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1.  A novel link between p53 and ROS 
Cell Cycle  2013;12(2):201.
doi:10.4161/cc.23418
PMCID: PMC3575445  PMID: 23287470
p53; ROS; NCF2/p67phox; NADPH oxidase; NOX2
2.  Refining the definition of hypereosinophilic syndrome 
Because of advances in our understanding of the hypereosinophilic syndrome (HES) and the availability of novel therapeutic agents, the original criteria defining these disorders are becoming increasingly problematic. Here, we discuss shortcomings with the current definition of HES and recent developments in the classification of these disorders. Despite significant progress in our understanding of the pathogenesis of some forms of HES, the current state of knowledge is still insufficient to formulate a new comprehensive etiologic definition of HESs. Nevertheless, we suggest a new working definition that overcomes some of the most obvious limitations with the original definition.
doi:10.1016/j.jaci.2010.03.042
PMCID: PMC3400024  PMID: 20639008
Definition; eosinophilia; eosinophilic leukemia; hypereosinophilic syndromes
3.  Caspase-8 is activated by cathepsin D initiating neutrophil apoptosis during the resolution of inflammation 
In the resolution of inflammatory responses, neutrophils rapidly undergo apoptosis. We describe a new proapoptotic pathway in which cathepsin D directly activates caspase-8. Cathepsin D is released from azurophilic granules in neutrophils in a caspase-independent but reactive oxygen species–dependent manner. Under inflammatory conditions, the translocation of cathepsin D in the cytosol is blocked. Pharmacological or genetic inhibition of cathepsin D resulted in delayed caspase activation and reduced neutrophil apoptosis. Cathepsin D deficiency or lack of its translocation in the cytosol prolongs innate immune responses in experimental bacterial infection and in septic shock. Thus, we identified a new function of azurophilic granules that is in addition to their role in bacterial defense mechanisms: to regulate the life span of neutrophils and, therefore, the duration of innate immune responses through the release of cathepsin D.
doi:10.1084/jem.20072152
PMCID: PMC2275389  PMID: 18299403
4.  Inflammation-associated Cell Cycle–independent Block of Apoptosis by Survivin in Terminally Differentiated Neutrophils 
The Journal of Experimental Medicine  2004;199(10):1343-1354.
Survivin has received great attention due to its expression in many human tumors and its potential as a therapeutic target in cancer. Survivin expression has been described to be cell cycle–dependent and restricted to the G2-M checkpoint, where it inhibits apoptosis in proliferating cells. In agreement with this current view, we found that survivin expression was high in immature neutrophils, which proliferate during differentiation. In contrast with immature cells, mature neutrophils contained only little or no survivin protein. Strikingly, these cells reexpressed survivin upon granulocyte/macrophage colony-stimulating factor (CSF) or granulocyte CSF stimulation in vitro and under inflammatory conditions in vivo. Moreover, survivin-deficient mature neutrophils were unable to increase their lifespan after survival factor exposure. Together, our findings demonstrate the following: (a) overexpression of survivin occurs in primary, even terminally differentiated cells and is not restricted to proliferating cells; and (b) survivin acts as an inhibitor of apoptosis protein in a cell cycle–independent manner. Therefore, survivin plays distinct and independent roles in the maintenance of the G2-M checkpoint and in apoptosis control, and its overexpression is not restricted to proliferating cells. These data provide new insights into the regulation and function of survivin and have important implications for the pathogenesis, diagnosis, and treatment of inflammatory diseases and cancer.
