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Abstract
Increases in the number of cell therapies in the preclinical and clinical phases have prompted the need for reliable and noninvasive assays to validate transplant function in clinical biomanufacturing. We developed a robust characterization methodology composed of quantitative bright-field absorbance microscopy (QBAM) and deep neural networks (DNNs) to noninvasively predict tissue function and cellular donor identity. The methodology was validated using clinical-grade induced pluripotent stem cell–derived retinal pigment epithelial cells (iPSC-RPE). QBAM images of iPSC-RPE were used to train DNNs that predicted iPSC-RPE monolayer transepithelial resistance, predicted polarized vascular endothelial growth factor (VEGF) secretion, and matched iPSC-RPE monolayers to the stem cell donors. DNN predictions were supplemented with traditional machine-learning algorithms that identified shape and texture features of single cells that were used to predict tissue function and iPSC donor identity. These results demonstrate noninvasive cell therapy characterization can be achieved with QBAM and machine learning.
Authors
Nicholas J. Schaub, Nathan A. Hotaling, Petre Manescu, Sarala Padi, Qin Wan, Ruchi Sharma, Aman George, Joe Chalfoun, Mylene Simon, Mohamed Ouladi, Carl G. Simon Jr., Peter Bajcsy, Kapil Bharti
Abstract
Hereditary hemorrhagic telangiectasia (HHT), a genetic bleeding disorder leading to systemic arteriovenous malformations (AVMs), is caused by loss-of-function mutations in the ALK1/ENG/Smad1/5/8 pathway. Evidence suggests that HHT pathogenesis strongly relies on overactivated PI3K/Akt/mTOR and VEGFR2 pathways in endothelial cells (ECs). In the BMP9/10-immunoblocked (BMP9/10ib) neonatal mouse model of HHT, we report here that the mTOR inhibitor, sirolimus, and the receptor tyrosine kinase inhibitor, nintedanib, could synergistically fully block, but also reversed, retinal AVMs to avert retinal bleeding and anemia. Sirolimus plus nintedanib prevented vascular pathology in the oral mucosa, lungs, and liver of the BMP9/10ib mice, as well as significantly reduced gastrointestinal bleeding and anemia in inducible ALK1-deficient adult mice. Mechanistically, in vivo in BMP9/10ib mouse ECs, sirolimus and nintedanib blocked the overactivation of mTOR and VEGFR2, respectively. Furthermore, we found that sirolimus activated ALK2-mediated Smad1/5/8 signaling in primary ECs — including in HHT patient blood outgrowth ECs — and partially rescued Smad1/5/8 activity in vivo in BMP9/10ib mouse ECs. These data demonstrate that the combined correction of endothelial Smad1/5/8, mTOR, and VEGFR2 pathways opposes HHT pathogenesis. Repurposing of sirolimus plus nintedanib might provide therapeutic benefit in patients with HHT.
Authors
Santiago Ruiz, Haitian Zhao, Pallavi Chandakkar, Julien Papoin, Hyunwoo Choi, Aya Nomura-Kitabayashi, Radhika Patel, Matthew Gillen, Li Diao, Prodyot K. Chatterjee, Mingzhu He, Yousef Al-Abed, Ping Wang, Christine N. Metz, S. Paul Oh, Lionel Blanc, Fabien Campagne, Philippe Marambaud
Abstract
Cancer immune evasion is achieved through multiple layers of immune tolerance mechanisms including immune editing, recruitment of tolerogenic immune cells, and secretion of immunosuppressive cytokines. Recent success with immune checkpoint inhibitors in cancer immunotherapy suggests a dysfunctional immune synapse as a pivotal tolerogenic mechanism. Tumor cells express immune synapse proteins to suppress the immune system, which is often modulated by epigenetic mechanisms. When the methylation status of key immune synapse genes was interrogated, we observed disproportionately hypermethylated costimulatory genes and hypomethylation of immune checkpoint genes, which were negatively associated with functional T cell recruitment to the tumor microenvironment. Therefore, the methylation status of immune synapse genes reflects tumor immunogenicity and correlates with survival.
Authors
Anders Berglund, Matthew Mills, Ryan M. Putney, Imène Hamaidi, James Mulé, Sungjune Kim
Abstract
The protein-protein interaction between menin and mixed lineage leukemia 1 (MLL1) plays a critical role in acute leukemias with translocations of the MLL1 gene or with mutations in the nucleophosmin 1 (NPM1) gene. As a step toward clinical translation of menin-MLL1 inhibitors, we report development of MI-3454, a highly potent and orally bioavailable inhibitor of the menin-MLL1 interaction. MI-3454 profoundly inhibited proliferation and induced differentiation in acute leukemia cells and primary patient samples with MLL1 translocations or NPM1 mutations. When applied as a single agent, MI-3454 induced complete remission or regression of leukemia in mouse models of MLL1-rearranged or NPM1-mutated leukemia, including patient-derived xenograft models, through downregulation of key genes involved in leukemogenesis. We also identified MEIS1 as a potential pharmacodynamic biomarker of treatment response with MI-3454 in leukemia, and demonstrated that this compound is well tolerated and did not impair normal hematopoiesis in mice. Overall, this study demonstrates, for the first time to our knowledge, profound activity of the menin-MLL1 inhibitor as a single agent in clinically relevant PDX models of leukemia. These data provide a strong rationale for clinical translation of MI-3454 or its analogs for leukemia patients with MLL1 rearrangements or NPM1 mutations.
