Human mutations in MCM4
False-colored transmission electron microscopy image of a steroid-secreting adrenal cell. In this issue of the
Image credit: Photo Researchers, Inc.
Starting with this issue, the Editorial duties for the JCI move to Duke University and the University of North Carolina at Chapel Hill. As we begin our five-year tenure at the helm of this prestigious journal, the tradition of excellence that these two schools typically display on the basketball court now enters the editorial boardroom.
Science In Medicine
Macrophage plasticity and polarization: in vivo veritas
Diversity and plasticity are hallmarks of cells of the monocyte-macrophage lineage. In response to IFNs, Toll-like receptor engagement, or IL-4/IL-13 signaling, macrophages undergo M1 (classical) or M2 (alternative) activation, which represent extremes of a continuum in a universe of activation states. Progress has now been made in defining the signaling pathways, transcriptional networks, and epigenetic mechanisms underlying M1-M2 or M2-like polarized activation. Functional skewing of mononuclear phagocytes occurs in vivo under physiological conditions (e.g., ontogenesis and pregnancy) and in pathology (allergic and chronic inflammation, tissue repair, infection, and cancer). However, in selected preclinical and clinical conditions, coexistence of cells in different activation states and unique or mixed phenotypes have been observed, a reflection of dynamic changes and complex tissue-derived signals. The identification of mechanisms and molecules associated with macrophage plasticity and polarized activation provides a basis for macrophage-centered diagnostic and therapeutic strategies.
Endothelium: from cellophane to orchestral maestro
Endothelial cells from human umbilical veins were first cultured nearly four decades ago, initiating explosive growth in research in vascular biology and leading to major insights into angiogenesis, vasculogenesis, and tumor biology. Recent studies now promise to open new horizons in regenerative medicine as well as organ engineering.
Unraveling human natural killer cell deficiency
NK cells are a component of the innate immune system identified in animals as serving an essential role in antiviral immunity. Establishing their role in human health has been challenging, with the most direct insight coming from the study of NK cell–deficient individuals. However, NK cell deficiencies are rare, and more research is needed. In this issue of the JCI, two independent groups of researchers have simultaneously identified the genetic cause of a human NK cell deficiency as mutation in the MCM4 gene, encoding minichromosome maintenance complex component 4. These reports suggest a critical role for the minichromosome maintenance helicase complex in NK cells and NK cell–mediated host defense.
Unraveling the mystery of the hygiene hypothesis through Helicobacter pylori infection
Epidemiological studies have revealed an inverse association between Helicobacter pylori infection and the incidence of allergic asthma. This association is consistent with the hygiene hypothesis, which posits that exposure to microbes early in life prevents the later development of allergic diseases, and has been reproduced in mouse models of asthma. In this issue of the JCI, Oertli and colleagues report that H. pylori infection in neonates elicits tolerogenic DCs that produce IL-18, which drive the generation of Tregs that subsequently protect the mice from allergic asthma. This finding strengthens the intriguing link between pathogen exposure and allergic disease.
NPs — heart hormones that regulate brown fat?
Thermogenesis in brown adipose tissue (BAT) is well characterized as being under the control of the sympathetic nervous system. The energy-burning capacity of BAT makes it an attractive target for anti-obesity therapies. However, previous attempts to manipulate BAT’s sympathetic activation have lacked specificity. In this issue of the JCI, Bordicchia et al. provide new data indicating that cardiac natriuretic peptides (NPs) are also able to activate thermogenic machinery in adipose tissue. Their findings suggest a novel strategy to increase energy dissipation in adipose tissue, independent of adrenergic receptors.
DYRK1A in Down syndrome: an oncogene or tumor suppressor?
Children with Down syndrome (DS) have a markedly increased risk of developing acute megakaryoblastic leukemia (AMKL) and acute lymphoblastic leukemia compared with that of children without DS. Despite recent breakthroughs, it is not clear which genes on chromosome 21, the chromosome that is trisomic in individuals with DS, cause this predisposition. In this issue of the JCI, Malinge et al. report their loss- and gain-of-function experiments in mouse and human cells that show that increased expression of the kinase encoded by the chromosome 21 gene DYRK1A suppresses the nuclear factor of activated T cells pathway and promotes AMKL. Interestingly, the same protein has been suggested to contribute to the reduced risk of epithelial cancers in adults with DS, leading to the possibility that it could be proleukemic in children and antitumorigenic in adults.
Popeye proteins: muscle for the aging sinus node
The electrical impulses that dictate the rhythm of the heartbeat in normal situations and during exercise or stress are initiated by a small number of sinus node pacemaker cells. Senescence and dysfunction of the sinus node affects many people later in life, causing physiologically inappropriate heart rates, but the underlying mechanisms are not well understood. In this issue of the JCI, Froese and colleagues show that deficiency in either Popeye domain containing 1 (Popdc1) or Popdc2 leads to sinus node dysfunction under stressed conditions in aged mice. The mechanism reported to underlie the effects of Popdc1/2 deficiency in mice may cause the stress-induced sinus node dysfunction found in many aged individuals and may point to new strategies for therapeutic intervention.
MCM4 mutation causes adrenal failure, short stature, and natural killer cell deficiency in humans
Claire R. Hughes, Leonardo Guasti, Eirini Meimaridou, Chen-Hua Chuang, John C. Schimenti, Peter J. King, Colm Costigan, Adrian J.L. Clark, Louise A. MetherellAbstract | Full text | PDF | Supplemental material (Page 814)
An interesting variant of familial glucocorticoid deficiency (FGD), an autosomal recessive form of adrenal failure, exists in a genetically isolated Irish population. In addition to hypocortisolemia, affected children show signs of growth failure, increased chromosomal breakage, and NK cell deficiency. Targeted exome sequencing in 8 patients identified a variant (c.71-1insG) in minichromosome maintenance–deficient 4 (MCM4) that was predicted to result in a severely truncated protein (p.Pro24ArgfsX4). Western blotting of patient samples revealed that the major 96-kDa isoform present in unaffected human controls was absent, while the presence of the minor 85-kDa isoform was preserved. Interestingly, histological studies with Mcm4-depleted mice showed grossly abnormal adrenal morphology that was characterized by non-steroidogenic GATA4- and Gli1-positive cells within the steroidogenic cortex, which reduced the number of steroidogenic cells in the zona fasciculata of the adrenal cortex. Since MCM4 is one part of a MCM2-7 complex recently confirmed as the replicative helicase essential for normal DNA replication and genome stability in all eukaryotes, it is possible that our patients may have an increased risk of neoplastic change. In summary, we have identified what we believe to be the first human mutation in MCM4 and have shown that it is associated with adrenal insufficiency, short stature, and NK cell deficiency.