doi:10.1084/jem.20032033
PMCID: PMC2211817  PMID: 15148334
antisense; cancer; cytokines; differentiation; infection
5.  Disruption of Fas Receptor Signaling by Nitric Oxide in Eosinophils  
It has been suggested that Fas ligand–Fas receptor interactions are involved in the regulation of eosinophil apoptosis and that dysfunctions in this system could contribute to the accumulation of these cells in allergic and asthmatic diseases. Here, we demonstrate that nitric oxide (NO) specifically prevents Fas receptor–mediated apoptosis in freshly isolated human eosinophils. In contrast, rapid acceleration of eosinophil apoptosis by activation of the Fas receptor occurs in the presence of eosinophil hematopoietins. Analysis of the intracellular mechanisms revealed that NO disrupts Fas receptor–mediated signaling events at the level of, or proximal to, Jun kinase (JNK), but distal to sphingomyelinase (SMase) activation and ceramide generation. In addition, activation of SMase occurs downstream of an interleukin 1 converting enzyme–like (ICE-like) protease(s) that is not blocked by NO. However, NO prevents activation of a protease that targets lamin B1. These findings suggest a role for an additional NO-sensitive apoptotic signaling pathway that amplifies the proteolytic cascade initialized by activation of the Fas receptor. Therefore, NO concentrations within allergic inflammatory sites may be important in determining whether an eosinophil survives or undergoes apoptosis upon Fas ligand stimulation.
PMCID: PMC2212112  PMID: 9449721
6.  Contemporary consensus proposal on criteria and classification of eosinophilic disorders and related syndromes 
Eosinophilia is an important indicator of various neoplastic and nonneoplastic conditions. Depending on the underlying disease and mechanisms, eosinophil infiltration can lead to organ dysfunction, clinical symptoms, or both. During the past 2 decades, several different classifications of eosinophilic disorders and related syndromes have been proposed in various fields of medicine. Although criteria and definitions are, in part, overlapping, no global consensus has been presented to date. The Year 2011 Working Conference on Eosinophil Disorders and Syndromes was organized to update and refine the criteria and definitions for eosinophilic disorders and to merge prior classifications in a contemporary multidisciplinary schema. A panel of experts from the fields of immunology, allergy, hematology, and pathology contributed to this project. The expert group agreed on unifying terminologies and criteria and a classification that delineates various forms of hypereosinophilia, including primary and secondary variants based on specific hematologic and immunologic conditions, and various forms of the hypereosinophilic syndrome. For patients in whom no underlying disease or hypereosinophilic syndrome is found, the term hypereosinophilia of undetermined significance is introduced. The proposed novel criteria, definitions, and terminologies should assist in daily practice, as well as in the preparation and conduct of clinical trials.
doi:10.1016/j.jaci.2012.02.019
PMCID: PMC4091810  PMID: 22460074
Hypereosinophilic syndrome; eosinophilic leukemia; criteria; classification; hypereosinophilia of undetermined significance
7.  Interactions between Siglec-7/9 receptors and ligands influence NK cell–dependent tumor immunosurveillance 
The Journal of Clinical Investigation  2014;124(4):1810-1820.
Alteration of the surface glycosylation pattern on malignant cells potentially affects tumor immunity by directly influencing interactions with glycan-binding proteins (lectins) on the surface of immunomodulatory cells. The sialic acid–binding Ig-like lectins Siglec-7 and -9 are MHC class I–independent inhibitory receptors on human NK cells that recognize sialic acid–containing carbohydrates. Here, we found that the presence of Siglec-9 defined a subset of cytotoxic NK cells with a mature phenotype and enhanced chemotactic potential. Interestingly, this Siglec-9+ NK cell population was reduced in the peripheral blood of cancer patients. Broad analysis of primary tumor samples revealed that ligands of Siglec-7 and -9 were expressed on human cancer cells of different histological types. Expression of Siglec-7 and -9 ligands was associated with susceptibility of NK cell–sensitive tumor cells and, unexpectedly, of presumably NK cell–resistant tumor cells to NK cell–mediated cytotoxicity. Together, these observations have direct implications for NK cell–based therapies and highlight the requirement to consider both MHC class I haplotype and tumor-specific glycosylation.
doi:10.1172/JCI65899
PMCID: PMC3973073  PMID: 24569453
9.  Workshop Report from the NIH Taskforce on the Research Needs of Eosinophil-Associated Diseases (TREAD) 
Background
Eosinophils are blood cells that are often found in high numbers in the tissues of allergic conditions and helminthic parasite infections. The pathophysiological roles that eosinophils may serve in other human ‘eosinophil-associated’ diseases remain obscure.