Authors
Szymon Klossowski, Hongzhi Miao, Katarzyna Kempinska, Tao Wu, Trupta Purohit, EunGi Kim, Brian M. Linhares, Dong Chen, Gloria Jih, Eric Perkey, Huang Huang, Miao He, Bo Wen, Yi Wang, Ke Yu, Stanley Chun-Wei Lee, Gwenn Danet-Desnoyers, Winifred Trotman, Malathi Kandarpa, Anitria Cotton, Omar Abdel-Wahab, Hongwei Lei, Yali Dou, Monica Guzman, Luke Peterson, Tanja Gruber, Sarah Choi, Duxin Sun, Pingda Ren, Lian-Sheng Li, Yi Liu, Francis Burrows, Ivan Maillard, Tomasz Cierpicki, Jolanta Grembecka
Abstract
Leptomeningeal anastomoses or pial collateral vessels play a critical role in cerebral blood flow (CBF) restoration following ischemic stroke. The magnitude of this adaptive response is postulated to be controlled by the endothelium, although the underlying molecular mechanisms remain under investigation. Here we demonstrated that endothelial genetic deletion, using EphA4fl/fl/Tie2-Cre and EphA4fl/fl/VeCahderin-CreERT2 mice and vessel painting strategies, implicated EphA4 receptor tyrosine kinase as a major suppressor of pial collateral remodeling, CBF, and functional recovery following permanent middle cerebral artery occlusion. Pial collateral remodeling is limited by the crosstalk between EphA4-Tie2 signaling in vascular endothelial cells, which is mediated through p-Akt regulation. Furthermore, peptide inhibition of EphA4 resulted in acceleration of the pial arteriogenic response. Our findings demonstrate that EphA4 is a negative regulator of Tie2 receptor signaling, which limits pial collateral arteriogenesis following cerebrovascular occlusion. Therapeutic targeting of EphA4 and/or Tie2 represents an attractive new strategy for improving collateral function, neural tissue health, and functional recovery following ischemic stroke.
Authors
Benjamin Okyere, William A. Mills III, Xia Wang, Michael Chen, Jiang Chen, Amanda Hazy, Yun Qian, John B. Matson, Michelle H. Theus
Abstract
BACKGROUND Chronic hepatitis C virus (HCV) infection is characterized by a severe impairment of HCV-specific CD4+ T cell help that is driven by chronic antigen stimulation. We aimed to study the fate of HCV-specific CD4+ T cells after virus elimination.METHODS HCV-specific CD4+ T cell responses were longitudinally analyzed using MHC class II tetramer technology, multicolor flow cytometry, and RNA sequencing in a cohort of patients chronically infected with HCV undergoing therapy with direct-acting antivirals. In addition, HCV-specific neutralizing antibodies and CXCL13 levels were analyzed.RESULTS We observed that the frequency of HCV-specific CD4+ T cells increased within 2 weeks after initiating direct-acting antiviral therapy. Multicolor flow cytometry revealed a downregulation of exhaustion and activation markers and an upregulation of memory-associated markers. Although cells with a Th1 phenotype were the predominant subset at baseline, cells with phenotypic and transcriptional characteristics of follicular T helper cells increasingly shaped the circulating HCV-specific CD4+ T cell repertoire, suggesting antigen-independent survival of this subset. These changes were accompanied by a decline of HCV-specific neutralizing antibodies and the germinal center activity.CONCLUSION We identified a population of HCV-specific CD4+ T cells with a follicular T helper cell signature that is maintained after therapy-induced elimination of persistent infection and may constitute an important target population for vaccination efforts to prevent reinfection and immunotherapeutic approaches for persistent viral infections.FUNDING Deutsche Forschungsgemeinschaft (DFG, German Research Foundation), the National Institute of Allergy and Infectious Diseases (NIAID), the European Union, the Berta-Ottenstein-Programme for Advanced Clinician Scientists, and the ANRS.
Authors
Maike Smits, Katharina Zoldan, Naveed Ishaque, Zuguang Gu, Katharina Jechow, Dominik Wieland, Christian Conrad, Roland Eils, Catherine Fauvelle, Thomas F. Baumert, Florian Emmerich, Bertram Bengsch, Christoph Neumann-Haefelin, Maike Hofmann, Robert Thimme, Tobias Boettler
Abstract
Signaling by the TGF-β superfamily is important in the regulation of hematopoiesis and is dysregulated in myelodysplastic syndromes (MDSs), contributing to ineffective hematopoiesis and clinical cytopenias. TGF-β, activins, and growth differentiation factors exert inhibitory effects on red cell formation by activating canonical SMAD2/3 pathway signaling. In this Review, we summarize evidence that overactivation of SMAD2/3 signaling pathways in MDSs causes anemia due to impaired erythroid maturation. We also describe the basis for biological activity of activin receptor ligand traps, novel fusion proteins such as luspatercept that are promising as erythroid maturation agents to alleviate anemia and related comorbidities in MDSs and other conditions characterized by impaired erythroid maturation.