Partial MCM4 deficiency in patients with growth retardation, adrenal insufficiency, and natural killer cell deficiency
Laure Gineau, Céline Cognet, Nihan Kara, Francis Peter Lach, Jean Dunne, Uma Veturi, Capucine Picard, Céline Trouillet, Céline Eidenschenk, Said Aoufouchi, Alexandre Alcaïs, Owen Smith, Frédéric Geissmann, Conleth Feighery, Laurent Abel, Agata Smogorzewska, Bruce Stillman, Eric Vivier, Jean-Laurent Casanova, Emmanuelle JouanguyAbstract | Full text | PDF | Supplemental material (Page 821)
Natural killer (NK) cells are circulating cytotoxic lymphocytes that exert potent and nonredundant antiviral activity and antitumoral activity in the mouse; however, their function in host defense in humans remains unclear. Here, we investigated 6 related patients with autosomal recessive growth retardation, adrenal insufficiency, and a selective NK cell deficiency characterized by a lack of the CD56dim NK subset. Using linkage analysis and fine mapping, we identified the disease-causing gene, MCM4, which encodes a component of the MCM2-7 helicase complex required for DNA replication. A splice-site mutation in the patients produced a frameshift, but the mutation was hypomorphic due to the creation of two new translation initiation methionine codons downstream of the premature termination codon. The patients’ fibroblasts exhibited genomic instability, which was rescued by expression of WT MCM4. These data indicate that the patients’ growth retardation and adrenal insufficiency likely reflect the ubiquitous but heterogeneous impact of the MCM4 mutation in various tissues. In addition, the specific loss of the NK CD56dim subset in patients was associated with a lower rate of NK CD56bright cell proliferation, and the maturation of NK CD56bright cells toward an NK CD56dim phenotype was tightly dependent on MCM4-dependent cell division. Thus, partial MCM4 deficiency results in a genetic syndrome of growth retardation with adrenal insufficiency and selective NK deficiency.
ChIP sequencing of cyclin D1 reveals a transcriptional role in chromosomal instability in mice
Mathew C. Casimiro, Marco Crosariol, Emanuele Loro, Adam Ertel, Zuoren Yu, William Dampier, Elizabeth A. Saria, Alex Papanikolaou, Timothy J. Stanek, Zhiping Li, Chenguang Wang, Paolo Fortina, Sankar Addya, Aydin Tozeren, Erik S. Knudsen, Andrew Arnold, Richard G. PestellAbstract | Full text | PDF | Supplemental material | Correction (Page 833)
Chromosomal instability (CIN) in tumors is characterized by chromosomal abnormalities and an altered gene expression signature; however, the mechanism of CIN is poorly understood. CCND1 (which encodes cyclin D1) is overexpressed in human malignancies and has been shown to play a direct role in transcriptional regulation. Here, we used genome-wide ChIP sequencing and found that the DNA-bound form of cyclin D1 occupied the regulatory region of genes governing chromosomal integrity and mitochondrial biogenesis. Adding cyclin D1 back to Ccnd1–/– mouse embryonic fibroblasts resulted in CIN gene regulatory region occupancy by the DNA-bound form of cyclin D1 and induction of CIN gene expression. Furthermore, increased chromosomal aberrations, aneuploidy, and centrosome abnormalities were observed in the cyclin D1–rescued cells by spectral karyotyping and immunofluorescence. To assess cyclin D1 effects in vivo, we generated transgenic mice with acute and continuous mammary gland–targeted cyclin D1 expression. These transgenic mice presented with increased tumor prevalence and signature CIN gene profiles. Additionally, interrogation of gene expression from 2,254 human breast tumors revealed that cyclin D1 expression correlated with CIN in luminal B breast cancer. These data suggest that cyclin D1 contributes to CIN and tumorigenesis by directly regulating a transcriptional program that governs chromosomal stability.
p27Kip1 controls cytokinesis via the regulation of citron kinase activation
Murielle P. Serres, Uta Kossatz, Yong Chi, James M. Roberts, Nisar P. Malek, Arnaud BessonAbstract | Full text | PDF | Supplemental material (Page 844)
p27Kip1 (p27) acts as a tumor suppressor by inhibiting cyclin–cyclin-dependent kinase (cyclin-CDK) activity. However, mice expressing a form of p27 that is unable to bind or inhibit cyclin-CDK complexes (p27CK–) have increased incidence of tumor development as compared with wild-type and p27–/– mice, revealing an oncogenic role for p27. Here, we identified a phenotype of multinucleation and polyploidy in p27CK– mice not present in p27–/– animals, suggesting a role for p27 in G2/M that is independent of cyclin-CDK regulation. Further analysis revealed that p27CK– expression caused a cytokinesis and abscission defect in mouse embryonic fibroblasts. We identified the Rho effector citron kinase (citron-K) as a p27-interacting protein in vitro and in vivo and found that p27 and citron-K colocalized at the contractile ring and mid-body during telophase and cytokinesis. Moreover, overexpression of the minimal p27-binding domain of citron-K was sufficient to rescue the phenotype caused by p27CK–. Conversely, expression of a mutant p27CK– unable to bind citron-K did not induce multinucleation. Finally, by binding to citron-K, p27 prevented the interaction of citron-K with its activator RhoA. Taken together, these data suggest a role for p27 during cytokinesis via the regulation of citron-K activity.
