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Engelmaier A, Zimmermann M, Butterweck HA, Weber A. Clinical Study Support by Long-Term Stability Studies of Alpha 1-Proteinase Inhibitor and Urea in Relevant Biological Matrices. Pharmaceuticals (Basel) 2025; 18:572. [PMID: 40284007 PMCID: PMC12030187 DOI: 10.3390/ph18040572] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/21/2025] [Revised: 04/10/2025] [Accepted: 04/12/2025] [Indexed: 04/29/2025] Open
Abstract
Background/Objectives: According to recent guidelines, including the guideline on bioanalytical method validation issued by the European Medicine Agency, the stability of clinical analytes should be known. We summarize human α1-proteinase inhibitor (A1PI) and urea stability data in relevant matrices, as these analytes are usually measured in clinical A1PI studies. Methods: Stability samples with appropriate A1PI concentrations were prepared in a citrated human reference plasma pool and a matrix mimicking bronchoalveolar lavage (BAL) solution. These samples were kept at -20 °C and -60 °C for up to 24 months. A1PI protein was measured with a nephelometric method and an enzyme-linked immunosorbent assay using paired commercially available polyclonal antibodies. A1PI elastase inhibitory activity was determined with an elastase complex formation immunosorbent assay that combines A1PI complex formation with a solid phase-immobilized elastase and immunological detection of the formed A1PI-elastase complex and urea in samples kept at -20 °C only with an enzymatic assay. Results: Overall, the stability criterion (100 ± 20%) was met for the analytes A1PI protein and A1PI activity at both temperatures during storage of BAL-mimicking and plasma samples for 15 and 24 months, respectively; urea was stable in both matrices at -20 °C for 18 months. Plasma samples showed smaller drops in concentration over storage time than BAL-mimicking samples. As expected, the reduction of A1PI elastase inhibitory activity was more pronounced than that of A1PI protein. Interestingly, the analyte concentration did not significantly influence the results in either of the sample matrices. Conclusions: The data confirmed the appropriate stability of the three analytes in the matrices of citrated plasma and BAL-mimicking samples for at least up to 15 months.
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Affiliation(s)
- Andrea Engelmaier
- Pharmaceutical Science, Baxalta Innovations GmbH, Part of Takeda, 1220 Vienna, Austria;
| | - Martin Zimmermann
- Plasma Derived Therapies R&D, Baxalta Innovations GmbH, Part of Takeda, 1220 Vienna, Austria; (M.Z.); (H.A.B.)
| | - Harald A. Butterweck
- Plasma Derived Therapies R&D, Baxalta Innovations GmbH, Part of Takeda, 1220 Vienna, Austria; (M.Z.); (H.A.B.)
| | - Alfred Weber
- Plasma Derived Therapies R&D, Baxalta Innovations GmbH, Part of Takeda, 1220 Vienna, Austria; (M.Z.); (H.A.B.)
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Korkmaz-Icöz S, Szabó G, Gieldon A, McDonald PP, Dashkevich A, Yildirim AÖ, Korkmaz B. Protective effects of neutrophil serine protease inhibition against ischemia-reperfusion injury in lung or heart transplantation. FEBS J 2025. [PMID: 39854149 DOI: 10.1111/febs.17411] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/19/2024] [Revised: 12/15/2024] [Accepted: 01/13/2025] [Indexed: 01/26/2025]
Abstract
Transplanted organs are inevitably exposed to ischemia-reperfusion (IR) injury, which is known to cause graft dysfunction. Functional and structural changes that follow IR tissue injury are mediated by neutrophils through the production of oxygen-derived free radicals, as well as from degranulation which entails the release of proteases and other pro-inflammatory mediators. Neutrophil serine proteases (NSPs) are believed to be the principal triggers of post-ischemic reperfusion damage. Extended preservation times for the transplanted donor organ correlate with heightened occurrences of vascular damage and graft dysfunction. Preservation with α1-antitrypsin, an endogenous inhibitor of NSPs, improves primary graft function after lung or heart transplantation. Furthermore, pre-operative pharmacological targeting of NSP activation in the recipient using chemical inhibitors suppresses neutrophilic inflammation in transplanted organs. Hence, effective control of NSPs in the graft and recipient is a promising strategy to prevent IR injury. In this review, we describe the pathological functions of NSPs in IR injury and discuss their pharmacological inhibition to prevent primary graft dysfunction in lung or heart transplantation.
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Affiliation(s)
- Sevil Korkmaz-Icöz
- Department of Cardiac Surgery, University Hospital Heidelberg, Germany
- Department of Cardiac Surgery, University Hospital Halle (Saale), Germany
| | - Gábor Szabó
- Department of Cardiac Surgery, University Hospital Heidelberg, Germany
- Department of Cardiac Surgery, University Hospital Halle (Saale), Germany
| | | | | | - Alexey Dashkevich
- University Department of Cardiac Surgery, Leipzig Heart Center, Leipzig, Germany
| | - Ali Önder Yildirim
- Institute of Lung Health and Immunity (LHI), Comprehensive Pneumology Center (CPC), Helmholtz Munich, Member of the German Center for Lung Research (DZL), Munich, Germany
- Institute of Experimental Pneumology, Ludwig-Maximilians University (LMU), Munich, Germany
| | - Brice Korkmaz
- INSERM UMR-1100, "Research Center for Respiratory Diseases (CEPR)", Tours, France
- Université de Tours, France
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Roberts BS, Mitra D, Abishek S, Beher R, Satpute-Krishnan P. The p24-family and COPII subunit SEC24C facilitate the clearance of alpha1-antitrypsin Z from the endoplasmic reticulum to lysosomes. Mol Biol Cell 2024; 35:ar45. [PMID: 38294851 PMCID: PMC10916869 DOI: 10.1091/mbc.e23-06-0257] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/03/2023] [Revised: 01/16/2024] [Accepted: 01/24/2024] [Indexed: 02/01/2024] Open
Abstract
A subpopulation of the alpha-1-antitrypsin misfolding Z mutant (ATZ) is cleared from the endoplasmic reticulum (ER) via an ER-to-lysosome-associated degradation (ERLAD) pathway. Here, we report that the COPII subunit SEC24C and the p24-family of proteins facilitate the clearance of ATZ via ERLAD. In addition to the previously reported ERLAD components calnexin and FAM134B, we discovered that ATZ coimmunoprecipitates with the p24-family members TMP21 and TMED9. This contrasts with wild type alpha1-antitrypsin, which did not coimmunoprecipitate with FAM134B, calnexin or the p24-family members. Live-cell imaging revealed that ATZ and the p24-family members traffic together from the ER to lysosomes. Using chemical inhibitors to block ER exit or autophagy, we demonstrated that p24-family members and ATZ co-accumulate at SEC24C marked ER-exit sites or in ER-derived compartments, respectively. Furthermore, depletion of SEC24C, TMP21, or TMED9 inhibited lysosomal trafficking of ATZ and resulted in the increase of intracellular ATZ levels. Conversely, overexpression of these p24-family members resulted in the reduction of ATZ levels. Intriguingly, the p24-family members coimmunoprecipitate with ATZ, FAM134B, and SEC24C. Thus, we propose a model in which the p24-family functions in an adaptor complex linking SEC24C with the ERLAD machinery for the clearance of ATZ.