Authors
Amit Verma, Rajasekhar N.V.S. Suragani, Srinivas Aluri, Nishi Shah, Tushar D. Bhagat, Mark J. Alexander, Rami Komrokji, Ravi Kumar
Abstract
The advent of human induced pluripotent stem cells (iPSCs) provided a means for avoiding ethical concerns associated with the use of cells isolated from human embryos. The number of labs now using iPSCs to generate photoreceptor, retinal pigmented epithelial (RPE), and more recently choroidal endothelial cells has grown exponentially. However, for autologous cell replacement to be effective, manufacturing strategies will need to change. Many tasks carried out by hand will need simplifying and automating. In this issue of the JCI, Schaub and colleagues combined quantitative brightfield microscopy and artificial intelligence (deep neural networks and traditional machine learning) to noninvasively monitor iPSC-derived graft maturation, predict donor cell identity, and evaluate graft function prior to transplantation. This approach allowed the authors to preemptively identify and remove abnormal grafts. Notably, the method is (a) transferable, (b) cost- and time effective, (c) high throughput, and (d) useful for primary product validation.
Authors
Budd A. Tucker, Robert F. Mullins, Edwin M. Stone
Abstract
Increased rates of locoregional recurrence are observed in patients with basal-like breast cancer (BC) despite the use of radiation therapy (RT); therefore, approaches that result in radiosensitization of basal-like BC are critically needed. Using patients’ tumor gene expression data from 4 independent data sets, we correlated gene expression with recurrence to find genes significantly correlated with early recurrence after RT. The highest-ranked gene, TTK, was most highly expressed in basal-like BC across multiple data sets. Inhibition of TTK by both genetic and pharmacologic methods enhanced radiosensitivity in multiple basal-like cell lines. Radiosensitivity was mediated, at least in part, through persistent DNA damage after treatment with TTK inhibition and RT. Inhibition of TTK impaired homologous recombination (HR) and repair efficiency, but not nonhomologous end-joining, and decreased the formation of Rad51 foci. Reintroduction of wild-type TTK rescued both radioresistance and HR repair efficiency after TTK knockdown, however, reintroduction of kinase-dead TTK did not. In vivo, TTK inhibition combined with RT led to a significant decrease in tumor growth in both heterotopic and orthotopic, including patient-derived xenograft, BC models. These data support the rationale for clinical development of TTK inhibition as a radiosensitizing strategy for patients with basal-like BC, and efforts toward this end are currently underway.
Authors
Benjamin C. Chandler, Leah Moubadder, Cassandra L. Ritter, Meilan Liu, Meleah Cameron, Kari Wilder-Romans, Amanda Zhang, Andrea M. Pesch, Anna R. Michmerhuizen, Nicole Hirsh, Marlie Androsiglio, Tanner Ward, Eric Olsen, Yashar S. Niknafs, Sofia Merajver, Dafydd G. Thomas, Powel H. Brown, Theodore S. Lawrence, Shyam Nyati, Lori J. Pierce, Arul Chinnaiyan, Corey Speers
Abstract
Antigen receptor–dependent (AgR-dependent) stimulation of the NF-κB transcription factor in lymphocytes is a required event during adaptive immune response, but dysregulated activation of this signaling pathway can lead to lymphoma. AgR stimulation promotes assembly of the CARMA1-BCL10-MALT1 complex, wherein MALT1 acts as (a) a scaffold to recruit components of the canonical NF-κB machinery, and (b) a protease to cleave and inactivate specific substrates, including negative regulators of NF-κB. In multiple lymphoma subtypes, malignant B cells hijack AgR signaling pathways to promote their own growth and survival, and inhibiting MALT1 reduces the viability and growth of these tumors. As such, MALT1 has emerged as a potential pharmaceutical target. Here, we identified G protein–coupled receptor kinase 2 (GRK2) as a new MALT1-interacting protein. We demonstrated that GRK2 binds the death domain of MALT1 and inhibits MALT1 scaffolding and proteolytic activities. We found that lower GRK2 levels in activated B cell–type diffuse large B cell lymphoma (ABC-DLBCL) are associated with reduced survival, and that GRK2 knockdown enhances ABC-DLBCL tumor growth in vitro and in vivo. Together, our findings suggest that GRK2 can function as a tumor suppressor by inhibiting MALT1 and provide a roadmap for developing new strategies to inhibit MALT1-dependent lymphomagenesis.
Authors
Jing Cheng, Linda R. Klei, Nathaniel E. Hubel, Ming Zhang, Rebekka Schairer, Lisa M. Maurer, Hanna B. Klei, Heejae Kang, Vincent J. Concel, Phillip C. Delekta, Eric V. Dang, Michelle A. Mintz, Mathijs Baens, Jason G. Cyster, Narayanan Parameswaran, Margot Thome, Peter C. Lucas, Linda M. McAllister-Lucas
Abstract
The corneocyte lipid envelope, composed of covalently bound ceramides and fatty acids, is important to the integrity of the permeability barrier in the stratum corneum, and its absence is a prime structural defect in various skin diseases associated with defective skin barrier function. SDR9C7 encodes a short-chain dehydrogenase/reductase family 9C member 7 (SDR9C7) recently found mutated in ichthyosis. In a patient with SDR9C7 mutation and a mouse Sdr9c7 knockout model, we show loss of covalent binding of epidermal ceramides to protein, a structural fault in the barrier. For reasons unresolved, protein binding requires lipoxygenase-catalyzed transformations of linoleic acid (18:2) esterified in ω-O-acylceramides. In Sdr9c7–/– epidermis, quantitative liquid chromatography–mass spectometry (LC-MS) assays revealed almost complete loss of a species of ω-O-acylceramide esterified with linoleate-9,10-trans-epoxy-11E-13-ketone; other acylceramides related to the lipoxygenase pathway were in higher abundance. Recombinant SDR9C7 catalyzed NAD+-dependent dehydrogenation of linoleate 9,10-trans-epoxy-11E-13-alcohol to the corresponding 13-ketone, while ichthyosis mutants were inactive. We propose, therefore, that the critical requirement for lipoxygenases and SDR9C7 is in producing acylceramide containing the 9,10-epoxy-11E-13-ketone, a reactive moiety known for its nonenzymatic coupling to protein. This suggests a mechanism for coupling of ceramide to protein and provides important insights into skin barrier formation and pathogenesis.