DYRK2 priming phosphorylation of c-Jun and c-Myc modulates cell cycle progression in human cancer cells
Naoe Taira, Rei Mimoto, Morito Kurata, Tomoko Yamaguchi, Masanobu Kitagawa, Yoshio Miki, Kiyotsugu YoshidaAbstract | Full text | PDF | Supplemental material (Page 859)
Dysregulation of the G1/S transition in the cell cycle contributes to tumor development. The oncogenic transcription factors c-Jun and c-Myc are indispensable regulators at this transition, and their aberrant expression is associated with many malignancies. Degradation of c-Jun/c-Myc is a critical process for the G1/S transition, which is initiated upon phosphorylation by glycogen synthase kinase 3 β (GSK3β). However, a specific kinase or kinases responsible for priming phosphorylation events that precede this GSK3β modification has not been definitively identified. Here, we found that the dual-specificity tyrosine phosphorylation–regulated kinase DYRK2 functions as a priming kinase of c-Jun and c-Myc. Knockdown of DYRK2 in human cancer cells shortened the G1 phase and accelerated cell proliferation due to escape of c-Jun and c-Myc from ubiquitination-mediated degradation. In concert with these results, silencing DYRK2 increased cell proliferation in human cancer cells, and this promotion was completely impeded by codeprivation of c-Jun or c-Myc in vivo. We also found marked attenuation of DYRK2 expression in multiple human tumor samples. Downregulation of DYRK2 correlated with high levels of unphosphorylated c-Jun and c-Myc and, importantly, with invasiveness of human breast cancers. These results reveal that DYRK2 regulates tumor progression through modulation of c-Jun and c-Myc.
Modulation of noncanonical TGF-β signaling prevents cleft palate in Tgfbr2 mutant mice
Jun-ichi Iwata, Joseph G. Hacia, Akiko Suzuki, Pedro A. Sanchez-Lara, Mark Urata, Yang ChaiAbstract | Full text | PDF | Supplemental material (Page 873)
Patients with mutations in either TGF-β receptor type I (TGFBR1) or TGF-β receptor type II (TGFBR2), such as those with Loeys-Dietz syndrome, have craniofacial defects and signs of elevated TGF-β signaling. Similarly, mutations in TGF-β receptor gene family members cause craniofacial deformities, such as cleft palate, in mice. However, it is unknown whether TGF-β ligands are able to elicit signals in Tgfbr2 mutant mice. Here, we show that loss of Tgfbr2 in mouse cranial neural crest cells results in elevated expression of TGF-β2 and TGF-β receptor type III (TβRIII); activation of a TβRI/TβRIII-mediated, SMAD-independent, TRAF6/TAK1/p38 signaling pathway; and defective cell proliferation in the palatal mesenchyme. Strikingly, Tgfb2, Tgfbr1 (also known as Alk5), or Tak1 haploinsufficiency disrupted TβRI/TβRIII-mediated signaling and rescued craniofacial deformities in Tgfbr2 mutant mice, indicating that activation of this noncanonical TGF-β signaling pathway was responsible for craniofacial malformations in Tgfbr2 mutant mice. Thus, modulation of TGF-β signaling may be beneficial for the prevention of congenital craniofacial birth defects.
PPARγ agonists enhance ET-743–induced adipogenic differentiation in a transgenic mouse model of myxoid round cell liposarcoma
Elizabeth Charytonowicz, Melissa Terry, Katherine Coakley, Leonid Telis, Fabrizio Remotti, Carlos Cordon-Cardo, Robert N. Taub, Igor MatushanskyAbstract | Full text | PDF | Supplemental material (Page 886)
Myxoid round cell liposarcoma (MRCLS) is a common liposarcoma subtype characterized by a translocation that results in the fusion protein TLS:CHOP as well as by mixed adipocytic histopathology. Both the etiology of MRCLS and the mechanism of action of TLS:CHOP remain poorly understood. It was previously shown that ET-743, an antitumor compound with an unclear mechanism of action, is highly effective in patients with MRCLS. To identify the cellular origin of MRCLS, we engineered a mouse model in which TLS:CHOP was expressed under the control of a mesodermally restricted promoter (Prx1) in a p53-depleted background. This model resembled MRCLS histologically as well as functionally in terms of its specific adipocytic differentiation–based response to ET-743. Specifically, endogenous mesenchymal stem cells (MSCs) expressing TLS:CHOP developed into MRCLS in vivo. Gene expression and microRNA analysis of these MSCs showed that they were committed to adipocytic differentiation, but unable to terminally differentiate. We also explored the method of action of ET-743. ET-743 downregulated TLS:CHOP expression, which correlated with CEBPα expression and adipocytic differentiation. Furthermore, PPARγ agonists enhanced the differentiation process initiated by ET-743. Our work highlights how clinical observations can lead to the generation of a mouse model that recapitulates human disease and may be used to develop rational treatment combinations, such as ET-743 plus PPARγ agonists, for the treatment of MRCLS.
Matrix architecture defines the preferential localization and migration of T cells into the stroma of human lung tumors
Hélène Salmon, Katarzyna Franciszkiewicz, Diane Damotte, Marie-Caroline Dieu-Nosjean, Pierre Validire, Alain Trautmann, Fathia Mami-Chouaib, Emmanuel DonnadieuAbstract | Full text | PDF | Supplemental material (Page 899)
Appropriate localization and migration of T cells is a prerequisite for antitumor immune surveillance. Studies using fixed tumor samples from human patients have shown that T cells accumulate more efficiently in the stroma than in tumor islets, but the mechanisms by which this occurs are unknown. By combining immunostaining and real-time imaging in viable slices of human lung tumors, we revealed that the density and the orientation of the stromal extracellular matrix likely play key roles in controlling the migration of T cells. Active T cell motility, dependent on chemokines but not on β1 or β2 integrins, was observed in loose fibronectin and collagen regions, whereas T cells migrated poorly in dense matrix areas. Aligned fibers in perivascular regions and around tumor epithelial cell regions dictated the migratory trajectory of T cells and restricted them from entering tumor islets. Consistently, matrix reduction with collagenase increased the ability of T cells to contact cancer cells. Thus, the stromal extracellular matrix influences antitumor immunity by controlling the positioning and migration of T cells. Understanding the mechanisms by which this collagen network is generated has the potential to aid in the development of new therapeutics.