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Affiliation(s)
| | - Debashree Mitra
- Uniformed Services University of the Health Sciences, Bethesda, MD 20814
| | - Sudhanshu Abishek
- Uniformed Services University of the Health Sciences, Bethesda, MD 20814
| | - Richa Beher
- Uniformed Services University of the Health Sciences, Bethesda, MD 20814
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Quaglia A, Roberts EA, Torbenson M. Developmental and Inherited Liver Disease. MACSWEEN'S PATHOLOGY OF THE LIVER 2024:122-294. [DOI: 10.1016/b978-0-7020-8228-3.00003-x] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/02/2025]
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Korkmaz-Icöz S, Abulizi S, Li K, Korkmaz B, Georgevici AI, Sayour AA, Loganathan S, Canoglu H, Karck M, Szabó G. Preservation solution Custodiol containing human alpha-1-antitrypsin improves graft recovery after prolonged cold ischemic storage in a rat model of heart transplantation. Front Immunol 2023; 14:1155343. [PMID: 37426668 PMCID: PMC10323193 DOI: 10.3389/fimmu.2023.1155343] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/01/2023] [Accepted: 06/08/2023] [Indexed: 07/11/2023] Open
Abstract
Introduction The shortage of available donor hearts and the risk of ischemia/reperfusion injury restrict heart transplantation (HTX). Alpha-1-antitrypsin (AAT), a well-characterized inhibitor of neutrophil serine protease, is used in augmentation therapy to treat emphysema due to severe AAT deficiency. Evidence demonstrates its additional anti-inflammatory and tissue-protective effects. We hypothesized that adding human AAT in a preservation solution reduces graft dysfunction in a rat model of HTX following extended cold ischemic storage. Methods The hearts from isogenic Lewis donor rats were explanted, stored for either 1h or 5h in cold Custodiol supplemented with either vehicle (1h ischemia, n=7 or 5h ischemia, n=7 groups) or 1 mg/ml AAT (1h ischemia+AAT, n=7 or 5h ischemia+AAT, n=9 groups) before heterotopic HTX. Left-ventricular (LV) graft function was evaluated in vivo 1.5h after HTX. Immunohistochemical detection of myeloperoxydase (MPO) was performed in myocardial tissue and expression of 88 gene quantified with PCR was analyzed both statistical and with machine-learning methods. Results After HTX, LV systolic function (dP/dtmax 1h ischemia+AAT 4197 ± 256 vs 1h ischemia 3123 ± 110; 5h ischemia+AAT 2858 ± 154 vs 5h ischemia 1843 ± 104mmHg/s, p<0.05) and diastolic function (dP/dtmin 5h ischemia+AAT 1516 ± 68 vs 5h ischemia 1095 ± 67mmHg/s, p<0.05) at an intraventricular volume of 90µl were improved in the AAT groups compared with the corresponding vehicle groups. In addition, the rate pressure product (1h ischemia+AAT 53 ± 4 vs 1h ischemia 26 ± 1; 5h ischemia+AAT 37 ± 3 vs 5h ischemia 21 ± 1mmHg*beats/min at an intraventricular volume of 90µl; p<0.05) was increased in the AAT groups compared with the corresponding vehicle groups. Moreover, the 5h ischemia+AAT hearts exhibited a significant reduction in MPO-positive cell infiltration in comparison to the 5h ischemia group. Our computational analysis shows that ischemia+AAT network displays higher homogeneity, more positive and fewer negative gene correlations than the ischemia+placebo network. Discussion We provided experimental evidence that AAT protects cardiac grafts from prolonged cold ischemia during HTX in rats.
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Affiliation(s)
- Sevil Korkmaz-Icöz
- Department of Cardiac Surgery, University Hospital Heidelberg, Heidelberg, Germany
- Department of Cardiac Surgery, University Hospital Halle (Saale), Halle, Germany
| | - Sophia Abulizi
- Department of Cardiac Surgery, University Hospital Heidelberg, Heidelberg, Germany
| | - Kunsheng Li
- Department of Cardiac Surgery, University Hospital Heidelberg, Heidelberg, Germany
| | - Brice Korkmaz
- INSERM UMR-1100, “Research Center for Respiratory Diseases” and University of Tours, Tours, France
| | - Adrian-Iustin Georgevici
- Department of Cardiac Surgery, University Hospital Halle (Saale), Halle, Germany
- Department of Anaesthesiology, St. Josef Hospital, Ruhr-University Bochum, Bochum, Germany
- Heart and Vascular Center, Semmelweis University, Budapest, Hungary
| | - Alex Ali Sayour
- Department of Cardiac Surgery, University Hospital Heidelberg, Heidelberg, Germany
| | - Sivakkanan Loganathan
- Department of Cardiac Surgery, University Hospital Heidelberg, Heidelberg, Germany
- Department of Cardiac Surgery, University Hospital Halle (Saale), Halle, Germany
| | - Hansa Canoglu
- Department of Cardiac Surgery, University Hospital Heidelberg, Heidelberg, Germany
| | - Matthias Karck
- Department of Cardiac Surgery, University Hospital Heidelberg, Heidelberg, Germany
| | - Gábor Szabó
- Department of Cardiac Surgery, University Hospital Heidelberg, Heidelberg, Germany
- Department of Cardiac Surgery, University Hospital Halle (Saale), Halle, Germany
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Zubeldia-Varela E, Barker-Tejeda TC, Mera-Berriatua L, Bazire R, Cabrera-Freitag P, Ubeda C, Barber D, Francino MP, Rojo D, Ibáñez-Sandín MD, Pérez-Gordo M. Further Insights into the Gut Microbiota of Cow's Milk Allergic Infants: Analysis of Microbial Functionality and Its Correlation with Three Fecal Biomarkers. Int J Mol Sci 2023; 24:ijms24119247. [PMID: 37298198 DOI: 10.3390/ijms24119247] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/04/2023] [Revised: 05/17/2023] [Accepted: 05/19/2023] [Indexed: 06/12/2023] Open
Abstract
Cow's milk allergy (CMA) is one of the most prevalent food allergies in children. Several studies have demonstrated that gut microbiota influences the acquisition of oral tolerance to food antigens at initial stages of life. Changes in the gut microbiota composition and/or functionality (i.e., dysbiosis) have been linked to inadequate immune system regulation and the emergence of pathologies. Moreover, omic sciences have become an essential tool for the analysis of the gut microbiota. On the other hand, the use of fecal biomarkers for the diagnosis of CMA has recently been reviewed, with fecal calprotectin, α-1 antitrypsin, and lactoferrin being the most relevant. This study aimed at evaluating functional changes in the gut microbiota in the feces of cow's milk allergic infants (AI) compared to control infants (CI) by metagenomic shotgun sequencing and at correlating these findings with the levels of fecal biomarkers (α-1 antitrypsin, lactoferrin, and calprotectin) by an integrative approach. We have observed differences between AI and CI groups in terms of fecal protein levels and metagenomic analysis. Our findings suggest that AI have altered glycerophospholipid metabolism as well as higher levels of lactoferrin and calprotectin that could be explained by their allergic status.