Authors
Takuya Takeichi, Tetsuya Hirabayashi, Yuki Miyasaka, Akane Kawamoto, Yusuke Okuno, Shijima Taguchi, Kana Tanahashi, Chiaki Murase, Hiroyuki Takama, Kosei Tanaka, William E. Boeglin, M. Wade Calcutt, Daisuke Watanabe, Michihiro Kono, Yoshinao Muro, Junko Ishikawa, Tamio Ohno, Alan R. Brash, Masashi Akiyama
Abstract
Pattern recognition receptors (PRRs) are crucial for responses to infections and tissue damage; however, their role in autoimmunity is less clear. Herein we demonstrate that 2 C-type lectin receptors (CLRs), Mcl and Mincle, play an important role in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). Congenic rats expressing lower levels of Mcl and Mincle on myeloid cells exhibited a drastic reduction in EAE incidence. In vivo silencing of Mcl and Mincle or blockade of their endogenous ligand SAP130 revealed that these receptors’ expression in the central nervous system is crucial for T cell recruitment and reactivation into a pathogenic Th17/GM-CSF phenotype. Consistent with this, we uncovered MCL- and MINCLE-expressing cells in brain lesions of MS patients and we further found an upregulation of the MCL/MINCLE signaling pathway and an increased response following MCL/MINCLE stimulation in peripheral blood mononuclear cells from MS patients. Together, these data support a role for CLRs in autoimmunity and implicate the MCL/MINCLE pathway as a potential therapeutic target in MS.
Authors
Marie N’diaye, Susanna Brauner, Sevasti Flytzani, Lara Kular, Andreas Warnecke, Milena Z. Adzemovic, Eliane Piket, Jin-Hong Min, Will Edwards, Filia Mela, Hoi Ying Choi, Vera Magg, Tojo James, Magdalena Linden, Holger M. Reichardt, Michael R. Daws, Jack van Horssen, Ingrid Kockum, Robert A. Harris, Tomas Olsson, Andre O. Guerreiro-Cacais, Maja Jagodic
Abstract
Successful infection by mucosal pathogens requires overcoming the mucus barrier. To better understand this key step, we performed a survey of the interactions between human respiratory mucus and the human pathogen Streptococcus pneumoniae. Pneumococcal adherence to adult human nasal fluid was seen only by isolates expressing pilus-1. Robust binding was independent of pilus-1 adhesive properties but required Fab-dependent recognition of RrgB, the pilus shaft protein, by naturally acquired secretory IgA (sIgA). Pilus-1 binding by specific sIgA led to bacterial agglutination, but adherence required interaction of agglutinated pneumococci and entrapment in mucus particles. To test the effect of these interactions in vivo, pneumococci were preincubated with human sIgA before intranasal challenge in a mouse model of colonization. sIgA treatment resulted in rapid immune exclusion of pilus-expressing pneumococci. Our findings predict that immune exclusion would select for nonpiliated isolates in individuals who acquired RrgB-specific sIgA from prior episodes of colonization with piliated strains. Accordingly, genomic data comparing isolates carried by mothers and their children showed that mothers are less likely to be colonized with pilus-expressing strains. Our study provides a specific example of immune exclusion involving naturally acquired antibody in the human host, a major factor driving pneumococcal adaptation.
Authors
Ulrike Binsker, John A. Lees, Alexandria J. Hammond, Jeffrey N. Weiser
Abstract
Glucocorticoids (GCs) are a central component of therapy for patients with T cell acute lymphoblastic leukemia (T-ALL), and although resistance to GCs is a strong negative prognostic indicator in T-ALL, the mechanisms of GC resistance remain poorly understood. Using diagnostic samples from patients enrolled in the frontline Children’s Oncology Group (COG) T-ALL clinical trial AALL1231, we demonstrated that one-third of primary T-ALLs were resistant to GCs when cells were cultured in the presence of IL-7, a cytokine that is critical for normal T cell function and that plays a well-established role in leukemogenesis. We demonstrated that in these T-ALLs and in distinct populations of normal developing thymocytes, GCs paradoxically induced their own resistance by promoting upregulation of IL-7 receptor (IL-7R) expression. In the presence of IL-7, this augmented downstream signal transduction, resulting in increased STAT5 transcriptional output and upregulation of the prosurvival protein BCL-2. Taken together, we showed that IL-7 mediates an intrinsic and physiologic mechanism of GC resistance in normal thymocyte development that is retained during leukemogenesis in a subset of T-ALLs and is reversible with targeted inhibition of the IL-7R/JAK/STAT5/BCL-2 axis.