Objective
NIH Institutes and the Office of Disease Prevention assembled an international taskforce of clinical and basic scientists with the charge to propose and prioritize unmet research needs in eosinophil-associated diseases.
Methods
The taskforce used an organ system approach to dissect out the different and common themes of eosinophil cell involvement in these diseases. In early 2012, a draft document was circulated for review. The document was amended and the prioritizations were set at a NIH-organized workshop in June 2012.
Results
The taskforce identified significant research needs. These needs cross disease entities but some are disease-specific. There are substantial shortcomings to the various preclinical animal models, as well as significant gaps in our epidemiologic, pathophysiologic, diagnostic, prognostic and therapeutic knowledge. The taskforce recognized that recent efforts by patient advocacy groups have played instrumental roles in improving the identification and characterization of these disorders. However, communication amongst the eosinophil interested communities, e.g., governmental funding and regulatory agencies, and industry and clinician scientists need to be more comprehensive.
Conclusions
Significant efforts are required to address our knowledge gaps in order to improve the outcomes of eosinophil-associated diseases. NIH Institutes, other federal agencies, lay organizations and the pharmaceutical industry should consider the taskforce’s recommendations in their future research activities.
doi:10.1016/j.jaci.2012.07.024
PMCID: PMC3432981  PMID: 22935587
eosinophil; apoptosis; therapeutics; research; discovery; fusion oncoproteins; hypereosinophilic syndromes; Churg-Strauss syndrome; coagulopathy
10.  Novel targeted therapies for eosinophilic disorders 
Hypereosinophilic syndromes (HESs) are a diverse group of conditions characterized by clinical manifestations attributable to eosinophilia and eosinophilic infiltration of tissues. HESs are chronic disorders with significant morbidity and mortality. Although the availability of targeted chemotherapeutic agents, including imatinib, has improved quality of life and survival in some patients with HESs, additional agents with increased efficacy and decreased toxicity are sorely needed. The purpose of this review is to provide an overview of eosinophil biology with an emphasis on potential targets of pharmacotherapy and to provide a summary of potential eosinophil-targeting agents, including those in development, in clinical trials, or approved for other disorders.
doi:10.1016/j.jaci.2012.07.027
PMCID: PMC3625625  PMID: 22935585
Hypereosinophilic syndromes; eosinophil-associated gastrointestinal disorders; eosinophilic esophagitis; Churg-Strauss syndrome; IL-5; mepolizumab; reslizumab
11.  Pathogenesis and classification of eosinophil disorders: a review of recent developments in the field 
Expert review of hematology  2012;5(2):157-176.
Eosinophils and their products play an essential role in the pathogenesis of various reactive and neoplastic disorders. Depending on the underlying disease, molecular defect and involved cytokines, hypereosinophilia may develop and may lead to organ damage. In other patients, persistent eosinophilia is accompanied by typical clinical findings, but the causative role and impact of eosinophilia remain uncertain. For patients with eosinophil-mediated organ pathology, early therapeutic intervention with agents reducing eosinophil counts can be effective in limiting or preventing irreversible organ damage. Therefore, it is important to approach eosinophil disorders and related syndromes early by using established criteria, to perform all appropriate staging investigations, and to search for molecular targets of therapy. In this article, we review current concepts in the pathogenesis and evolution of eosinophilia and eosinophil-related organ damage in neoplastic and non-neoplastic conditions. In addition, we discuss classifications of eosinophil disorders and related syndromes as well as diagnostic algorithms and standard treatment for various eosinophil-related disorders.
doi:10.1586/ehm.11.81
PMCID: PMC3625626  PMID: 22475285
classification; eosinophilic leukemia; FIP1L1-PDGFRA; hypereosinophilia; hypereosinophilic syndromes; targeted therapy
12.  Therapeutic Approaches to Patients with Hypereosinophilic Syndromes 
Seminars in Hematology  2012;49(2):160-170.
