CAR+ T cells containing the CD28 endodomain showed strikingly enhanced expansion and persistence compared with CAR+ T cells lacking this endodomain. These results demonstrate the superiority of CARs with dual signal domains and confirm a method of comparing CAR-modified T cells within individual patients, thereby avoiding patient-to-patient variability and accelerating the development of optimal T cell immunotherapies.
Barbara Savoldo, Carlos Almeida Ramos, Enli Liu, Martha P. Mims, Michael J. Keating, George Carrum, Rammurti T. Kamble, Catherine M. Bollard, Adrian P. Gee, Zhuyong Mei, Hao Liu, Bambi Grilley, Cliona M. Rooney, Helen E. Heslop, Malcolm K. Brenner, Gianpietro Dotti
Human cancer cells frequently have regions of their DNA hypermethylated, which results in transcriptional silencing of affected genes and promotion of tumor formation. However, it is still unknown whether cancer-associated aberrant DNA methylation is targeted to specific genomic regions, whether this methylation also occurs in noncancerous cells, and whether these epigenetic events are maintained in the absence of the initiating cause. Here we have addressed some of these issues by demonstrating that transgenic expression of DNA methyltransferase 3b (Dnmt3b) in the mouse colon initiates de novo DNA methylation of genes that are similar to genes that become methylated in human colon cancer. This is consistent with the notion that aberrant methylation in cancer may be attributable to targeting of specific sequences by Dnmt3b rather than to random methylation followed by clonal selection. We also showed that Dnmt3b-induced aberrant DNA methylation was maintained in regenerating tissue, even in the absence of continuous Dnmt3b expression. This supports the concept that transient stressors can cause permanent epigenetic changes in somatic stem cells and that these accumulate over the lifetime of an organism in analogy to DNA mutations.
Eveline J. Steine, Mathias Ehrich, George W. Bell, Arjun Raj, Seshamma Reddy, Alexander van Oudenaarden, Rudolf Jaenisch, Heinz G. Linhart
Vascular-disrupting agents (VDAs) such as combretastatin A4 phosphate (CA4P) selectively disrupt blood vessels in tumors and induce tumor necrosis. However, tumors rapidly repopulate after treatment with such compounds. Here, we show that CA4P-induced vessel narrowing, hypoxia, and hemorrhagic necrosis in murine mammary tumors were accompanied by elevated tumor levels of the chemokine CXCL12 and infiltration by proangiogenic TIE2-expressing macrophages (TEMs). Inhibiting TEM recruitment to CA4P-treated tumors either by interfering pharmacologically with the CXCL12/CXCR4 axis or by genetically depleting TEMs in tumor-bearing mice markedly increased the efficacy of CA4P treatment. These data suggest that TEMs limit VDA-induced tumor injury and represent a potential target for improving the clinical efficacy of VDA-based therapies.
Abigail F. Welford, Daniela Biziato, Seth B. Coffelt, Silvia Nucera, Matthew Fisher, Ferdinando Pucci, Clelia Di Serio, Luigi Naldini, Michele De Palma, Gillian M. Tozer, Claire E. Lewis
Huntington disease (HD) is a degenerative disorder caused by expanded CAG repeats in exon 1 of the huntingtin gene (HTT). Patients with late-stage HD are known to have abnormal auditory processing, but the peripheral auditory functions of HD patients have yet to be thoroughly assessed. In this study, 19 HD patients (aged 40–59 years) were assessed for hearing impairment using pure-tone audiometry and assessment of auditory brainstem responses (ABRs). PTA thresholds were markedly elevated in HD patients. Consistent with this, elevated ABR thresholds were also detected in two mouse models of HD. Hearing loss thus appears to be an authentic symptom of HD. Immunohistochemical analyses demonstrated the presence of mutant huntingtin that formed intranuclear inclusions in the organ of Corti of HD mice, which might interfere with normal auditory function. Quantitative RT-PCR and Western blot analyses further revealed reduced expression of brain creatine kinase (CKB), a major enzyme responsible for ATP regeneration via the phosphocreatine–creatine kinase (PCr-CK) system, in the cochlea of HD mice. Treatment with creatine supplements ameliorated the hearing impairment of HD mice, suggesting that the impaired PCr-CK system in the cochlea of HD mice may contribute to their hearing impairment. These data also suggest that creatine may be useful for treating the hearing abnormalities of patients with HD.