Authors
Lauren K. Meyer, Benjamin J. Huang, Cristina Delgado-Martin, Ritu P. Roy, Aaron Hechmer, Anica M. Wandler, Tiffaney L. Vincent, Paolo Fortina, Adam B. Olshen, Brent L. Wood, Terzah M. Horton, Kevin M. Shannon, David T. Teachey, Michelle L. Hermiston
Abstract
Parkinson’s disease (PD) is a neurodegenerative disorder associated with loss of striatal dopamine, secondary to degeneration of midbrain dopamine (mDA) neurons in the substantia nigra, rendering cell transplantation a promising therapeutic strategy. To establish human induced pluripotent stem cell–based (hiPSC-based) autologous cell therapy, we report a platform of core techniques for the production of mDA progenitors as a safe and effective therapeutic product. First, by combining metabolism-regulating microRNAs with reprogramming factors, we developed a method to more efficiently generate clinical grade iPSCs, as evidenced by genomic integrity and unbiased pluripotent potential. Second, we established a “spotting”-based in vitro differentiation methodology to generate functional and healthy mDA cells in a scalable manner. Third, we developed a chemical method that safely eliminates undifferentiated cells from the final product. Dopaminergic cells thus produced express high levels of characteristic mDA markers, produce and secrete dopamine, and exhibit electrophysiological features typical of mDA cells. Transplantation of these cells into rodent models of PD robustly restores motor function and reinnervates host brain, while showing no evidence of tumor formation or redistribution of the implanted cells. We propose that this platform is suitable for the successful implementation of human personalized autologous cell therapy for PD.
Authors
Bin Song, Young Cha, Sanghyeok Ko, Jeha Jeon, Nayeon Lee, Hyemyung Seo, Kyung-Joon Park, In-Hee Lee, Claudia Lopes, Melissa Feitosa, María José Luna, Jin Hyuk Jung, Jisun Kim, Dabin Hwang, Bruce M. Cohen, Martin H. Teicher, Pierre Leblanc, Bob S. Carter, Jeffrey H. Kordower, Vadim Y. Bolshakov, Sek Won Kong, Jeffrey S. Schweitzer, Kwang-Soo Kim
Abstract
Oncogene-targeted and immune checkpoint therapies have revolutionized the clinical management of malignant melanoma and now offer hope to patients with advanced disease. Intimately connected to patients’ overall clinical risk is whether the initial primary melanoma lesion will metastasize and cause advanced disease, but underlying mechanisms are not entirely understood. A subset of melanomas display heightened peroxisome proliferator–activated receptor γ coactivator 1-α (PGC1α) expression that maintains cell survival cues by promoting mitochondrial function, but also suppresses metastatic spread. Here, we show that PGC1α expression in melanoma cells was silenced by chromatin modifications that involve promoter H3K27 trimethylation. Pharmacological EZH2 inhibition diminished H3K27me3 histone markers, increased PGC1α expression, and functionally suppressed invasion within PGC1α-silenced melanoma cells. Mechanistically, PGC1α silencing activated transcription factor 12 (TCF12), to increase expression of WNT5A, which in turn stabilized YAP protein levels to promote melanoma migration and metastasis. Accordingly, inhibition of components of this transcription-signaling axis, including TCF12, WNT5A, or YAP, blocked melanoma migration in vitro and metastasis in vivo. These results indicate that epigenetic control of melanoma metastasis involved altered expression of PGC1α and an association with the inherent metabolic state of the tumor.
Authors
Chi Luo, Eduardo Balsa, Elizabeth A. Perry, Jiaxin Liang, Clint D. Tavares, Francisca Vazquez, Hans R. Widlund, Pere Puigserver
Abstract
Authors
Susan E. Pacheco
Abstract
Technological advances in rapid data acquisition have transformed medical biology into a data mining field, where new data sets are routinely dissected and analyzed by statistical models of ever-increasing complexity. Many hypotheses can be generated and tested within a single large data set, and even small effects can be statistically discriminated from a sea of noise. On the other hand, the development of therapeutic interventions moves at a much slower pace. They are determined from carefully randomized and well-controlled experiments with explicitly stated outcomes as the principal mechanism by which a single hypothesis is tested. In this paradigm, only a small fraction of interventions can be tested, and an even smaller fraction are ultimately deemed therapeutically successful. In this Review, we propose strategies to leverage large-cohort data to inform the selection of targets and the design of randomized trials of novel therapeutics. Ultimately, the incorporation of big data and experimental medicine approaches should aim to reduce the failure rate of clinical trials as well as expedite and lower the cost of drug development.