Heparan sulfate sulfatase SULF2 regulates PDGFRα signaling and growth in human and mouse malignant glioma
Joanna J. Phillips, Emmanuelle Huillard, Aaron E. Robinson, Anna Ward, David H. Lum, Mei-Yin Polley, Steven D. Rosen, David H. Rowitch, Zena WerbAbstract | Full text | PDF | Supplemental material (Page 911)
Glioblastoma (GBM), a uniformly lethal brain cancer, is characterized by diffuse invasion and abnormal activation of multiple receptor tyrosine kinase (RTK) signaling pathways, presenting a major challenge to effective therapy. The activation of many RTK pathways is regulated by extracellular heparan sulfate proteoglycans (HSPG), suggesting these molecules may be effective targets in the tumor microenvironment. In this study, we demonstrated that the extracellular sulfatase, SULF2, an enzyme that regulates multiple HSPG-dependent RTK signaling pathways, was expressed in primary human GBM tumors and cell lines. Knockdown of SULF2 in human GBM cell lines and generation of gliomas from Sulf2–/– tumorigenic neurospheres resulted in decreased growth in vivo in mice. We found a striking SULF2 dependence in activity of PDGFRα, a major signaling pathway in GBM. Ablation of SULF2 resulted in decreased PDGFRα phosphorylation and decreased downstream MAPK signaling activity. Interestingly, in a survey of SULF2 levels in different subtypes of GBM, the proneural subtype, characterized by aberrations in PDGFRα, demonstrated the strongest SULF2 expression. Therefore, in addition to its potential as an upstream target for therapy of GBM, SULF2 may help identify a subset of GBMs that are more dependent on exogenous growth factor–mediated signaling. Our results suggest the bioavailability of growth factors from the microenvironment is a significant contributor to tumor growth in a major subset of human GBM.
Essential role of gastric gland mucin in preventing gastric cancer in mice
Fumitoshi Karasawa, Akira Shiota, Yukinobu Goso, Motohiro Kobayashi, Yoshiko Sato, Junya Masumoto, Maiko Fujiwara, Shuichi Yokosawa, Takashi Muraki, Shinichi Miyagawa, Masatsugu Ueda, Michiko N. Fukuda, Minoru Fukuda, Kazuhiko Ishihara, Jun NakayamaAbstract | Full text | PDF | Supplemental material (Page 923)
Gastric gland mucin secreted from the lower portion of the gastric mucosa contains unique O-linked oligosaccharides (O-glycans) having terminal α1,4-linked N-acetylglucosamine residues (αGlcNAc). Previously, we identified human α1,4-N-acetylglucosaminyltransferase (α4GnT), which is responsible for the O-glycan biosynthesis and characterized αGlcNAc function in suppressing Helicobacter pylori in vitro. In the present study, we engineered A4gnt–/– mice to better understand its role in vivo. A4gnt–/– mice showed complete lack of αGlcNAc expression in gastric gland mucin. Surprisingly, all the mutant mice developed gastric adenocarcinoma through a hyperplasia-dysplasia-carcinoma sequence in the absence of H. pylori infection. Microarray and quantitative RT-PCR analysis revealed upregulation of genes encoding inflammatory chemokine ligands, proinflammatory cytokines, and growth factors, such as Ccl2, Il-11, and Hgf in the gastric mucosa of A4gnt–/– mice. Further supporting an important role for this O-glycan in cancer progression, we also observed significantly reduced αGlcNAc in human gastric adenocarcinoma and adenoma. Our results demonstrate that the absence of αGlcNAc triggers gastric tumorigenesis through inflammation-associated pathways in vivo. Thus, αGlcNAc-terminated gastric mucin plays dual roles in preventing gastric cancer by inhibiting H. pylori infection and also suppressing tumor-promoting inflammation.
The intersection of genetic and chemical genomic screens identifies GSK-3α as a target in human acute myeloid leukemia
Versha Banerji, Stacey M. Frumm, Kenneth N. Ross, Loretta S. Li, Anna C. Schinzel, Cynthia K. Hahn, Rose M. Kakoza, Kwan T. Chow, Linda Ross, Gabriela Alexe, Nicola Tolliday, Haig Inguilizian, Ilene Galinsky, Richard M. Stone, Daniel J. DeAngelo, Giovanni Roti, Jon C. Aster, William C. Hahn, Andrew L. Kung, Kimberly StegmaierAbstract | Full text | PDF | Supplemental material (Page 935)
Acute myeloid leukemia (AML) is the most common form of acute leukemia in adults. Long-term survival of patients with AML has changed little over the past decade, necessitating the identification and validation of new AML targets. Integration of genomic approaches with small-molecule and genetically based high-throughput screening holds the promise of improved discovery of candidate targets for cancer therapy. Here, we identified a role for glycogen synthase kinase 3α (GSK-3α) in AML by performing 2 independent small-molecule library screens and an shRNA screen for perturbations that induced a differentiation expression signature in AML cells. GSK-3 is a serine-threonine kinase involved in diverse cellular processes, including differentiation, signal transduction, cell cycle regulation, and proliferation. We demonstrated that specific loss of GSK-3α induced differentiation in AML by multiple measurements, including induction of gene expression signatures, morphological changes, and cell surface markers consistent with myeloid maturation. GSK-3α–specific suppression also led to impaired growth and proliferation in vitro, induction of apoptosis, loss of colony formation in methylcellulose, and anti-AML activity in vivo. Although the role of GSK-3β has been well studied in cancer development, these studies support a role for GSK-3α in AML.
Increased dosage of the chromosome 21 ortholog Dyrk1a promotes megakaryoblastic leukemia in a murine model of Down syndrome
Sébastien Malinge, Meghan Bliss-Moreau, Gina Kirsammer, Lauren Diebold, Timothy Chlon, Sandeep Gurbuxani, John D. CrispinoAbstract | Full text | PDF | Supplemental material (Page 948)
Individuals with Down syndrome (DS; also known as trisomy 21) have a markedly increased risk of leukemia in childhood but a decreased risk of solid tumors in adulthood. Acquired mutations in the transcription factor–encoding GATA1 gene are observed in nearly all individuals with DS who are born with transient myeloproliferative disorder (TMD), a clonal preleukemia, and/or who develop acute megakaryoblastic leukemia (AMKL). Individuals who do not have DS but bear germline GATA1 mutations analogous to those detected in individuals with TMD and DS-AMKL are not predisposed to leukemia. To better understand the functional contribution of trisomy 21 to leukemogenesis, we used mouse and human cell models of DS to reproduce the multistep pathogenesis of DS-AMKL and to identify chromosome 21 genes that promote megakaryoblastic leukemia in children with DS. Our results revealed that trisomy for only 33 orthologs of human chromosome 21 (Hsa21) genes was sufficient to cooperate with GATA1 mutations to initiate megakaryoblastic leukemia in vivo. Furthermore, through a functional screening of the trisomic genes, we demonstrated that DYRK1A, which encodes dual-specificity tyrosine-(Y)-phosphorylation–regulated kinase 1A, was a potent megakaryoblastic tumor–promoting gene that contributed to leukemogenesis through dysregulation of nuclear factor of activated T cells (NFAT) activation. Given that calcineurin/NFAT pathway inhibition has been implicated in the decreased tumor incidence in adults with DS, our results show that the same pathway can be both proleukemic in children and antitumorigenic in adults.