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Affiliation(s)
- Elisa Zubeldia-Varela
- Departamento de Ciencias Médicas Básicas, Instituto de Medicina Molecular Aplicada (IMMA) Nemesio Díez, Facultad de Medicina, Universidad San Pablo-CEU, CEU Universities, 28668 Boadilla del Monte, Spain
| | - Tomás Clive Barker-Tejeda
- Departamento de Ciencias Médicas Básicas, Instituto de Medicina Molecular Aplicada (IMMA) Nemesio Díez, Facultad de Medicina, Universidad San Pablo-CEU, CEU Universities, 28668 Boadilla del Monte, Spain
- Centro de Metabolómica y Bioanálisis (CEMBIO), Facultad de Farmacia, Universidad San Pablo-CEU, CEU Universities, 28668 Boadilla del Monte, Spain
| | - Leticia Mera-Berriatua
- Departamento de Ciencias Médicas Básicas, Instituto de Medicina Molecular Aplicada (IMMA) Nemesio Díez, Facultad de Medicina, Universidad San Pablo-CEU, CEU Universities, 28668 Boadilla del Monte, Spain
| | - Raphaëlle Bazire
- Department of Allergy, H. Infantil Universitario Niño Jesús, FibHNJ, ARADyAL-RETICs Instituto de Salud Carlos III, IIS-P, 28031 Madrid, Spain
| | - Paula Cabrera-Freitag
- Allergy Paediatric Unit, Allergy Service, Hospital General Universitario Gregorio Marañón, Gregorio Marañón Health Research Institute (IiSGM), 28007 Madrid, Spain
| | - Carles Ubeda
- Fundació per al Foment de la Investigació Sanitària i Biomèdica de la Comunitat Valenciana (FISABIO), 46020 Valencia, Spain
- CIBER en Epidemiología y Salud Pública, 28029 Madrid, Spain
| | - Domingo Barber
- Departamento de Ciencias Médicas Básicas, Instituto de Medicina Molecular Aplicada (IMMA) Nemesio Díez, Facultad de Medicina, Universidad San Pablo-CEU, CEU Universities, 28668 Boadilla del Monte, Spain
| | - María Pilar Francino
- CIBER en Epidemiología y Salud Pública, 28029 Madrid, Spain
- Joint Research Unit in Genomics and Health, Fundació per al Foment de la Investigació Sanitària i Biomèdica de la Comunitat Valenciana (FISABIO) and Institut de Biologia Integrativa de Sistemes (Universitat de València/Consejo Superior de Investigaciones Científicas), Avda. Catalunya 21, 46020 València, Spain
| | - David Rojo
- Centro de Metabolómica y Bioanálisis (CEMBIO), Facultad de Farmacia, Universidad San Pablo-CEU, CEU Universities, 28668 Boadilla del Monte, Spain
| | - María Dolores Ibáñez-Sandín
- Department of Allergy, H. Infantil Universitario Niño Jesús, FibHNJ, ARADyAL-RETICs Instituto de Salud Carlos III, IIS-P, 28031 Madrid, Spain
| | - Marina Pérez-Gordo
- Departamento de Ciencias Médicas Básicas, Instituto de Medicina Molecular Aplicada (IMMA) Nemesio Díez, Facultad de Medicina, Universidad San Pablo-CEU, CEU Universities, 28668 Boadilla del Monte, Spain
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Miranda-Galvis M, Carneiro Soares C, Moretto Carnielli C, Ramalho Buttura J, Sales de Sá R, Kaminagakura E, Marchi FA, Paes Leme AF, Lópes Pinto CA, Santos-Silva AR, Moraes Castilho R, Kowalski LP, Squarize CH. New Insights into the Impact of Human Papillomavirus on Oral Cancer in Young Patients: Proteomic Approach Reveals a Novel Role for S100A8. Cells 2023; 12:cells12091323. [PMID: 37174723 PMCID: PMC10177374 DOI: 10.3390/cells12091323] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/08/2023] [Revised: 04/29/2023] [Accepted: 05/02/2023] [Indexed: 05/15/2023] Open
Abstract
Human papillomavirus (HPV) infection has recently been linked to a subset of cancers affecting the oral cavity. However, the molecular mechanisms underlying HPV-driven oral squamous cell carcinoma (OSCC) onset and progression are poorly understood. METHODS We performed MS-based proteomics profiling based on HPV status in OSCC in young patients, following biological characterization and cell assays to explore the proteome functional landscape. RESULTS Thirty-nine proteins are differentially abundant between HPV (+) and HPV (-) OSCC. Among them, COPS3, DYHC1, and S100A8 are unfavorable for tumor recurrence and survival, in contrast to A2M and Serpine1, low levels of which show an association with better DFS. Remarkably, S100A8 is considered an independent prognostic factor for lower survival rates, and at high levels, it alters tumor-associated immune profiling, showing a lower proportion of M1 macrophages and dendritic cells. HPV (+) OSCC also displayed the pathogen-associated patterns receptor that, when activated, triggered the S100A8 and NFκB inflammatory responses. CONCLUSION HPV (+) OSCC has a peculiar microenvironment pattern distinctive from HPV (-), involving the expression of pathogen-associated pattern receptors, S100A8 overexpression, and NFκB activation and responses, which has important consequences in prognosis and may guide therapeutic decisions.