Authors
Eugene Melamud, D. Leland Taylor, Anurag Sethi, Madeleine Cule, Anastasia Baryshnikova, Danish Saleheen, Nick van Bruggen, Garret A. FitzGerald
Abstract
Posttraumatic stress disorder (PTSD) can develop after exposure to severe psychological trauma, leaving patients with disabling anxiety, nightmares, and flashbacks. Current treatments are only partially effective, and development of better treatments is hampered by limited knowledge of molecular mechanisms underlying PTSD. We have discovered that the glucocorticoid receptor (GR) and FK506 binding protein 51 (FKBP51) form a protein complex that is elevated in PTSD patients compared with unaffected control subjects, subjects exposed to trauma without PTSD, and patients with major depressive disorder (MDD). The GR-FKBP51 complex is also elevated in fear-conditioned mice, an aversive learning paradigm that models some aspects of PTSD. Both PTSD patients and fear-conditioned mice had decreased GR phosphorylation, decreased nuclear GR, and lower expression of 14-3-3ε, a gene regulated by GR. We created a peptide that disrupts GR-FKBP51 binding and reverses behavioral and molecular changes induced by fear conditioning. This peptide reduces freezing time and increases GR phosphorylation, GR-FKBP52 binding, GR nuclear translocation, and 14-3-3ε expression in fear-conditioned mice. These experiments demonstrate a molecular mechanism contributing to PTSD and suggest that the GR-FKBP51 complex may be a diagnostic biomarker and a potential therapeutic target for preventing or treating PTSD.
Authors
Haiyin Li, Ping Su, Terence K.Y. Lai, Anlong Jiang, Jing Liu, Dongxu Zhai, Charlie T.G. Campbell, Frankie H.F. Lee, WeiDong Yong, Suvercha Pasricha, Shupeng Li, Albert H.C. Wong, Kerry J. Ressler, Fang Liu
Abstract
Risk for childhood asthma is conferred by alleles within the 17q21 locus affecting ORMDL sphingolipid biosynthesis regulator 3 (ORMDL3) expression. ORMDL3 inhibits sphingolipid de novo synthesis. Although the effects of 17q21 genotypes on sphingolipid synthesis in human asthma remain unclear, both decreased sphingolipid synthesis and ORMDL3 overexpression are linked to airway hyperreactivity. To characterize the relationship of genetic asthma susceptibility with sphingolipid synthesis, we analyzed asthma-associated 17q21 genotypes (rs7216389, rs8076131, rs4065275, rs12603332, and rs8067378) in both children with asthma and those without asthma, quantified plasma and whole-blood sphingolipids, and assessed sphingolipid de novo synthesis in peripheral blood cells by measuring the incorporation of stable isotope–labeled serine (substrate) into sphinganine and sphinganine-1-phosphate. Whole-blood dihydroceramides and ceramides were decreased in subjects with the 17q21 asthma–risk alleles rs7216389 and rs8076131. Children with nonallergic asthma had lower dihydroceramides, ceramides, and sphingomyelins than did controls. Children with allergic asthma had higher dihydroceramides, ceramides, and sphingomyelins compared with children with nonallergic asthma. Additionally, de novo sphingolipid synthesis was lower in children with asthma compared with controls. These findings connect genetic 17q21 variations that are associated with asthma risk and higher ORMDL3 expression to lower sphingolipid synthesis in humans. Altered sphingolipid synthesis may therefore be a critical factor in asthma pathogenesis and may guide the development of future therapeutics.
Authors
Jennie G. Ono, Benjamin I. Kim, Yize Zhao, Paul J. Christos, Yohannes Tesfaigzi, Tilla S. Worgall, Stefan Worgall
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Abstract
Despite the effective clinical use of steroids for the treatment of Diamond Blackfan anemia (DBA), the mechanistic bases via which glucocorticoids regulate human erythropoiesis remain poorly understood. Here, we report that the sensitivity of erythroid differentiation to dexamethasone (Dex) is dependent on the developmental origin of human CD34+ progenitor cells, specifically increasing the expansion of CD34+ progenitors from peripheral blood (PB) but not cord blood (CB). Dexamethasone treatment of erythroid-differentiated PB, but not CB, CD34+ progenitors resulted in the expansion of a novel CD34+CD36+CD71hiCD105med immature colony-forming unit-erythroid (CFU-E) population. Furthermore, proteomics analyses revealed the induction of distinct proteins in dexamethasone-treated PB and CB erythroid progenitors. Dexamethasone treatment of PB progenitors resulted in the specific upregulation of p57Kip2, a Cip/Kip cyclin-dependent kinase inhibitor, and we identified this induction as critical; shRNA-mediated downregulation of p57Kip2, but not the related p27Kip1, significantly attenuated the impact of dexamethasone on erythroid differentiation and inhibited the expansion of the immature CFU-E subset. Notably, in the context of DBA, we found that steroid resistance was associated with a dysregulated p57Kip2 expression. Altogether, these data identify a novel glucocorticoid-responsive human erythroid progenitor and provide new insights into glucocorticoid-based therapeutic strategies for the treatment of patients with DBA.