doi:10.1053/j.seminhematol.2012.01.002
PMCID: PMC3314228  PMID: 22449626
14.  ICON: Eosinophil Disorders 
doi:10.1097/WOX.0b013e31827f4192
PMCID: PMC3651188  PMID: 23282419
eosinophil disorders; hypereosinophilic syndrome (HES); global consensus; classification
15.  Eosinophil and neutrophil extracellular DNA traps in human allergic asthmatic airways 
Background
Asthma is a heterogeneous inflammatory airway disorder which involves eosinophilic and non-eosinophilic phenotypes. Unlike in normal lungs, eosinophils are often present in atopic asthmatic airways although a subpopulation of asthmatic subjects predominantly develops neutrophilic inflammation. Recently, it has been demonstrated that eosinophils and neutrophils generate bactericidal extracellular traps consisting of DNA and cytotoxic granule proteins.
Objective
To explore if living eosinophils and neutrophils infiltrating human atopic asthmatic airways actively form extracellular DNA traps in vivo.
Methods
Quantitative analysis of eosinophils-releasing DNA was performed in endobronchial biopsies from 20 mild human atopic asthmatics at baseline and after local allergen challenge, and 10 normal subjects. DNA was stained with propidium iodine and major basic protein (MBP) with specific antibody. Differential cell counts and cytokines/chemokines were assessed in bronchoalveolar fluids.
Results
Asthmatic airways were infiltrated with a significantly higher number of eosinophils than normal airways (39.3±4.6 vs. 0.4±0.9, p<0.0001). All asthmatics but only one control subject expressed eosinophils releasing DNA that colocalized with MBP (33.65±20.33 vs. 0.3±0.9 per hpf, p<0.0001). Four asthmatics mostly expressed neutrophilic inflammation and neutrophil DNA traps. Allergen challenge had no significant quantitative effect on eosinophil or neutrophil DNA traps. Airway eosinophils or DNA traps did not correlate with either BAL levels of IL-5, IFN-γ, or eotaxin, or the provoking doses of methacholine or allergen in asthmatics.
Conclusions
Extracellular DNA traps are generated by eosinophils and neutrophils in human atopic asthmatic airways in vivo. The mechanism and role of this new finding will necessitate further investigation.
Clinical implications
Eosinophil extracellular traps (EETs) (1) are frequently seen in atopic asthma, (2) consist of DNA and granule proteins, and (3) might be a new useful biomarker reflecting eosinophil activation.
doi:10.1016/j.jaci.2010.12.1103
PMCID: PMC3085562  PMID: 21315435
Atopic asthma; BAL fluid; biomarker; eosinophils; extracellular DNA traps; human; neutrophils
16.  A Differential Concentration-Dependent Effect of IVIg on Neutrophil Functions: Relevance for Anti-Microbial and Anti-Inflammatory Mechanisms 
PLoS ONE  2011;6(10):e26469.
Background
Polymorphonuclear neutrophils (PMN) play a key role in host defences against invading microorganisms but can also potentiate detrimental inflammatory reactions in case of excessive or misdirected responses. Intravenous immunoglobulins (IVIg) are used to treat patients with immune deficiencies and, at higher doses, in autoimmune, allergic and systemic inflammatory disorders.
Methodology/Principal Findings
We used flow cytometry to examine the effects of IVIg on PMN functions and survival, using whole-blood conditions in order to avoid artifacts due to isolation procedures. IVIg at low concentrations induced PMN activation, as reflected by decreased L-selectin and increased CD11b expression at the PMN surface, oxidative burst enhancement, and prolonged cell survival. In contrast, IVIg at higher concentrations inhibited LPS-induced CD11b degranulation and oxidative burst priming, and counteracted LPS-induced PMN lifespan prolongation.
Conclusions/Significance
IVIg appears to have differential, concentration-dependent effects on PMN, possibly supporting the use of IVIg as either an anti-microbial or an anti-inflammatory agent.
doi:10.1371/journal.pone.0026469
PMCID: PMC3204983  PMID: 22065996
17.  Systems medicine and integrated care to combat chronic noncommunicable diseases 
Genome Medicine  2011;3(7):43.