Divergent requirement for Gαs and cAMP in the differentiation and inflammatory profile of distinct mouse Th subsets
Xiangli Li, Fiona Murray, Naoki Koide, Jonathan Goldstone, Sara M. Dann, Jianzhong Chen, Samuel Bertin, Guo Fu, Lee S. Weinstein, Min Chen, Maripat Corr, Lars Eckmann, Paul A. Insel, Eyal RazAbstract | Full text | PDF | Supplemental material (Page 963)
cAMP, the intracellular signaling molecule produced in response to GPCR signaling, has long been recognized as an immunosuppressive agent that inhibits T cell receptor activation and T cell function. However, recent studies show that cAMP also promotes T cell–mediated immunity. Central to cAMP production downstream of GPCR activation is the trimeric G protein Gs. In order to reconcile the reports of divergent effects of cAMP in T cells and to define the direct effect of cAMP in T cells, we engineered mice in which the stimulatory Gα subunit of Gs (Gαs) could be deleted in T cells using CD4-Cre (GnasΔCD4). GnasΔCD4 CD4+ T cells had reduced cAMP accumulation and Ca2+ influx. In vitro and in vivo, GnasΔCD4 CD4+ T cells displayed impaired differentiation to specific Th subsets: Th17 and Th1 cells were reduced or absent, but Th2 and regulatory T cells were unaffected. Furthermore, GnasΔCD4 CD4+ T cells failed to provoke colitis in an adoptive transfer model, indicating reduced inflammatory function. Restoration of cAMP levels rescued the impaired phenotype of GnasΔCD4 CD4+ T cells, reinstated the PKA-dependent influx of Ca2+, and enhanced the ability of these cells to induce colitis. Our findings thus define an important role for cAMP in the differentiation of Th subsets and their subsequent inflammatory responses, and provide evidence that altering cAMP levels in CD4+ T cells could provide an immunomodulatory approach targeting specific Th subsets.
Cxcr2 and Cxcl5 regulate the IL-17/G-CSF axis and neutrophil homeostasis in mice
Junjie Mei, Yuhong Liu, Ning Dai, Christian Hoffmann, Kristin M. Hudock, Peggy Zhang, Susan H. Guttentag, Jay K. Kolls, Paula M. Oliver, Frederic D. Bushman, G. Scott WorthenAbstract | Full text | PDF | Supplemental material (Page 974)
Neutrophils are essential for maintaining innate immune surveillance under normal conditions, but also represent a major contributor to tissue damage during inflammation. Neutrophil homeostasis is therefore tightly regulated. Cxcr2 plays a critical role in neutrophil homeostasis, as Cxcr2–/– mice demonstrate mild neutrophilia and severe neutrophil hyperplasia in the bone marrow. The mechanisms underlying these phenotypes, however, are unclear. We report here that Cxcr2 on murine neutrophils inhibits the IL-17A/G-CSF axis that regulates neutrophil homeostasis. Furthermore, enterocyte-derived Cxcl5 in the gut regulates IL-17/G-CSF levels and contributes to Cxcr2-dependent neutrophil homeostasis. Conversely, G-CSF was required for Cxcl5-dependent regulation of neutrophil homeostasis, and inhibition of IL-17A reduced plasma G-CSF concentrations and marrow neutrophil numbers in both Cxcl5–/– and Cxcr2–/– mice. Cxcr2–/– mice constitutively expressed IL-17A and showed increased numbers of IL-17A–producing cells in the lung, terminal ileum, and spleen. Most IL-17–producing splenocytes were responsive to IL-1β plus IL-23 in vitro. Depletion of commensal microbes by antibiotic treatment in Cxcr2–/– mice markedly decreased IL-17A and G-CSF expression, neutrophilia, and marrow myeloid hyperplasia. These data suggest a critical role for Cxcr2, Cxcl5, and commensal bacteria in regulation of the IL-17/G-CSF axis and neutrophil homeostasis at mucosal sites and have implications for the development of treatments for pathologies resulting from either excessive or ineffective neutrophil responses.
Protective antifungal memory CD8+ T cells are maintained in the absence of CD4+ T cell help and cognate antigen in mice
Som G. Nanjappa, Erika Heninger, Marcel Wüthrich, Thomas Sullivan, Bruce KleinAbstract | Full text | PDF | Supplemental material (Page 987)
Individuals who are immunocompromised, including AIDS patients with few CD4+ T cells, are at increased risk for opportunistic fungal infections. The incidence of such infections is increasing worldwide, meaning that the need for antifungal vaccines is increasing. Although CD4+ T cells play a dominant role in resistance to many pathogenic fungal infections, we have previously shown that vaccination can induce protective antifungal CD8+ T cell immunity in the absence of CD4+ T cells. However, it has not been determined whether vaccine-induced antifungal CD8+ T cell memory can be maintained in the absence of CD4+ T cell help. Here, we have shown in a mouse model of vaccination against blastomycosis that antifungal memory CD8+ T cells are maintained in the absence of CD4+ T cells without loss of numbers or function for at least 6 months and that the cells protect against infection. Using a system that enabled us to induce and track antigen-specific, antifungal CD8+ T cells, we found that such cells were maintained for at least 5 months upon transfer into naive mice lacking both CD4+ T cells and persistent fungal antigen. Additionally, fungal vaccination induced a profile of transcription factors functionally linked with persistent memory in CD8+ T cells. Thus, unlike bacteria and viruses, fungi elicit long-term CD8+ T cell memory that is maintained without CD4+ T cell help or persistent antigen. This has implications for the development of novel antifungal vaccine strategies effective in immunocompromised patients.