Yow-Sien Lin, Chiung-Mei Chen, Bing-wen Soong, Yih-Ru Wu, Hui-Mei Chen, Wen-Ying Yeh, Dai-Rong Wu, Yi-Jun Lin, Paul Wai-Fung Poon, Mei-Ling Cheng, Chih-Hung Wang, Yijuang Chern
Pilocytic astrocytoma (PA) is the most common type of primary brain tumor in children and the second most frequent cancer in childhood. Children with incompletely resected PA represent a clinically challenging patient cohort for whom conventional adjuvant therapies are only moderately effective. This has produced high clinical demand for testing of new molecularly targeted treatments. However, the development of new therapeutics for PA has been hampered by the lack of an adequate in vivo tumor model. Recent studies have identified activation of MAPK signaling, mainly by oncogenic BRAF activation, as a hallmark genetic event in the pathogenesis of human PA. Using in vivo retroviral somatic gene transfer into mouse neural progenitor cells, we have shown here that ectopic expression of the activated BRAF kinase domain is sufficient to induce PA in mice. Further in vitro analyses demonstrated that overexpression of activated BRAF led to increased proliferation of primary mouse astrocytes that could be inhibited by treatment with the kinase inhibitor sorafenib. Our in vivo model for PA shows that the activated BRAF kinase domain is sufficient to induce PA and highlights its role as a potential therapeutic target.
Jan Gronych, Andrey Korshunov, Josephine Bageritz, Till Milde, Manfred Jugold, Dolores Hambardzumyan, Marc Remke, Christian Hartmann, Hendrik Witt, David T.W. Jones, Olaf Witt, Sabine Heiland, Martin Bendszus, Eric C. Holland, Stefan Pfister, Peter Lichter
Hemolytic transfusion reactions (HTRs) can produce serious and potentially life-threatening complications in sickle cell disease (SCD) patients; however, the mechanisms underlying these complications remain undetermined. We established a model of alloimmune, IgG-mediated HTRs in a well-characterized humanized murine model of SCD. HTRs induced acute vaso-occlusive crisis (VOC), resulting in shortened survival of SCD mice. Acute VOC was associated with elevated circulating inflammatory chemokine levels, including striking elevation of the levels of the neutrophil chemoattractant CXCL1. Recombinant CXCL1 administration was sufficient to induce acute VOC in SCD mice, characterized by leukocyte recruitment in venules, capture of circulating red blood cells, reduction of venular flow, and shortened survival. In contrast, blockade of the CXCL1 receptor, CXCR2, prevented HTR-elicited acute VOC and prolonged survival in SCD mice. These results indicate that CXCL1 is a key inflammatory mediator of acute VOC in SCD mice. Targeted inhibition of CXCL1 and/or CXCR2 may therefore represent a new therapeutic approach for acute VOC in SCD patients.
Jung-Eun Jang, Eldad A. Hod, Steven L. Spitalnik, Paul S. Frenette
Several different neuronal populations are involved in regulating energy homeostasis. Among these, agouti-related protein (AgRP) neurons are thought to promote feeding and weight gain; however, the evidence supporting this view is incomplete. Using designer receptors exclusively activated by designer drugs (DREADD) technology to provide specific and reversible regulation of neuronal activity in mice, we have demonstrated that acute activation of AgRP neurons rapidly and dramatically induces feeding, reduces energy expenditure, and ultimately increases fat stores. All these effects returned to baseline after stimulation was withdrawn. In contrast, inhibiting AgRP neuronal activity in hungry mice reduced food intake. Together, these findings demonstrate that AgRP neuron activity is both necessary and sufficient for feeding. Of interest, activating AgRP neurons potently increased motivation for feeding and also drove intense food-seeking behavior, demonstrating that AgRP neurons engage brain sites controlling multiple levels of feeding behavior. Due to its ease of use and suitability for both acute and chronic regulation, DREADD technology is ideally suited for investigating the neural circuits hypothesized to regulate energy balance.