We propose an innovative, integrated, cost-effective health system to combat major non-communicable diseases (NCDs), including cardiovascular, chronic respiratory, metabolic, rheumatologic and neurologic disorders and cancers, which together are the predominant health problem of the 21st century. This proposed holistic strategy involves comprehensive patient-centered integrated care and multi-scale, multi-modal and multi-level systems approaches to tackle NCDs as a common group of diseases. Rather than studying each disease individually, it will take into account their intertwined gene-environment, socio-economic interactions and co-morbidities that lead to individual-specific complex phenotypes. It will implement a road map for predictive, preventive, personalized and participatory (P4) medicine based on a robust and extensive knowledge management infrastructure that contains individual patient information. It will be supported by strategic partnerships involving all stakeholders, including general practitioners associated with patient-centered care. This systems medicine strategy, which will take a holistic approach to disease, is designed to allow the results to be used globally, taking into account the needs and specificities of local economies and health systems.
doi:10.1186/gm259
PMCID: PMC3221551  PMID: 21745417
18.  Hypereosinophilic syndromes: a multicenter, retrospective analysis of clinical characteristics and response to therapy 
Background
Hypereosinophilic syndromes (HES) are a heterogeneous group of rare disorders defined by persistent blood eosinophilia ≥1.5 × 109/L, absence of a secondary cause, and evidence of eosinophil-associated pathology. With the exception of a recent multicenter trial of mepolizumab (anti-IL-5 monoclonal antibody), published therapeutic experience has been restricted to case reports and small case series.
Objective
The purpose of the study was to collect and summarize baseline demographic, clinical and laboratory characteristics in a large, diverse cohort of patients with HES and to review responses to treatment with conventional and novel therapies.
Methods
Clinical and laboratory data from 188 patients with HES, seen between January 2001 and December 2006 at eleven institutions in the United States and Europe, were collected retrospectively by chart review.
Results
Eighteen of 161 patients (11%) tested were FIP1L1-PDGFRA mutation-positive and 29/168 patients tested (17%) had a demonstrable aberrant or clonal T cell population. Corticosteroid monotherapy induced complete or partial responses at 1 month in 85% (120/141) of patients with most remaining on maintenance doses (median 10 mg prednisone equivalent daily for 2 months-20 years). Hydroxyurea and interferon-alpha (used in 64 and 46 patients, respectively) were also effective, but their use was limited by toxicity. Imatinib (used in 68 patients) was more effective in patients with the FIP1L1-PDGFRA mutation (88%) than in those without (23%; p<0.001).
Conclusion
This study, the largest clinical analysis of patients with HES to date, not only provides useful information for clinicians but should stimulate prospective trials to optimize treatment of HES.
doi:10.1016/j.jaci.2009.09.022
PMCID: PMC2829669  PMID: 19910029
eosinophil; hypereosinophilic syndrome; FIP1L1-PDGFRA
19.  Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes 