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Affiliation(s)
- Marisol Miranda-Galvis
- Laboratory of Epithelial Biology, Department of Periodontics and Oral Medicine, University of Michigan School of Dentistry, Ann Arbor, MI 48109, USA
- Oral Diagnosis Department, Piracicaba Dental School, University of Campinas (UNICAMP), Piracicaba 13414-903, SP, Brazil
| | - Carolina Carneiro Soares
- Oral Diagnosis Department, Piracicaba Dental School, University of Campinas (UNICAMP), Piracicaba 13414-903, SP, Brazil
- Department of Microbiology, Immune Biology, and Genetics, Center for Molecular Biology, University of Vienna, 1030 Vienna, Austria
| | - Carolina Moretto Carnielli
- Brazilian Biosciences National Laboratory (LNBio), Brazilian Center for Research in Energy and Materials (CNPEM), Campinas 13083-970, SP, Brazil
| | - Jaqueline Ramalho Buttura
- Laboratory of Bioinformatics and Computational Biology, A.C.Camargo Cancer Center (CIPE), São Paulo 01508-010, SP, Brazil
| | - Raisa Sales de Sá
- Oral Diagnosis Department, Piracicaba Dental School, University of Campinas (UNICAMP), Piracicaba 13414-903, SP, Brazil
| | - Estela Kaminagakura
- Department of Bioscience and Oral Diagnosis, Science and Technology Institute, University of São Paulo State (UNESP), São José dos Campos 01049-010, SP, Brazil
| | - Fabio Albuquerque Marchi
- Center for Translational Research in Oncology, Cancer Institute of the State of São Paulo (ICESP), São Paulo 01246-000, SP, Brazil
- Comprehensive Center for Precision Oncology, University of São Paulo, São Paulo 05508-900, SP, Brazil
| | - Adriana Franco Paes Leme
- Brazilian Biosciences National Laboratory (LNBio), Brazilian Center for Research in Energy and Materials (CNPEM), Campinas 13083-970, SP, Brazil
| | - Clóvis A Lópes Pinto
- Department of Anatomic Pathology, A.C.Camargo Cancer Center, São Paulo 01509-001, SP, Brazil
| | - Alan Roger Santos-Silva
- Oral Diagnosis Department, Piracicaba Dental School, University of Campinas (UNICAMP), Piracicaba 13414-903, SP, Brazil
| | - Rogerio Moraes Castilho
- Laboratory of Epithelial Biology, Department of Periodontics and Oral Medicine, University of Michigan School of Dentistry, Ann Arbor, MI 48109, USA
- Rogel Cancer Center, University of Michigan, Ann Arbor, MI 48109, USA
| | - Luiz Paulo Kowalski
- Head and Neck Surgery Department, Medical School, University of São Paulo, São Paulo 05508-900, SP, Brazil
- Department of Head and Neck Surgery and Otorhinolaryngology, A.C.Camargo Cancer Center, São Paulo 01509-001, SP, Brazil
| | - Cristiane Helena Squarize
- Laboratory of Epithelial Biology, Department of Periodontics and Oral Medicine, University of Michigan School of Dentistry, Ann Arbor, MI 48109, USA
- Rogel Cancer Center, University of Michigan, Ann Arbor, MI 48109, USA
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Ding Q, Loganathan S, Zhou P, Sayour AA, Brlecic P, Radovits T, Domain R, Korkmaz B, Karck M, Szabó G, Korkmaz-Icöz S. Alpha-1-Antitrypsin Protects Vascular Grafts of Brain-Dead Rats Against Ischemia/Reperfusion Injury. J Surg Res 2023; 283:953-964. [PMID: 36915024 DOI: 10.1016/j.jss.2022.11.047] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/12/2021] [Revised: 11/04/2022] [Accepted: 11/20/2022] [Indexed: 12/14/2022]
Abstract
INTRODUCTION Endothelial dysfunction is a potential side effect of brain death (BD). Ischemia/reperfusion (IR) injury during heart transplantation may lead to further endothelial damage. Protective effects of alpha-1-antitrypsin (AAT), a human neutrophil serine protease inhibitor, have been demonstrated against IR injury. We hypothesized that AAT protects brain-dead rats' vascular grafts from IR injury. METHODS Donor rats were subjected to BD by inflation of a subdural balloon. After 5.5 h, aortic rings were immediately mounted in organ baths (BD, n = 6 rats) or preserved in saline, supplemented either with vehicle (BD-IR, n = 8 rats) or AAT (BD-IR + AAT, n = 14 rats) for 24 h. During organ bath experiment, rings from both IR groups were exposed to hypochlorite to simulate warm reperfusion-associated endothelial injury. Endothelial function was measured ex vivo. Immunohistochemical staining for caspases was carried out and DNA-strand breaks were evaluated using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling. Data are presented as median (interquartile range). RESULTS AAT improved IR-induced decreased maximum endothelium-dependent vasorelaxation to acetylcholine in the BD-IR + AAT aortas compared to the BD-IR group (BD: 83 (9-28) % versus BD-IR: 49 (39-60) % versus BD-IR + AAT: 64 (24-42) %, P < 0.05). Additionally, an increase in the rings' sensitivity to acetylcholine was noted after AAT (pD2-value: BD-IR + AAT: 7.35 (7.06-7.89) versus BD-IR: 6.96 (6.65-7.21), P < 0.05). Caspase-3, -8, -9, and -12 immunoreactivity and the number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive cells were significantly decreased by AAT. CONCLUSIONS AAT alleviates endothelial dysfunction, prevents increased caspase-3, -8, -9, and -12 levels, and decreases apoptotic DNA breakage due to BD and IR injury. This suggests that AAT treatment may be therapeutically beneficial to reduce IR-induced vascular damage.