Michael J. Krashes, Shuichi Koda, ChianPing Ye, Sarah C. Rogan, Andrew C. Adams, Daniel S. Cusher, Eleftheria Maratos-Flier, Bryan L. Roth, Bradford B. Lowell
Human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs) are promising candidate cell sources for regenerative medicine. However, despite the common ability of hiPSCs and hESCs to differentiate into all 3 germ layers, their functional equivalence at the single cell level remains to be demonstrated. Moreover, single cell heterogeneity amongst stem cell populations may underlie important cell fate decisions. Here, we used single cell analysis to resolve the gene expression profiles of 362 hiPSCs and hESCs for an array of 42 genes that characterize the pluripotent and differentiated states. Comparison between single hESCs and single hiPSCs revealed markedly more heterogeneity in gene expression levels in the hiPSCs, suggesting that hiPSCs occupy an alternate, less stable pluripotent state. hiPSCs also displayed slower growth kinetics and impaired directed differentiation as compared with hESCs. Our results suggest that caution should be exercised before assuming that hiPSCs occupy a pluripotent state equivalent to that of hESCs, particularly when producing differentiated cells for regenerative medicine aims.
Kazim H. Narsinh, Ning Sun, Veronica Sanchez-Freire, Andrew S. Lee, Patricia Almeida, Shijun Hu, Taha Jan, Kitchener D. Wilson, Denise Leong, Jarrett Rosenberg, Mylene Yao, Robert C. Robbins, Joseph C. Wu
To be of therapeutic use, autologous stem cells derived from patients with inherited genetic disorders require genetic modification via gene repair or insertion. Here, we present proof of principle that, for diseases associated with dominant alleles (gain-of-function or haploinsufficient loss-of-function), disease allele–free ES cells can be derived from afflicted individuals without genome manipulation. This approach capitalizes on the derivation of uniparental cells, such as parthenogenetic (PG) ES cell lines from disease allele–free gametes. Diploid mammalian uniparental embryos with only maternally (oocyte-) or paternally (sperm-)derived genomes fail early in development due to the nonequivalence of parental genomes caused by genomic imprinting. However, these uniparental embryos develop to the blastocyst stage, allowing the derivation of ES cell lines. Using a mouse model for dominant beta-thalassemia, we developed disease allele–free PG ES cell lines from the oocytes of affected animals. Phenotype correction was obtained in donor-genotype recipients after transplantation of in vitro hematopoietic ES cell derivatives. This genetic correction strategy without gene targeting is potentially applicable to any dominant disease. It could also be the sole approach for larger or more complex mutations that cannot be corrected by homologous recombination.
Sigrid Eckardt, N. Adrian Leu, Ashley Yanchik, Seigo Hatada, Michael Kyba, K. John McLaughlin
Type 1A diabetes (T1D) is an autoimmune disease characterized by leukocyte infiltration of the pancreatic islets of Langerhans. A major impediment to advances in understanding, preventing, and curing T1D has been the inability to “see” the disease initiate, progress, or regress, especially during the occult phase. Here, we report the development of a noninvasive method to visualize T1D at the target organ level in patients with active insulitis. Specifically, we visualized islet inflammation, manifest by microvascular changes and monocyte/macrophage recruitment and activation, using magnetic resonance imaging of magnetic nanoparticles (MNPs). As a proof of principle for this approach, imaging of infused ferumoxtran-10 nanoparticles permitted effective visualization of the pancreas and distinction of recent-onset diabetes patients from nondiabetic controls. The observation that MNPs accumulate in the pancreas of T1D patients opens the door to exploiting this noninvasive imaging method to follow T1D progression and monitoring the ability of immunomodulatory agents to clear insulitis.