Direct leptin action on POMC neurons regulates glucose homeostasis and hepatic insulin sensitivity in mice
Eric D. Berglund, Claudia R. Vianna, Jose Donato Jr., Mi Hwa Kim, Jen-Chieh Chuang, Charlotte E. Lee, Danielle A. Lauzon, Peagan Lin, Laura J. Brule, Michael M. Scott, Roberto Coppari, Joel K. ElmquistAbstract | Full text | PDF | Supplemental material (Page 1000)
Leptin action on its receptor (LEPR) stimulates energy expenditure and reduces food intake, thereby lowering body weight. One leptin-sensitive target cell mediating these effects on energy balance is the proopiomelanocortin (POMC) neuron. Recent evidence suggests that the action of leptin on POMC neurons regulates glucose homeostasis independently of its effects on energy balance. Here, we have dissected the physiological impact of direct leptin action on POMC neurons using a mouse model in which endogenous LEPR expression was prevented by a LoxP-flanked transcription blocker (loxTB), but could be reactivated by Cre recombinase. Mice homozygous for the LeprloxTB allele were obese and exhibited defects characteristic of LEPR deficiency. Reexpression of LEPR only in POMC neurons in the arcuate nucleus of the hypothalamus did not reduce food intake, but partially normalized energy expenditure and modestly reduced body weight. Despite the moderate effects on energy balance and independent of changes in body weight, restoring LEPR in POMC neurons normalized blood glucose and ameliorated hepatic insulin resistance, hyperglucagonemia, and dyslipidemia. Collectively, these results demonstrate that direct leptin action on POMC neurons does not reduce food intake, but is sufficient to normalize glucose and glucagon levels in mice otherwise lacking LEPR.
FOSL2 promotes leptin gene expression in human and mouse adipocytes
Christiane D. Wrann, Jun Eguchi, Aline Bozec, Zhao Xu, Tarjei Mikkelsen, Jeffrey Gimble, Heike Nave, Erwin F. Wagner, Shao-En Ong, Evan D. RosenAbstract | Full text | PDF | Supplemental material (Page 1010)
The adipocyte-derived hormone leptin is a critical regulator of many physiological functions, ranging from satiety to immunity. Surprisingly, very little is known about the transcriptional pathways that regulate adipocyte-specific expression of leptin. Here, we report studies in which we pursued a strategy integrating BAC transgenic reporter mice, reporter assays, and chromatin state mapping to locate an adipocyte-specific cis-element upstream of the leptin (LEP) gene in human fat cells. Quantitative proteomics with affinity enrichment of protein-DNA complexes identified the transcription factor FOS-like antigen 2 (FOSL2) as binding specifically to the identified region, a result that was confirmed by ChIP. Knockdown of FOSL2 in human adipocytes decreased LEP expression, and overexpression of Fosl2 increased Lep expression in mouse adipocytes. Moreover, the elevated LEP expression observed in obesity correlated well with increased FOSL2 levels in mice and humans, and adipocyte-specific genetic deletion of Fosl2 in mice reduced Lep expression. Taken together, these data identify FOSL2 as a critical regulator of leptin expression in adipocytes.
Cardiac natriuretic peptides act via p38 MAPK to induce the brown fat thermogenic program in mouse and human adipocytes
Marica Bordicchia, Dianxin Liu, Ez-Zoubir Amri, Gerard Ailhaud, Paolo Dessì-Fulgheri, Chaoying Zhang, Nobuyuki Takahashi, Riccardo Sarzani, Sheila CollinsAbstract | Full text | PDF | Supplemental material | Correction (Page 1022)
The ability of mammals to resist body fat accumulation is linked to their ability to expand the number and activity of “brown adipocytes” within white fat depots. Activation of β-adrenergic receptors (β-ARs) can induce a functional “brown-like” adipocyte phenotype. As cardiac natriuretic peptides (NPs) and β-AR agonists are similarly potent at stimulating lipolysis in human adipocytes, we investigated whether NPs could induce human and mouse adipocytes to acquire brown adipocyte features, including a capacity for thermogenic energy expenditure mediated by uncoupling protein 1 (UCP1). In human adipocytes, atrial NP (ANP) and ventricular NP (BNP) activated PPARγ coactivator-1α (PGC-1α) and UCP1 expression, induced mitochondriogenesis, and increased uncoupled and total respiration. At low concentrations, ANP and β-AR agonists additively enhanced expression of brown fat and mitochondrial markers in a p38 MAPK–dependent manner. Mice exposed to cold temperatures had increased levels of circulating NPs as well as higher expression of NP signaling receptor and lower expression of the NP clearance receptor (Nprc) in brown adipose tissue (BAT) and white adipose tissue (WAT). NPR-C–/– mice had markedly smaller WAT and BAT depots but higher expression of thermogenic genes such as Ucp1. Infusion of BNP into mice robustly increased Ucp1 and Pgc-1α expression in WAT and BAT, with corresponding elevation of respiration and energy expenditure. These results suggest that NPs promote “browning” of white adipocytes to increase energy expenditure, defining the heart as a central regulator of adipose tissue biology.
Anti-ganglioside antibody internalization attenuates motor nerve terminal injury in a mouse model of acute motor axonal neuropathy
Simon N. Fewou, Angie Rupp, Lauren E. Nickolay, Kathryn Carrick, Kay N. Greenshields, John Pediani, Jaap J. Plomp, Hugh J. WillisonAbstract | Full text | PDF | Supplemental material (Page 1037)
In the Guillain-Barré syndrome subform acute motor axonal neuropathy (AMAN), Campylobacter jejuni enteritis triggers the production of anti-ganglioside Abs (AGAbs), leading to immune-mediated injury of distal motor nerves. An important question has been whether injury to the presynaptic neuron at the neuromuscular junction is a major factor in AMAN. Although disease modeling in mice exposed to AGAbs indicates that complement-mediated necrosis occurs extensively in the presynaptic axons, evidence in humans is more limited, in comparison to the extensive injury seen at nodes of Ranvier. We considered that rapid AGAb uptake at the motor nerve terminal membrane might attenuate complement-mediated injury. We found that PC12 rat neuronal cells rapidly internalized AGAb, which were trafficked to recycling endosomes and lysosomes. Consequently, complement-mediated cytotoxicity was attenuated. Importantly, we observed the same AGAb endocytosis and protection from cytotoxicity in live mouse nerve terminals. AGAb uptake was attenuated following membrane cholesterol depletion in vitro and ex vivo, indicating that this process may be dependent upon cholesterol-enriched microdomains. In contrast, we observed minimal AGAb uptake at nodes of Ranvier, and this structure thus remained vulnerable to complement-mediated injury. These results indicate that differential endocytic processing of AGAbs by different neuronal and glial membranes might be an important modulator of site-specific injury in acute AGAb-mediated Guillain-Barré syndrome subforms and their chronic counterparts.