Authors
Ryan J. Ashley, Hongxia Yan, Nan Wang, John Hale, Brian M Dulmovits, Julien Papoin, Meagan E. Olive, Namrata D Udeshi, Steven A. Carr, Adrianna Vlachos, Jeffrey M. Lipton, Lydie Da Costa, Christopher D. Hillyer, Sandrina Kinet, Naomi Taylor, Narla Mohandas, Anupama Narla, Lionel Blanc
Abstract
BACKGROUND. Glucose-6-phosphate dehydrogenase (G6PD) deficiency decreases the ability of red blood cells (RBCs) to withstand oxidative stress. Refrigerated storage of RBCs induces oxidative stress. We hypothesized that G6PD-deficient donor RBCs would have inferior storage quality for transfusion as compared to G6PD-normal RBCs. METHODS. Male volunteers were screened for G6PD deficiency; 27 control and 10 G6PD-deficient volunteers each donated one RBC unit. After 42 days of refrigerated storage, autologous 51-Chromium 24-hour post-transfusion RBC recovery (PTR) studies were performed. Metabolomics analyses of these RBC units were also performed. RESULTS. The mean 24-hour PTR for G6PD-deficient subjects was 78.5 ± 8.4% (mean ± SD), which was significantly lower than that for G6PD-normal RBCs (85.3 ± 3.2%; P = 0.0009). None of the G6PD-normal volunteers (0/27) and three G6PD-deficient volunteers (3/10) had PTR results below 75%, a key FDA acceptability criterion for stored donor RBCs. As expected, fresh G6PD-deficient RBCs demonstrated defects in the oxidative phase of the pentose phosphate pathway. During refrigerated storage, G6PD-deficient RBCs demonstrated increased glycolysis, impaired glutathione homeostasis, and increased purine oxidation, as compared with G6PD-normal RBCs. In addition, there were significant correlations between PTR and specific metabolites in these pathways. CONCLUSIONS. Based on current FDA criteria, RBCs from G6PD-deficient donors would not meet the requirements for storage quality. Metabolomics assessment identified markers of PTR and G6PD deficiency (e.g., pyruvate/lactate ratios), along with potential compensatory pathways that could be leveraged to ameliorate the metabolic needs of G6PD-deficient RBCs. REGISTRATION. ClinicalTrials.gov NCT04081272. FUNDING. The Harold Amos Medical Faculty Development Program, Robert Wood Johnson Foundation Grant 71590, the National Blood Foundation, NIH grant UL1 TR000040, the Webb-Waring Early Career Award 2017 by the Boettcher Foundation and the NHLBI grant R01HL14644 and R01HL148151.
Authors
Richard O. Francis, Angelo D’Alessandro, Andrew Eisenberger, Mark Soffing, Randy Yeh, Esther Coronel, Arif Sheikh, Francesca Rapido, Francesca La Carpia, Julie A. Reisz, Sarah Gehrke, Travis Nemkov, Tiffany Thomas, Joseph Schwartz, Chaitanya Divgi, Debra A. Kessler, Beth H. Shaz, Yelena Ginzburg, James C. Zimring, Steven L. Spitalnik, Eldad A. Hod
Abstract
BACKGROUND. Beige adipose tissue is associated with improved glucose homeostasis in mice. Adipose tissue contains β3 adrenergic receptors (β3-AR), and this study was intended to determine whether the treatment of obese, insulin-resistant humans with the β3AR agonist mirabegron, which stimulates beige adipose formation in subcutaneous white adipose tissue (SC WAT), would induce other beneficial changes in fat and muscle, and improve metabolic homeostasis. METHODS. Before and after β3AR agonist treatment, oral glucose tolerance tests and euglycemic clamps were performed, and histochemistry and gene expression profiling were performed from fat and muscle biopsies. PET CT scans quantified brown adipose tissue volume and activity and we conducted in vitro studies with primary cultures of differentiated human adipocytes and muscle.RESULTS. Clinical effects of mirabegron treatment included improved oral glucose tolerance (P<0.01), reduced hemoglobin A1c (P=0.01), and improved insulin sensitivity (P=0.03) and β-cell function (P=0.01). In SC WAT, mirabegron treatment stimulated lipolysis, reduced fibrotic gene expression and increased alternatively activated macrophages. Subjects with the most SC WAT beiging demonstrated the most improvement in β-cell function. In skeletal muscle, mirabegron reduced triglycerides, increased expression of PGC1A (P<0.05), and increased type I fibers (P<0.01). Conditioned media from adipocytes treated with mirabegron stimulated muscle fiber PGC1A expression in vitro (P<0.001). CONCLUSION. Mirabegron treatment significantly improves glucose tolerance in obese, insulin resistant humans. Since β-cells and skeletal muscle do not express β3-ARs, these data suggest that the beiging of SC WAT by mirabegron reduces adipose tissue dysfunction, which enhances muscle oxidative capacity and improves β-cell function. TRIAL REGISTRATION. Clinicaltrials.gov NCT02919176.FUNDING. NIH (DK112282, P30GM127211, DK 71349, and CTSA grant UL1TR001998).
Authors
Brian S. Finlin, Hasiyet Memetimin, Beibei Zhu, Amy L. Confides, Hemendra J. Vekaria, Riham H. El Khouli, Zachary R. Johnson, Philip M. Westgate, Jianzhong Chen, Andrew J. Morris, Patrick G. Sullivan, Esther E. Dupont-Versteegden, Philip A. Kern
Abstract
Cystic fibrosis (CF) lung disease is characterized by an inflammatory response that can lead to terminal respiratory failure. The cystic fibrosis transmembrane regulator (CFTR) is mutated in CF and we hypothesized that dysfunctional CFTR in platelets, which are key participants in immune responses, is a central determinant of CF inflammation. We found that deletion of CFTR in platelets produced exaggerated acute lung inflammation and platelet activation after intratracheal LPS or Pseudomonas aeruginosa challenge. CFTR loss of function in mouse or human platelets resulted in agonist-induced hyperactivation and increased calcium entry into platelets. Inhibition of the transient receptor potential cation channel 6 (TRPC6) reduced platelet activation and calcium flux, and reduced lung injury in CF mice after intratracheal LPS or Pseudomonas aeruginosa challenge. CF subjects receiving CFTR modulator therapy showed partial restoration of CFTR function in platelets, which may be a convenient approach to monitoring biological responses to CFTR modulators. We conclude that CFTR dysfunction in platelets produces aberrant TRPC6-dependent platelet activation, which is a major driver of CF lung inflammation and impaired bacterial clearance. Platelets, and TRPC6, are what we believe to be novel therapeutic targets in the treatment of CF lung disease.