Jason L. Gaglia, Alexander R. Guimaraes, Mukesh Harisinghani, Stuart E. Turvey, Richard Jackson, Christophe Benoist, Diane Mathis, Ralph Weissleder
Recent genome-wide association studies have identified a genetic locus at human chromosome 8q24 as having minor alleles associated with lower levels of plasma triglyceride (TG) and LDL cholesterol (LDL-C), higher levels of HDL-C, as well as decreased risk for myocardial infarction. This locus contains only one annotated gene, tribbles homolog 1 (TRIB1), which has not previously been implicated in lipoprotein metabolism. Here we demonstrate a role for Trib1 as a regulator of lipoprotein metabolism in mice. Hepatic-specific overexpression of Trib1 reduced levels of plasma TG and cholesterol by reducing VLDL production; conversely, Trib1-knockout mice showed elevated levels of plasma TG and cholesterol due to increased VLDL production. Hepatic Trib1 expression was inversely associated with the expression of key lipogenic genes and measures of lipogenesis. Thus, we provide functional evidence for what we believe to be a novel gene regulating hepatic lipogenesis and VLDL production in mice that influences plasma lipids and risk for myocardial infarction in humans.
Ralph Burkhardt, Sue-Anne Toh, William R. Lagor, Andrew Birkeland, Michael Levin, Xiaoyu Li, Megan Robblee, Victor D. Fedorov, Masahiro Yamamoto, Takashi Satoh, Shizuo Akira, Sekar Kathiresan, Jan L. Breslow, Daniel J. Rader
Thrombopoiesis, the process by which circulating platelets arise from megakaryocytes, remains incompletely understood. Prior studies suggest that megakaryocytes shed platelets in the pulmonary vasculature. To better understand thrombopoiesis and to develop a potential platelet transfusion strategy that is not dependent upon donors, of which there remains a shortage, we examined whether megakaryocytes infused into mice shed platelets. Infused megakaryocytes led to clinically relevant increases in platelet numbers. The released platelets were normal in size, displayed appropriate surface markers, and had a near-normal circulating half-life. The functionality of the donor-derived platelets was also demonstrated in vivo. The infused megakaryocytes mostly localized to the pulmonary vasculature, where they appeared to shed platelets. These data suggest that it may be unnecessary to generate platelets from ex vivo grown megakaryocytes to achieve clinically relevant increases in platelet numbers.
Rudy Fuentes, Yuhuan Wang, Jessica Hirsch, Cheng Wang, Lubica Rauova, G. Scott Worthen, M. Anna Kowalska, Mortimer Poncz
MicroRNAs inhibit mRNA translation or promote mRNA degradation by binding complementary sequences in 3′ untranslated regions of target mRNAs. MicroRNA-21 (miR-21) is upregulated in response to cardiac stress, and its inhibition by a cholesterol-modified antagomir has been reported to prevent cardiac hypertrophy and fibrosis in rodents in response to pressure overload. In contrast, we have shown here that miR-21–null mice are normal and, in response to a variety of cardiac stresses, display cardiac hypertrophy, fibrosis, upregulation of stress-responsive cardiac genes, and loss of cardiac contractility comparable to wild-type littermates. Similarly, inhibition of miR-21 through intravenous delivery of a locked nucleic acid–modified (LNA-modified) antimiR oligonucleotide also failed to block the remodeling response of the heart to stress. We therefore conclude that miR-21 is not essential for pathological cardiac remodeling.