Klionsky, Daniel J. | Abeliovich, Hagai | Agostinis, Patrizia | Agrawal, Devendra K. | Aliev, Gjumrakch | Askew, David S. | Baba, Misuzu | Baehrecke, Eric H. | Bahr, Ben A. | Ballabio, Andrea | Bamber, Bruce A. | Bassham, Diane C. | Bergamini, Ettore | Bi, Xiaoning | Biard-Piechaczyk, Martine | Blum, Janice S. | Bredesen, Dale E. | Brodsky, Jeffrey L. | Brumell, John H. | Brunk, Ulf T. | Bursch, Wilfried | Camougrand, Nadine | Cebollero, Eduardo | Cecconi, Francesco | Chen, Yingyu | Chin, Lih-Shen | Choi, Augustine | Chu, Charleen T. | Chung, Jongkyeong | Clarke, Peter G.H. | Clark, Robert S.B. | Clarke, Steven G. | Clavé, Corinne | Cleveland, John L. | Codogno, Patrice | Colombo, María I. | Coto-Montes, Ana | Cregg, James M. | Cuervo, Ana Maria | Debnath, Jayanta | Demarchi, Francesca | Dennis, Patrick B. | Dennis, Phillip A. | Deretic, Vojo | Devenish, Rodney J. | Di Sano, Federica | Dice, J. Fred | DiFiglia, Marian | Dinesh-Kumar, Savithramma | Distelhorst, Clark W. | Djavaheri-Mergny, Mojgan | Dorsey, Frank C. | Dröge, Wulf | Dron, Michel | Dunn, William A. | Duszenko, Michael | Eissa, N. Tony | Elazar, Zvulun | Esclatine, Audrey | Eskelinen, Eeva-Liisa | Fésüs, László | Finley, Kim D. | Fuentes, José M. | Fueyo, Juan | Fujisaki, Kozo | Galliot, Brigitte | Gao, Fen-Biao | Gewirtz, David A. | Gibson, Spencer B. | Gohla, Antje | Goldberg, Alfred L. | Gonzalez, Ramon | González-Estévez, Cristina | Gorski, Sharon | Gottlieb, Roberta A. | Häussinger, Dieter | He, You-Wen | Heidenreich, Kim | Hill, Joseph A. | Høyer-Hansen, Maria | Hu, Xun | Huang, Wei-Pang | Iwasaki, Akiko | Jäättelä, Marja | Jackson, William T. | Jiang, Xuejun | Jin, Shengkan | Johansen, Terje | Jung, Jae U. | Kadowaki, Motoni | Kang, Chanhee | Kelekar, Ameeta | Kessel, David H. | Kiel, Jan A.K.W. | Kim, Hong Pyo | Kimchi, Adi | Kinsella, Timothy J. | Kiselyov, Kirill | Kitamoto, Katsuhiko | Knecht, Erwin | Komatsu, Masaaki | Kominami, Eiki | Kondo, Seiji | Kovács, Attila L. | Kroemer, Guido | Kuan, Chia-Yi | Kumar, Rakesh | Kundu, Mondira | Landry, Jacques | Laporte, Marianne | Le, Weidong | Lei, Huan-Yao | Lenardo, Michael J. | Levine, Beth | Lieberman, Andrew | Lim, Kah-Leong | Lin, Fu-Cheng | Liou, Willisa | Liu, Leroy F. | Lopez-Berestein, Gabriel | López-Otín, Carlos | Lu, Bo | Macleod, Kay F. | Malorni, Walter | Martinet, Wim | Matsuoka, Ken | Mautner, Josef | Meijer, Alfred J. | Meléndez, Alicia | Michels, Paul | Miotto, Giovanni | Mistiaen, Wilhelm P. | Mizushima, Noboru | Mograbi, Baharia | Monastyrska, Iryna | Moore, Michael N. | Moreira, Paula I. | Moriyasu, Yuji | Motyl, Tomasz | Münz, Christian | Murphy, Leon O. | Naqvi, Naweed I. | Neufeld, Thomas P. | Nishino, Ichizo | Nixon, Ralph A. | Noda, Takeshi | Nürnberg, Bernd | Ogawa, Michinaga | Oleinick, Nancy L. | Olsen, Laura J. | Ozpolat, Bulent | Paglin, Shoshana | Palmer, Glen E. | Papassideri, Issidora | Parkes, Miles | Perlmutter, David H. | Perry, George | Piacentini, Mauro | Pinkas-Kramarski, Ronit | Prescott, Mark | Proikas-Cezanne, Tassula | Raben, Nina | Rami, Abdelhaq | Reggiori, Fulvio | Rohrer, Bärbel | Rubinsztein, David C. | Ryan, Kevin M. | Sadoshima, Junichi | Sakagami, Hiroshi | Sakai, Yasuyoshi | Sandri, Marco | Sasakawa, Chihiro | Sass, Miklós | Schneider, Claudio | Seglen, Per O. | Seleverstov, Oleksandr | Settleman, Jeffrey | Shacka, John J. | Shapiro, Irving M. | Sibirny, Andrei | Silva-Zacarin, Elaine C.M. | Simon, Hans-Uwe | Simone, Cristiano | Simonsen, Anne | Smith, Mark A. | Spanel-Borowski, Katharina | Srinivas, Vickram | Steeves, Meredith | Stenmark, Harald | Stromhaug, Per E. | Subauste, Carlos S. | Sugimoto, Seiichiro | Sulzer, David | Suzuki, Toshihiko | Swanson, Michele S. | Tabas, Ira | Takeshita, Fumihiko | Talbot, Nicholas