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Affiliation(s)
- Qingwei Ding
- Department of Cardiac Surgery, University Hospital Heidelberg, Heidelberg, Germany
| | - Sivakkanan Loganathan
- Department of Cardiac Surgery, University Hospital Heidelberg, Heidelberg, Germany; Department of Cardiac Surgery, University Hospital Halle (Saale), Halle, Germany
| | - Pengyu Zhou
- Department of Cardiac Surgery, University Hospital Heidelberg, Heidelberg, Germany
| | - Alex Ali Sayour
- Department of Cardiac Surgery, University Hospital Heidelberg, Heidelberg, Germany; Heart and Vascular Center, Semmelweis University, Budapest, Hungary
| | - Paige Brlecic
- Department of Cardiac Surgery, University Hospital Heidelberg, Heidelberg, Germany
| | - Tamás Radovits
- Heart and Vascular Center, Semmelweis University, Budapest, Hungary
| | - Roxane Domain
- INSERM UMR-1100, "Research Center for Respiratory Diseases" and University of Tours, Tours, France
| | - Brice Korkmaz
- INSERM UMR-1100, "Research Center for Respiratory Diseases" and University of Tours, Tours, France
| | - Matthias Karck
- Department of Cardiac Surgery, University Hospital Heidelberg, Heidelberg, Germany
| | - Gábor Szabó
- Department of Cardiac Surgery, University Hospital Heidelberg, Heidelberg, Germany; Department of Cardiac Surgery, University Hospital Halle (Saale), Halle, Germany
| | - Sevil Korkmaz-Icöz
- Department of Cardiac Surgery, University Hospital Heidelberg, Heidelberg, Germany; Department of Cardiac Surgery, University Hospital Halle (Saale), Halle, Germany.
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Discovery of New 3,3-Diethylazetidine-2,4-dione Based Thiazoles as Nanomolar Human Neutrophil Elastase Inhibitors with Broad-Spectrum Antiproliferative Activity. Int J Mol Sci 2022; 23:ijms23147566. [PMID: 35886913 PMCID: PMC9321231 DOI: 10.3390/ijms23147566] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/07/2022] [Revised: 07/02/2022] [Accepted: 07/06/2022] [Indexed: 01/03/2023] Open
Abstract
A series of 3,3-diethylazetidine-2,4-dione based thiazoles 3a–3j were designed and synthesized as new human neutrophil elastase (HNE) inhibitors in nanomolar range. The representative compounds 3c, 3e, and 3h exhibit high HNE inhibitory activity with IC50 values of 35.02–44.59 nM, with mixed mechanism of action. Additionally, the most active compounds 3c and 3e demonstrate high stability under physiological conditions. The molecular docking study showed good correlation of the binding energies with the IC50 values, suggesting that the inhibition properties are largely dependent on the stage of ligand alignment in the binding cavity. The inhibition properties are correlated with the energy level of substrates of the reaction of ligand with Ser195. Moreover, most compounds showed high and broad-spectrum antiproliferative activity against human leukemia (MV4-11), human lung carcinoma (A549), human breast adenocarcinoma (MDA-MB-231), and urinary bladder carcinoma (UMUC-3), with IC50 values of 4.59–9.86 μM. Additionally, compounds 3c and 3e can induce cell cycle arrest at the G2/M phase and apoptosis via caspase-3 activation, leading to inhibition of A549 cell proliferation. These findings suggest that these new types of drugs could be used to treat cancer and other diseases in which immunoreactive HNE is produced.
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Koyuturk I, Kedia S, Robotham A, Star A, Brochu D, Sauvageau J, Kelly J, Gilbert M, Durocher Y. High-level production of wild-type and oxidation-resistant recombinant alpha-1-antitrypsin in glycoengineered CHO cells. Biotechnol Bioeng 2022; 119:2331-2344. [PMID: 35508753 DOI: 10.1002/bit.28129] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/21/2022] [Revised: 04/24/2022] [Accepted: 05/03/2022] [Indexed: 11/10/2022]
Abstract
Alpha-1-antitrypsin (A1AT) is a serine protease inhibitor which blocks the activity of serum proteases including neutrophil elastase to protect the lungs. Its deficiency is known to increase the risk of pulmonary emphysema as well as chronic obstructive pulmonary disease. Currently, the only treatment for patients with A1AT deficiency is weekly injection of plasma-purified A1AT. There is still today no commercial source of therapeutic recombinant A1AT, likely due to significant differences in expression host-specific glycosylation profile and/or high costs associated with the huge therapeutic dose needed. Accordingly, we aimed to produce high levels of recombinant wild-type A1AT, as well as a mutated protein (mutein) version for increased oxidation resistance, with N-glycans analogous to human plasma-derived A1AT. To achieve this, we disrupted two endogenous glycosyltransferase genes controlling core α-1,6-fucosylation (Fut8) and α-2,3-sialylation (ST3Gal4) in CHO cells using CRISPR/Cas9 technology, followed by overexpression of human α-2,6-sialyltransferase (ST6Gal1) using a cumate-inducible expression system. Volumetric A1AT productivity obtained from stable CHO pools was 2.5- to 6.5-fold higher with the cumate-inducible CR5 promoter compared to five strong constitutive promoters. Using the CR5 promoter, glycoengineered stable CHO pools were able to produce over 2.1 g/L and 2.8 g/L of wild-type and mutein forms of A1AT, respectively, with N-glycans analogous to the plasma-derived clinical product Prolastin-C. Supplementation of N-acetylmannosamine to the cell culture media during production increased the overall sialylation of A1AT as well as the proportion of bi-antennary and disialylated A2G2S2 N-glycans. These purified recombinant A1AT proteins showed in vitro inhibitory activity equivalent to Prolastin-C and substitution of methionine residues 351 and 358 with valines rendered A1AT significantly more resistant to oxidation. The recombinant A1AT mutein bearing an improved oxidation-resistance described in this study could represent a viable biobetter drug, offering a safe and more stable alternative for augmentation therapy. This article is protected by copyright. All rights reserved.