David M. Patrick, Rusty L. Montgomery, Xiaoxia Qi, Susanna Obad, Sakari Kauppinen, Joseph A. Hill, Eva van Rooij, Eric N. Olson
Accumulating evidence points to inflammation as a promoter of carcinogenesis. MyD88 is an adaptor molecule in TLR and IL-1R signaling that was recently implicated in tumorigenesis through proinflammatory mechanisms. Here we have shown that MyD88 is also required in a cell-autonomous fashion for RAS-mediated carcinogenesis in mice in vivo and for MAPK activation and transformation in vitro. Mechanistically, MyD88 bound to the key MAPK, Erk, and prevented its inactivation by its phosphatase, MKP3, thereby amplifying the activation of the canonical RAS pathway. The relevance of this mechanism to human neoplasia was suggested by the finding that MyD88 was overexpressed and interacted with activated Erk in primary human cancer tissues. Collectively, these results show that in addition to its role in inflammation, MyD88 plays what we believe to be a crucial direct role in RAS signaling, cell-cycle control, and cell transformation.
Isabelle Coste, Katy Le Corf, Alain Kfoury, Isabelle Hmitou, Sabine Druillennec, Pierre Hainaut, Alain Eychene, Serge Lebecque, Toufic Renno
Patients with Kallmann syndrome (KS) have hypogonadotropic hypogonadism caused by a deficiency of gonadotropin-releasing hormone (GnRH) and a defective sense of smell related to olfactory bulb aplasia. Based on the findings in a fetus affected by the X chromosome–linked form of the disease, it has been suggested that hypogonadism in KS results from the failed embryonic migration of neuroendocrine GnRH1 cells from the nasal epithelium to the forebrain. We asked whether this singular observation might extend to other developmental disorders that also include arrhinencephaly. We therefore studied the location of GnRH1 cells in fetuses affected by different arrhinencephalic disorders, specifically X-linked KS, CHARGE syndrome, trisomy 13, and trisomy 18, using immunohistochemistry. Few or no neuroendocrine GnRH1 cells were detected in the preoptic and hypothalamic regions of all arrhinencephalic fetuses, whereas large numbers of these cells were present in control fetuses. In all arrhinencephalic fetuses, many GnRH1 cells were present in the frontonasal region, the first part of their migratory path, as were interrupted olfactory nerve fibers that formed bilateral neuromas. Our findings define a pathological sequence whereby a lack of migration of neuroendocrine GnRH cells stems from the primary embryonic failure of peripheral olfactory structures. This can occur either alone, as in isolated KS, or as part of a pleiotropic disease, such as CHARGE syndrome, trisomy 13, and trisomy 18.
Luis Teixeira, Fabien Guimiot, Catherine Dodé, Catherine Fallet-Bianco, Robert P. Millar, Anne-Lise Delezoide, Jean-Pierre Hardelin
HDL has anti-atherogenic properties, and plasma levels of HDL cholesterol correlate inversely with risk of coronary artery disease. HDL reportedly functions as a cofactor to the anticoagulant activated protein C (APC) in the degradation of factor Va (FVa). The aim of the present study was to elucidate the mechanism by which HDL functions as cofactor to APC. Consistent with a previous report, HDL isolated from human plasma by ultracentrifugation was found to stimulate APC-mediated degradation of FVa. However, further purification of HDL by gel filtration revealed that the stimulating activity was not a property of HDL. Instead, the stimulating activity eluted completely separately from HDL in the high-molecular-weight void volume fractions. The active portion of these fractions stimulated FVa degradation by APC and supported the assembly of factor Xa and FVa into a functional prothrombinase complex. Both the procoagulant and anticoagulant activities were blocked by addition of annexin V, suggesting that the active portion was negatively charged phospholipid membranes. These results demonstrate that HDL does not stimulate the APC/protein S effect and that the activity previously reported to be a property of HDL is instead caused by contaminating negatively charged phospholipid membranes.
Cecilia Oslakovic, Eva Norstrøm, Björn Dahlbäck
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