J. | Tallóczy, Zsolt | Tanaka, Keiji | Tanaka, Kozo | Tanida, Isei | Taylor, Graham S. | Taylor, J. Paul | Terman, Alexei | Tettamanti, Gianluca | Thompson, Craig B. | Thumm, Michael | Tolkovsky, Aviva M. | Tooze, Sharon A. | Truant, Ray | Tumanovska, Lesya V. | Uchiyama, Yasuo | Ueno, Takashi | Uzcátegui, Néstor L. | van der Klei, Ida | Vaquero, Eva C. | Vellai, Tibor | Vogel, Michael W. | Wang, Hong-Gang | Webster, Paul | Wiley, John W. | Xi, Zhijun | Xiao, Gutian | Yahalom, Joachim | Yang, Jin-Ming | Yap, George | Yin, Xiao-Ming | Yoshimori, Tamotsu | Yu, Li | Yue, Zhenyu | Yuzaki, Michisuke | Zabirnyk, Olga | Zheng, Xiaoxiang | Zhu, Xiongwei | Deter, Russell L.
Autophagy  2007;4(2):151-175.
Research in autophagy continues to accelerate,1 and as a result many new scientists are entering the field. Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms. Recent reviews have described the range of assays that have been used for this purpose.2,3 There are many useful and convenient methods that can be used to monitor macroautophagy in yeast, but relatively few in other model systems, and there is much confusion regarding acceptable methods to measure macroautophagy in higher eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers of autophagosomes versus those that measure flux through the autophagy pathway; thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from fully functional autophagy that includes delivery to, and degradation within, lysosomes (in most higher eukaryotes) or the vacuole (in plants and fungi). Here, we present a set of guidelines for the selection and interpretation of the methods that can be used by investigators who are attempting to examine macroautophagy and related processes, as well as by reviewers who need to provide realistic and reasonable critiques of papers that investigate these processes. This set of guidelines is not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to verify an autophagic response.
PMCID: PMC2654259  PMID: 18188003
autolysosome; autophagosome; flux; lysosome; phagophore; stress; vacuole
20.  T cell–mediated Fas-induced keratinocyte apoptosis plays a key pathogenetic role in eczematous dermatitis 
Clinical and histologic similarities between various eczematous disorders point to a common efferent pathway. We demonstrate here that activated T cells infiltrating the skin in atopic dermatitis (AD) and allergic contact dermatitis (ACD) induce keratinocyte (KC) apoptosis. KCs normally express low levels of Fas receptor (FasR) that can be substantially enhanced by the presence of IFN-γ. KCs are rendered susceptible to apoptosis by IFN-γ when FasR numbers reach a threshold of approximately 40,000 per KC. Subsequently, KCs undergo apoptosis induced by anti-FasR mAb’s, soluble Fas ligand, supernatants from activated T cells, or direct contact between T cells and KCs. Apoptotic KCs show typical DNA fragmentation and membrane phosphatidylserine expression. KC apoptosis was demonstrated in situ in lesional skin affected by AD, ACD, and patch tests. Using numerous cytokines and anti-cytokine neutralizing mAb’s, we found no evidence that cytokines other than IFN-γ participate in this process. In addition, apoptosis-inducing pathways other than FasR triggering were ruled out by blocking T cell–induced KC apoptosis by caspase inhibitors and soluble Fas-Fc protein. Responses of normal human skin and cultured skin equivalents to activated T cells demonstrated that KC apoptosis caused by skin-infiltrating T cells is a key event in the pathogenesis of eczematous dermatitis.