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Affiliation(s)
- Izel Koyuturk
- Department of Biochemistry and Molecular Medicine, Faculty of Medicine, Université de Montréal, Qc, Canada, H3C 3J7.,Life Sciences, Human Health Therapeutics Research Centre, Building Montreal-Royalmount, National Research Council Canada, Montréal, Qc, Canada, H4P 2R2
| | - Surbhi Kedia
- Department of Parasitology, Faculty of Agricultural and Environmental Sciences, McGill University, Qc, Canada, H9X 3V9
| | - Anna Robotham
- Life Sciences, Human Health Therapeutics Research Centre, 100 Sussex Drive, National Research Council Canada, Ottawa, Ontario, Canada, K1A OR6
| | - Alexandra Star
- Life Sciences, Human Health Therapeutics Research Centre, 100 Sussex Drive, National Research Council Canada, Ottawa, Ontario, Canada, K1A OR6
| | - Denis Brochu
- Life Sciences, Human Health Therapeutics Research Centre, 100 Sussex Drive, National Research Council Canada, Ottawa, Ontario, Canada, K1A OR6
| | - Janelle Sauvageau
- Life Sciences, Human Health Therapeutics Research Centre, 100 Sussex Drive, National Research Council Canada, Ottawa, Ontario, Canada, K1A OR6
| | - John Kelly
- Life Sciences, Human Health Therapeutics Research Centre, 100 Sussex Drive, National Research Council Canada, Ottawa, Ontario, Canada, K1A OR6
| | - Michel Gilbert
- Life Sciences, Human Health Therapeutics Research Centre, 100 Sussex Drive, National Research Council Canada, Ottawa, Ontario, Canada, K1A OR6
| | - Yves Durocher
- Department of Biochemistry and Molecular Medicine, Faculty of Medicine, Université de Montréal, Qc, Canada, H3C 3J7.,Life Sciences, Human Health Therapeutics Research Centre, Building Montreal-Royalmount, National Research Council Canada, Montréal, Qc, Canada, H4P 2R2
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11
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Im H, Lim J. Antioxidant Responses are Crucial for Defense against Misfolded Human
Z-Type α1-Antitrypsin. Protein Pept Lett 2022; 29:384-391. [DOI: 10.2174/0929866529666220321151913] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/20/2021] [Revised: 12/18/2021] [Accepted: 02/07/2022] [Indexed: 11/22/2022]
Abstract
Backgrounds:
The Z-type variant of human α1-antitrypsin is involved in liver cirrhosis
and pulmonary emphysema. Due to its slow folding characteristics, this variant accumulates folding
intermediates and forms protein aggregates within hepatocytes. Misfolded proteins may induce
oxidative stress and subsequent cell death.
Objective:
The potential application of antioxidant response signaling pathway and antioxidants to
cope with Z-type α1-antitrypsin-induced oxidative stress was evaluated.
Methods:
Overexpression of Z-type α1-antitrypsin in Saccharomyces cerevisiae provoked oxidative
stress and increased susceptibility to oxidative challenges such as hydrogen peroxide treatment.
Deletion of antioxidant-response genes, including yap1, skn7, sod2, tsa1, and pst2, exacerbated the
slow growth phenotype of Z-type α1-antitrypsin-expressing cells. Antioxidant treatment alleviated
oxidative stress and cytotoxicity induced by Z-type α1-antitrypsin.
Results:
Our results show that cellular antioxidant capacity is crucial to protection against
misfolded Z-type α1-antitrypsin.
Conclusion:
The information obtained here may be used to prevent oxidative stress caused by
misfolded proteins, which are associated with several degenerative diseases, including amyotrophic
lateral sclerosis and Parkinson’s disease.
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Affiliation(s)
- Hana Im
- Department of Integrative Bioscience and Biotechnology, Sejong University, Seoul, Republic of Korea
| | - Jaeyeon Lim
- Department of Integrative Bioscience and Biotechnology, Sejong University, Seoul, Republic of Korea
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12
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McElvaney OJ, McEvoy NL, Boland F, McElvaney OF, Hogan G, Donnelly K, Friel O, Browne E, Fraughen DD, Murphy MP, Clarke J, Choileáin ON, O'Connor E, McGuinness R, Boylan M, Kelly A, Hayden JC, Collins AM, Cullen A, Hyland D, Carroll TP, Geoghegan P, Laffey JG, Hennessy M, Martin-Loeches I, McElvaney NG, Curley GF. A randomized, double-blind, placebo-controlled trial of intravenous alpha-1 antitrypsin for acute respiratory distress syndrome secondary to COVID-19. MED 2022; 3:233-248.e6. [PMID: 35291694 PMCID: PMC8913266 DOI: 10.1016/j.medj.2022.03.001] [Citation(s) in RCA: 15] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/13/2022] [Revised: 02/11/2022] [Accepted: 03/07/2022] [Indexed: 11/16/2022]
Abstract
Background Patients with severe coronavirus disease 2019 (COVID-19) develop a febrile pro-inflammatory cytokinemia with accelerated progression to acute respiratory distress syndrome (ARDS). Here we report the results of a phase 2, multicenter, randomized, double-blind, placebo-controlled trial of intravenous (IV) plasma-purified alpha-1 antitrypsin (AAT) for moderate to severe ARDS secondary to COVID-19 (EudraCT 2020-001391-15). Methods Patients (n = 36) were randomized to receive weekly placebo, weekly AAT (Prolastin, Grifols, S.A.; 120 mg/kg), or AAT once followed by weekly placebo. The primary endpoint was the change in plasma interleukin (IL)-6 concentration at 1 week. In addition to assessing safety and tolerability, changes in plasma levels of IL-1β, IL-8, IL-10, and soluble tumor necrosis factor receptor 1 (sTNFR1) and clinical outcomes were assessed as secondary endpoints. Findings Treatment with IV AAT resulted in decreased inflammation and was safe and well tolerated. The study met its primary endpoint, with decreased circulating IL-6 concentrations at 1 week in the treatment group. This was in contrast to the placebo group, where IL-6 was increased. Similarly, plasma sTNFR1 was substantially decreased in the treatment group while remaining unchanged in patients receiving placebo. IV AAT did not definitively reduce levels of IL-1β, IL-8, and IL-10. No difference in mortality or ventilator-free days was observed between groups, although a trend toward decreased time on ventilator was observed in AAT-treated patients. Conclusions In patients with COVID-19 and moderate to severe ARDS, treatment with IV AAT was safe, feasible, and biochemically efficacious. The data support progression to a phase 3 trial and prompt further investigation of AAT as an anti-inflammatory therapeutic. Funding ECSA-2020-009; Elaine Galwey Research Bursary.