Cdc42 and Rab8a are critical for intestinal stem cell division, survival, and differentiation in mice
Ryotaro Sakamori, Soumyashree Das, Shiyan Yu, Shanshan Feng, Ewa Stypulkowski, Yinzheng Guan, Veronique Douard, Waixing Tang, Ronaldo P. Ferraris, Akihiro Harada, Cord Brakebusch, Wei Guo, Nan GaoAbstract | Full text | PDF | Supplemental material (Page 1052)
The constant self renewal and differentiation of adult intestinal stem cells maintains a functional intestinal mucosa for a lifetime. However, the molecular mechanisms that regulate intestinal stem cell division and epithelial homeostasis are largely undefined. We report here that the small GTPases Cdc42 and Rab8a are critical regulators of these processes in mice. Conditional ablation of Cdc42 in the mouse intestinal epithelium resulted in the formation of large intracellular vacuolar structures containing microvilli (microvillus inclusion bodies) in epithelial enterocytes, a phenotype reminiscent of human microvillus inclusion disease (MVID), a devastating congenital intestinal disorder that results in severe nutrient deprivation. Further analysis revealed that Cdc42-deficient stem cells had cell division defects, reduced capacity for clonal expansion and differentiation into Paneth cells, and increased apoptosis. Cdc42 deficiency impaired Rab8a activation and its association with multiple effectors, and prevented trafficking of Rab8a vesicles to the midbody. This impeded cytokinesis, triggering crypt apoptosis and disrupting epithelial morphogenesis. Rab8a was also required for Cdc42-GTP activity in the intestinal epithelium, where continued cell division takes place. Furthermore, mice haploinsufficient for both Cdc42 and Rab8a in the intestine demonstrated abnormal crypt morphogenesis and epithelial transporter physiology, further supporting their functional interaction. These data suggest that defects of the stem cell niche can cause MVID. This hypothesis represents a conceptual departure from the conventional view of this disease, which has focused on the affected enterocytes, and suggests stem cell–based approaches could be beneficial to infants with this often lethal condition.
Stimulation of natural killer cells with a CD137-specific antibody enhances trastuzumab efficacy in xenotransplant models of breast cancer
Holbrook E. Kohrt, Roch Houot, Kipp Weiskopf, Matthew J. Goldstein, Ferenc Scheeren, Debra Czerwinski, A. Dimitrios Colevas, Wen-Kai Weng, Michael F. Clarke, Robert W. Carlson, Frank E. Stockdale, Joseph A. Mollick, Lieping Chen, Ronald LevyAbstract | Full text | PDF | Supplemental material (Page 1066)
Trastuzumab, a monoclonal antibody targeting human epidermal growth factor receptor 2 (HER2; also known as HER-2/neu), is indicated for the treatment of women with either early stage or metastatic HER2+ breast cancer. It kills tumor cells by several mechanisms, including antibody-dependent cellular cytotoxicity (ADCC). Strategies that enhance the activity of ADCC effectors, including NK cells, may improve the efficacy of trastuzumab. Here, we have shown that upon encountering trastuzumab-coated, HER2-overexpressing breast cancer cells, human NK cells become activated and express the costimulatory receptor CD137. CD137 activation, which was dependent on NK cell expression of the FcγRIII receptor, occurred both in vitro and in the peripheral blood of women with HER2-expressing breast cancer after trastuzumab treatment. Stimulation of trastuzumab-activated human NK cells with an agonistic mAb specific for CD137 killed breast cancer cells (including an intrinsically trastuzumab-resistant cell line) more efficiently both in vitro and in vivo in xenotransplant models of human breast cancer, including one using a human primary breast tumor. The enhanced cytotoxicity was restricted to antibody-coated tumor cells. This sequential antibody strategy, combining a tumor-targeting antibody with a second antibody that activates the host innate immune system, may improve the therapeutic effects of antibodies against breast cancer and other HER2-expressing tumors.
Brief Report Direct and efficient cellular transformation of primary rat mesenchymal precursor cells by KSHV
Tiffany Jones, Fengchun Ye, Roble Bedolla, Yufei Huang, Jia Meng, Liwu Qian, Hongyi Pan, Fuchun Zhou, Rosalie Moody, Brent Wagner, Mazen Arar, Shou-Jiang GaoAbstract | Full text | PDF | Supplemental material (Page 1076)
Infections by viruses are associated with approximately 12% of human cancer. Kaposi’s sarcoma-associated herpesvirus (KSHV) is causally linked to several malignancies commonly found in AIDS patients. The mechanism of KSHV-induced oncogenesis remains elusive, due in part to the lack of an adequate experimental system for cellular transformation of primary cells. Here, we report efficient infection and cellular transformation of primary rat embryonic metanephric mesenchymal precursor cells (MM cells) by KSHV. Cellular transformation occurred at as early as day 4 after infection and in nearly all infected cells. Transformed cells expressed hallmark vascular endothelial, lymphatic endothelial, and mesenchymal markers and efficiently induced tumors in nude mice. KSHV established latent infection in MM cells, and lytic induction resulted in low levels of detectable infectious virions despite robust expression of lytic genes. Most KSHV-induced tumor cells were in a latent state, although a few showed heterogeneous expression of lytic genes. This efficient system for KSHV cellular transformation of primary cells might facilitate the study of growth deregulation mechanisms resulting from KSHV infections.
DC-derived IL-18 drives Treg differentiation, murine Helicobacter pylori–specific immune tolerance, and asthma protection
Mathias Oertli, Malin Sundquist, Iris Hitzler, Daniela B. Engler, Isabelle C. Arnold, Sebastian Reuter, Joachim Maxeiner, Malin Hansson, Christian Taube, Marianne Quiding-Järbrink, Anne MüllerAbstract | Full text | PDF | Supplemental material (Page 1082)
Persistent colonization with the gastric bacterial pathogen Helicobacter pylori causes gastritis and predisposes infected individuals to gastric cancer. Conversely, it is also linked to protection from allergic, chronic inflammatory, and autoimmune diseases. We demonstrate here that H. pylori inhibits LPS-induced maturation of DCs and reprograms DCs toward a tolerance-promoting phenotype. Our results showed that DCs exposed to H. pylori in vitro or in vivo failed to induce T cell effector functions. Instead, they efficiently induced expression of the forkhead transcription factor FoxP3, the master regulator of Tregs, in naive T cells. Depletion of DCs in mice infected with H. pylori during the neonatal period was sufficient to break H. pylori–specific tolerance. DC depletion resulted in improved control of the infection but also aggravated T cell–driven immunopathology. Consistent with the mouse data, DCs infiltrating the gastric mucosa of human H. pylori carriers exhibited a semimature DC-SIGN+HLA–DRhiCD80loCD86lo phenotype. Mechanistically, the tolerogenic activity of H. pylori–experienced DCs was shown to require IL-18 in vitro and in vivo; DC-derived IL-18 acted directly on T cells to drive their conversion to Tregs. CD4+CD25+ Tregs from infected wild-type mice but not Il18–/– or Il18r1–/– mice prevented airway inflammation and hyperresponsiveness in an experimental model of asthma. Taken together, our results indicate that tolerogenic reprogramming of DCs ensures the persistence of H. pylori and protects against allergic asthma in a process that requires IL-18.