PMCID: PMC517909  PMID: 10880045
21.  Mesenchymal stem cells derived from human induced pluripotent stem cells modulate T-cell phenotypes in allergic rhinitis 
Allergy  2012;67(10):1215-1222.
Background
Human induced pluripotent stem cells (iPSCs) possess remarkable self-renewal capacity and the potential to differentiate into novel cell types, such as mesenchymal stem cells (MSCs). iPSC-MSCs have been shown to enhance tissue regeneration and attenuate tissue ischaemia; however, their contribution to the immune regulation of Th2-skewed allergic rhinitis (AR) and asthma remains unclear.
Objective
This study compared the immunomodulatory effects of iPSC-MSCs and bone marrow-derived MSCs (BM-MSCs) on lymphocyte proliferation, T-cell phenotypes and cytokine production in peripheral blood mononuclear cells (PBMCs) in patients with AR, and investigated the possible molecular mechanisms underlying the immunomodulatory properties of iPSC-MSCs.
Methods
In co-cultures of PBMCs with iPSC-MSCs or BM-MSCs, lymphocyte proliferation was evaluated using 3H-thymidine (3H-TdR) uptake, carboxyfluorescein diacetate, succinimidyl ester (CFDA-SE) assays; the regulatory T-cell (Treg) phenotype was determined by flow cytometry, and cytokine levels were measured using an enzyme-linked immunosorbent assay. The immunomodulatory properties of both MSCs were further evaluated using NS398 and transwell experiments.
Results
Similar to BM-MSCs, we determined that iPSC-MSCs significantly inhibit lymphocyte proliferation and promote Treg response in PBMCs (P < 0.05). Accordingly, the cytokine milieu (IFN-γ, IL-4, IL-5, IL-10 and IL-13) in the supernatants of PBMCs changed significantly (P < 0.05). The immunomodulatory properties of iPSC-MSCs and BM-MSCs were associated with prostaglandin E2 (PGE2) production and cell–cell contact.
Conclusions
These data demonstrate that iPSC-MSCs are capable of modulating T-cell phenotypes towards Th2 suppression through inducing Treg expansion, suggesting that iPSC-MSCs can be used as an alternative candidate to adult MSCs to treat allergic airway diseases.
doi:10.1111/j.1398-9995.2012.02875.x.
PMCID: PMC3555482  PMID: 22882409
allergic rhinitis; immunomodulation; induced pluripotent stem cells; mesenchymal stem cells; T cell
22.  ATG5 is induced by DNA-damaging agents and promotes mitotic catastrophe independent of autophagy 
Nature Communications  2013;4:2130.
Anticancer drug therapy activates both molecular cell death and autophagy pathways. Here we show that even sublethal concentrations of DNA-damaging drugs, such as etoposide and cisplatin, induce the expression of autophagy-related protein 5 (ATG5), which is both necessary and sufficient for the subsequent induction of mitotic catastrophe. We demonstrate that ATG5 translocates to the nucleus, where it physically interacts with survivin in response to DNA-damaging agents both in vitro and in carcinoma tissues obtained from patients who had undergone radiotherapy and/or chemotherapy. As a consequence, elements of the chromosomal passenger complex are displaced during mitosis, resulting in chromosome misalignment and segregation defects. Pharmacological inhibition of autophagy does not prevent ATG5-dependent mitotic catastrophe, but shifts the balance to an early caspase-dependent cell death. Our data suggest a dual role for ATG5 in response to drug-induced DNA damage, where it acts in two signalling pathways in two distinct cellular compartments, the cytosol and the nucleus.
The protein ATG5 is known to be involved in the formation of autophagosomes. Here, Maskey et al. identify a new role of ATG5 in response to drug-induced DNA damage whereby ATG5 translocates to the nucleus, leading to chromosome misalignment and mitotic catastrophe.
doi:10.1038/ncomms3130
PMCID: PMC3753548  PMID: 23945651

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