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Affiliation(s)
- Oliver J McElvaney
- Department of Medicine, Royal College of Surgeons in Ireland, Dublin, Ireland
- Beaumont Hospital, Dublin, Ireland
| | - Natalie L McEvoy
- Department of Anaesthesia and Critical Care, Royal College of Surgeons in Ireland, Dublin, Ireland
| | - Fiona Boland
- Data Science Centre, Division of Biostatistics and Population Health Sciences, Royal College of Surgeons in Ireland, Dublin, Ireland
| | - Oisín F McElvaney
- Department of Medicine, Royal College of Surgeons in Ireland, Dublin, Ireland
- Beaumont Hospital, Dublin, Ireland
| | - Grace Hogan
- Department of Anaesthesia and Critical Care, Royal College of Surgeons in Ireland, Dublin, Ireland
| | | | | | | | - Daniel D Fraughen
- Department of Medicine, Royal College of Surgeons in Ireland, Dublin, Ireland
- Beaumont Hospital, Dublin, Ireland
| | - Mark P Murphy
- Department of Medicine, Royal College of Surgeons in Ireland, Dublin, Ireland
| | - Jennifer Clarke
- Beaumont Hospital, Dublin, Ireland
- Department of Anaesthesia and Critical Care, Royal College of Surgeons in Ireland, Dublin, Ireland
| | | | | | | | | | | | - John C Hayden
- School of Pharmacy and Biomolecular Sciences, Royal College of Surgeons in Ireland, Dublin, Ireland
| | - Ann M Collins
- RCSI Education and Research Centre, Beaumont Hospital, Dublin, Ireland
| | - Ailbhe Cullen
- RCSI Education and Research Centre, Beaumont Hospital, Dublin, Ireland
| | - Deirdre Hyland
- RCSI Education and Research Centre, Beaumont Hospital, Dublin, Ireland
| | - Tomás P Carroll
- Department of Medicine, Royal College of Surgeons in Ireland, Dublin, Ireland
| | | | - John G Laffey
- Department of Anaesthesia, Galway University Hospitals, SAOLTA University Health Group, Galway, Ireland
| | - Martina Hennessy
- Department of Critical Care Medicine, St. James' Hospital, Dublin, Ireland
| | | | - Noel G McElvaney
- Department of Medicine, Royal College of Surgeons in Ireland, Dublin, Ireland
- Beaumont Hospital, Dublin, Ireland
| | - Gerard F Curley
- Beaumont Hospital, Dublin, Ireland
- Department of Anaesthesia and Critical Care, Royal College of Surgeons in Ireland, Dublin, Ireland
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13
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Hussein H, Kishen A. Application of Proteomics in Apical Periodontitis. FRONTIERS IN DENTAL MEDICINE 2022. [DOI: 10.3389/fdmed.2022.814603] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
Abstract
Apical periodontitis is an inflammatory reaction of the periradicular tissues as a consequence of multispecies microbial communities organized as biofilms within the root canal system. Periradicular tissue changes at the molecular level initiate and orchestrate the inflammatory process and precede the presentation of clinical symptoms. Inflammatory mediators have been studied at either the proteomic, metabolomic, or transcriptomic levels. Analysis at the protein level is the most common approach used to identify and quantify analytes from diseased periradicular tissues during root canal treatment, since it is more representative of definitive and active periradicular inflammatory mediator than its transcript expression level. In disease, proteins expressed in an altered manner could be utilized as biomarkers. Biomarker proteins in periradicular tissues have been qualitatively and quantitatively assessed using antibodies (immunoassays and immunostaining) or mass spectrometry-based approaches. Herein, we aim to provide a comprehensive understanding of biomarker proteins identified in clinical studies investigating periradicular lesions and pulp tissue associated with apical periodontitis using proteomics. The high throughput mass spectrometry-based proteomics has the potential to improve the current methods of monitoring inflammation while distinguishing between progressive, stable, and healing lesions for the identification of new diagnostic and therapeutic targets. This method would provide more objective tools to (a) discover biomarkers related to biological processes for better clinical case selection, and (b) determine tissue response to novel therapeutic interventions for more predictable outcomes in endodontic treatment.
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14
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Engelmaier A, Weber A. Sensitive and specific measurement of alpha 1-antitrypsin activity with an elastase complex formation immunosorbent assay (ECFISA). J Pharm Biomed Anal 2021; 209:114476. [PMID: 34838346 DOI: 10.1016/j.jpba.2021.114476] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/15/2021] [Revised: 11/10/2021] [Accepted: 11/11/2021] [Indexed: 11/16/2022]
Abstract
Functionally active alpha1-antitrypsin (AAT) is measured predominantly with a chromogenic elastase inhibition assay, where the concentration of AAT activity inversely correlates with the levels of residual elastase. This standard assay has moderate sensitivity as it hardly allows the measurement of samples containing less than 10 µg of functionally active AAT per mL. To overcome this drawback, we developed a new assay format for the measurement of functionally active AAT, which we termed the elastase complex formation immunosorbent assay (ECFISA). The ECFISA uses plate-bound, still proteolytically active elastase, which attacks functionally active AAT under irreversible formation of a stable stochiometric 1 + 1 complex. This complex is then detected and measured by an anti-AAT peroxidase conjugate. Using three different approaches for the preparation of functionally inactive AAT - heating, oxidation, and complex formation with elastase - we confirmed beyond doubt that the ECFISA exclusively measures functionally active AAT and that these measurements are unimpaired by the presence of high concentrations of functionally inactive AAT. Studies addressing the coating procedure demonstrated that adequate and robust conditions had been defined for this essential first step of the ECFISA. Possible interference caused by the presence of important plasma proteinase inhibitors in the test samples could be excluded for the most abundant inhibitors. Even a 1.5-times molar excess of alpha2-macroglobulin over AAT was shown to have no impact, which is not the case for a conventional chromogenic activity assay. Functional activities determined with the ECFISA and validated chromogenic elastase inhibition assay matched well with a mean absolute bias of 0.64% calculated for the 25 samples measured. The results of the bioanalytical assay validation complied with the acceptance criteria for ligand-binding assays as given by current guidelines on validation of bioanalytical methods. Overall, the data obtained demonstrated the ECFISA as an accurate, precise, selective, and very sensitive method for AAT activity measurement at low levels previously inaccessible for direct measurement.
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Affiliation(s)
- Andrea Engelmaier
- Analytical Development, Pharmaceutical Science, Baxalta Innovations GmbH, a Takeda Company, Industriestrasse 67A-1221 Vienna Wien, Austria.
| | - Alfred Weber
- R&D Plasma Derived Therapies, Baxalta Innovations GmbH, a Takeda Company, Austria.