A microRNA-21 surge facilitates rapid cyclin D1 translation and cell cycle progression in mouse liver regeneration
Raymond Ng, Guisheng Song, Garrett R. Roll, Niels M. Frandsen, Holger WillenbringAbstract | Full text | PDF | Supplemental material (Page 1097)
MicroRNA-21 (miR-21) is thought to be an oncomir because it promotes cancer cell proliferation, migration, and survival. miR-21 is also expressed in normal cells, but its physiological role is poorly understood. Recently, it has been found that miR-21 expression is rapidly induced in rodent hepatocytes during liver regeneration after two-thirds partial hepatectomy (2/3 PH). Here, we investigated the function of miR-21 in regenerating mouse hepatocytes by inhibiting it with an antisense oligonucleotide. To maintain normal hepatocyte viability and function, we antagonized the miR-21 surge induced by 2/3 PH while preserving baseline expression. We found that knockdown of miR-21 impaired progression of hepatocytes into S phase of the cell cycle, mainly through a decrease in levels of cyclin D1 protein, but not Ccnd1 mRNA. Mechanistically, we discovered that increased miR-21 expression facilitated cyclin D1 translation in the early phase of liver regeneration by relieving Akt1/mTOR complex 1 signaling (and thus eIF-4F–mediated translation initiation) from suppression by Rhob. Our findings reveal that miR-21 enables rapid hepatocyte proliferation during liver regeneration by accelerating cyclin D1 translation.
Metabolic stress–induced activation of FoxO1 triggers diabetic cardiomyopathy in mice
Pavan K. Battiprolu, Berdymammet Hojayev, Nan Jiang, Zhao V. Wang, Xiang Luo, Myriam Iglewski, John M. Shelton, Robert D. Gerard, Beverly A. Rothermel, Thomas G. Gillette, Sergio Lavandero, Joseph A. HillAbstract | Full text | PDF | Supplemental material (Page 1109)
The leading cause of death in diabetic patients is cardiovascular disease; diabetic cardiomyopathy is typified by alterations in cardiac morphology and function, independent of hypertension or coronary disease. However, the molecular mechanism that links diabetes to cardiomyopathy is incompletely understood. Insulin resistance is a hallmark feature of diabetes, and the FoxO family of transcription factors, which regulate cell size, viability, and metabolism, are established targets of insulin and growth factor signaling. Here, we set out to evaluate a possible role of FoxO proteins in diabetic cardiomyopathy. We found that FoxO proteins were persistently activated in cardiac tissue in mice with diabetes induced either genetically or by high-fat diet (HFD). FoxO activity was critically linked with development of cardiomyopathy: cardiomyocyte-specific deletion of FoxO1 rescued HFD-induced declines in cardiac function and preserved cardiomyocyte insulin responsiveness. FoxO1-depleted cells displayed a shift in their metabolic substrate usage, from free fatty acids to glucose, associated with decreased accumulation of lipids in the heart. Furthermore, we found that FoxO1-dependent downregulation of IRS1 resulted in blunted Akt signaling and insulin resistance. Together, these data suggest that activation of FoxO1 is an important mediator of diabetic cardiomyopathy and is a promising therapeutic target for the disease.
Popeye domain containing proteins are essential for stress-mediated modulation of cardiac pacemaking in mice
Alexander Froese, Stephanie S. Breher, Christoph Waldeyer, Roland F.R. Schindler, Viacheslav O. Nikolaev, Susanne Rinné, Erhard Wischmeyer, Jan Schlueter, Jan Becher, Subreena Simrick, Franz Vauti, Juliane Kuhtz, Patrick Meister, Sonja Kreissl, Angela Torlopp, Sonja K. Liebig, Sandra Laakmann, Thomas D. Müller, Joachim Neumann, Juliane Stieber, Andreas Ludwig, Sebastian K. Maier, Niels Decher, Hans-Henning Arnold, Paulus Kirchhof, Larissa Fabritz, Thomas BrandAbstract | Full text | PDF | Supplemental material (Page 1119)
Cardiac pacemaker cells create rhythmic pulses that control heart rate; pacemaker dysfunction is a prevalent disorder in the elderly, but little is known about the underlying molecular causes. Popeye domain containing (Popdc) genes encode membrane proteins with high expression levels in cardiac myocytes and specifically in the cardiac pacemaking and conduction system. Here, we report the phenotypic analysis of mice deficient in Popdc1 or Popdc2. ECG analysis revealed severe sinus node dysfunction when freely roaming mutant animals were subjected to physical or mental stress. In both mutants, bradyarrhythmia developed in an age-dependent manner. Furthermore, we found that the conserved Popeye domain functioned as a high-affinity cAMP-binding site. Popdc proteins interacted with the potassium channel TREK-1, which led to increased cell surface expression and enhanced current density, both of which were negatively modulated by cAMP. These data indicate that Popdc proteins have an important regulatory function in heart rate dynamics that is mediated, at least in part, through cAMP binding. Mice with mutant Popdc1 and Popdc2 alleles are therefore useful models for the dissection of the mechanisms causing pacemaker dysfunction and could aid in the development of strategies for therapeutic intervention.
Synergy of understanding dermatologic disease and epidermal biology
Sphingosine-1-phosphate receptor-2 deficiency leads to inhibition of macrophage proinflammatory activities and atherosclerosis in apoE-deficient mice
Fei Wang, Yasuo Okamoto, Isao Inoki, Kazuaki Yoshioka, Wa Du, Xun Qi, Noriko Takuwa, Koichi Gonda, Yasuhiko Yamamoto, Ryunosuke Ohkawa, Takumi Nishiuchi, Naotoshi Sugimoto, Yutaka Yatomi, Kunitoshi Mitsumori, Masahide Asano, Makoto Kinoshita, Yoh TakuwaFull text | PDF | Original article (Page 1131)