Authors
Guadalupe Ortiz-Munoz, Michelle A. Yu, Emma Lefrançais, Benat Mallavia, Colin Valet, Jennifer J. Tian, Serena Ranucci, Kristin M. Wang, Zhe Liu, Nicholas Kwaan, Diana Dawson, Mary Ellen Kleinhenz, Fadi T. Khasawneh, Peter M. Haggie, Alan S. Verkman, Mark R. Looney
Abstract
BACKGROUND. Mirabegron is a β3-adrenergic receptor (β3-AR) agonist approved only for the treatment of overactive bladder. Encouraging preclinical results suggest that β3-AR agonists could also improve obesity-related metabolic disease by increasing brown adipose tissue (BAT) thermogenesis, white adipose tissue (WAT) lipolysis, and insulin sensitivity. METHODS. We treated 14 healthy women of diverse ethnicity, 27.5 ± 1.1 y, BMI 25.4 ± 1.2 kg/m2, with 100 mg mirabegron (Myrbetriq extended-release tablet, Astellas Pharma) for four weeks, open-label. The primary endpoint was the change in BAT metabolic activity as measured by [18F]-2-fluoro-D-2-deoxy-D-glucose (18F-FDG) positron emission tomography/computed tomography (PET/CT). Secondary endpoints included resting energy expenditure (REE), plasma metabolites, and glucose and insulin metabolism as assessed by frequently sampled intravenous glucose tolerance test. RESULTS. Chronic mirabegron therapy increased BAT metabolic activity. Whole-body REE was higher, without changes in body weight or composition. Additionally, there were elevations in plasma levels of the beneficial lipoprotein biomarkers high-density lipoprotein (HDL) and ApoA1, as well as total bile acids. Adiponectin, a WAT-derived hormone that has anti-diabetic and anti-inflammatory capabilities, increased with acute treatment and was 35% higher at study completion. Finally, an intravenous glucose tolerance test demonstrated higher insulin sensitivity, glucose effectiveness, and insulin secretion. CONCLUSION. These findings indicate that human BAT metabolic activity can be increased after chronic pharmacological stimulation with mirabegron and support the investigation of β3-AR agonists as a treatment for metabolic disease. TRIAL REGISTRATION. Clinicaltrials.gov NCT03049462. FUNDING. This work was supported by the Intramural Research Program of the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), DK075112, DK075116, DK071013, and DK071014.
Authors
Alana E. O'Mara, James W. Johnson, Joyce D. Linderman, Robert J. Brychta, Suzanne McGehee, Laura A. Fletcher, Yael A. Fink, Devika Kapuria, Thomas M. Cassimatis, Nathan Kelsey, Cheryl Cero, Zahraa Abdul-Sater, Francesca Piccinini, Alison S. Baskin, Brooks P. Leitner, Hongyi Cai, Corina M. Millo, William Dieckmann, Mary Walter, Norman B. Javitt, Yaron Rotman, Peter J. Walter, Marilyn Ader, Richard N. Bergman, Peter Herscovitch, Kong Y. Chen, Aaron M. Cypess
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January 2020
On the cover:
CD4+ T cell response to human RSV infection
January 2020 Issue
On the cover:
CD4+ T cell response to human RSV infection
CD4+ T cells play an important role in clearing respiratory syncytial virus (RSV), and better characterization of their responses may be critical to designing effective vaccines. In this issue, Guvenel et al. inoculated healthy volunteers with RSV to track T cell activation, proliferation, and recruitment prior to infection and throughout the course of disease. The study, which details a number of insights into the activity of bronchial-resident CD4+ T cells and their recognition of RSV antigens, lays the groundwork for developing more effective long-term RSV vaccination strategies. The cover image is an artistic rendering of RSV, with fusion proteins and attachment proteins depicted in purple and red, respectively. Image credit: Kateryna Kon/Shutterstock.
JCI This Month
January 2020 JCI This Month
JCI This Month is a digest of the research, reviews, and other features published each month.
Review Series - More
Series edited by Philipp E. Scherer
Mechanisms Underlying the Metabolic Syndrome
Series edited by Philipp E. Scherer
Obesity often occurs with a quintessential array of metabolic abnormalities: elevations in blood pressure, visceral fat, and circulating blood lipids, and, importantly, insulin resistance. Together, this constellation of conditions constitutes the metabolic syndrome and forecasts an individual’s increased risk of developing cardiovascular diseases and type 2 diabetes. Although metabolic syndrome presents as dysfunction across multiple tissues, its onset stems from pathological increases in adipose tissue. The 9 review in this series, conceptualized by series editor Philipp Scherer, delve into the complex biology underlying the metabolic syndrome. These reviews address adipocyte and beta cell dysfunction in the metabolic syndrome; the functions of adipose tissue fibrosis, the microbiome, and exosomal communication in obesity; and the concepts we use to define metabolic health.
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ISSN: 0021-9738 (print), 1558-8238 (online)