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15
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Zubeldia-Varela E, Barker-Tejeda TC, Blanco-Pérez F, Infante S, Zubeldia JM, Pérez-Gordo M. Non-IgE-Mediated Gastrointestinal Food Protein-Induced Allergic Disorders. Clinical Perspectives and Analytical Approaches. Foods 2021; 10:foods10112662. [PMID: 34828942 PMCID: PMC8623505 DOI: 10.3390/foods10112662] [Citation(s) in RCA: 12] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/13/2021] [Accepted: 10/27/2021] [Indexed: 12/21/2022] Open
Abstract
Non-IgE-mediated gastrointestinal food allergy (non-IgE-GI-FA) is the name given to a series of pathologies whose main entities are food protein-induced allergic proctocolitis (FPIAP), food protein-induced enteropathy (FPE), and food protein-induced enterocolitis syndrome (FPIES). These are more uncommon than IgE-mediated food allergies, their mechanisms remain largely unknown, and their diagnosis is mainly done by clinical history, due to the lack of specific biomarkers. In this review, we present the latest advances found in the literature about clinical aspects, the current diagnosis, and treatment options of non-IgE-GI-FAs. We discuss the use of animal models, the analysis of gut microbiota, omics techniques, and fecal proteins with a focus on understanding the pathophysiological mechanisms of these pathologies and obtaining possible diagnostic and/or prognostic biomarkers. Finally, we discuss the unmet needs that researchers should tackle to advance in the knowledge of these barely explored pathologies.
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Affiliation(s)
- Elisa Zubeldia-Varela
- Department of Basic Medical Sciences, Facultad de Medicina, Institute of Applied Molecular Medicine (IMMA), Universidad San Pablo-CEU, CEU Universities, ARADyAL, 28660 Madrid, Spain; (E.Z.-V.); (T.C.B.-T.)
- Centre for Metabolomics and Bioanalysis (CEMBIO), Department of Chemistry and Biochemistry, Facultad de Farmacia, Universidad San Pablo-CEU, CEU Universities, Urbanización Montepríncipe, Boadilla del Monte, 28660 Madrid, Spain
| | - Tomás Clive Barker-Tejeda
- Department of Basic Medical Sciences, Facultad de Medicina, Institute of Applied Molecular Medicine (IMMA), Universidad San Pablo-CEU, CEU Universities, ARADyAL, 28660 Madrid, Spain; (E.Z.-V.); (T.C.B.-T.)
- Centre for Metabolomics and Bioanalysis (CEMBIO), Department of Chemistry and Biochemistry, Facultad de Farmacia, Universidad San Pablo-CEU, CEU Universities, Urbanización Montepríncipe, Boadilla del Monte, 28660 Madrid, Spain
| | - Frank Blanco-Pérez
- VPr1 Research Group “Molecular Allergology”, Paul-Ehrlich-Institut, Federal Institute for Vaccines and Biomedicines, 63225 Langen, Germany;
| | - Sonsoles Infante
- Allergy Paediatric Unit, Allergy Service, Hospital General Universitario Gregorio Marañón, 28007 Madrid, Spain; (S.I.); (J.M.Z.)
- Gregorio Marañón Health Research Institute (IiSGM), 28007 Madrid, Spain
| | - José M. Zubeldia
- Allergy Paediatric Unit, Allergy Service, Hospital General Universitario Gregorio Marañón, 28007 Madrid, Spain; (S.I.); (J.M.Z.)
- Gregorio Marañón Health Research Institute (IiSGM), 28007 Madrid, Spain
- Rare Diseases Networking Biomedical Research Centre (CIBERER, U-761), Carlos III Institute of Health, 28029 Madrid, Spain
| | - Marina Pérez-Gordo
- Department of Basic Medical Sciences, Facultad de Medicina, Institute of Applied Molecular Medicine (IMMA), Universidad San Pablo-CEU, CEU Universities, ARADyAL, 28660 Madrid, Spain; (E.Z.-V.); (T.C.B.-T.)
- Correspondence: ; Tel.: +34-91-372-4700 (ext. 14675)
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16
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Higgins NR, Greenslade JE, Wu JJ, Miranda E, Galliciotti G, Monteiro MJ. Serpin neuropathology in the P497S UBQLN2 mouse model of ALS/FTD. Brain Pathol 2021; 31:e12948. [PMID: 33780087 PMCID: PMC8387369 DOI: 10.1111/bpa.12948] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Revised: 02/17/2021] [Accepted: 03/08/2021] [Indexed: 01/12/2023] Open
Abstract
Accumulating evidence suggests X-linked dominant mutations in UBQLN2 cause amyotrophic lateral sclerosis (ALS) with frontotemporal dementia (FTD) through both loss- and gain-of-function mechanisms. However, the mechanisms by which the mutations cause disease are still unclear. The goal of the study was to uncover the possible pathomechanism(s) by which UBQLN2 mutations cause ALS/FTD. An analysis of proteomic changes in neuronal tissue was used to identify proteins with altered accumulation in the P497S UBQLN2 transgenic mouse model of ALS/FTD. We then used immunocytochemistry and biochemical techniques to confirm protein changes in the mutant P497S mice. Additionally, we used cell lines inactivated of UBQLN2 expression to determine whether its loss underlies the alteration in the proteins seen in P497S mice. The proteome screen identified a dramatic alteration of serine protease inhibitor (serpin) proteins in the mutant P497S animals. Double immunofluorescent staining of brain and spinal cord tissues of the mutant and control mice revealed an age-dependent change in accumulation of Serpin A1, C1, and I1 in puncta whose staining colocalized with UBQLN2 puncta in the mutant P497S mice. Serpin A1 aggregation in P497S animals was confirmed by biochemical extraction and filter retardation assays. A similar phenomenon of serpin protein aggregation was found in HeLa and NSC34 motor neuron cells with inactivated UBQLN2 expression. We found aberrant aggregation of serpin proteins, particularly Serpin A1, in the brain and spinal cord of the P497S UBQLN2 mouse model of ALS/FTD. Similar aggregation of serpin proteins was found in UBQLN2 knockout cells suggesting that serpin aggregation in the mutant P497S animals may stem from loss of UBQLN2 function. Because serpin aggregation is known to cause disease through both loss- and gain-of-function mechanisms, we speculate that their accumulation in the P497S mouse model of ALS/FTD may contribute to disease pathogenesis through similar mechanism(s).
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Affiliation(s)
- Nicole R. Higgins
- Program in Molecular MedicineUniversity of Maryland School of MedicineBaltimoreMDUSA
- Center for Biomedical Engineering and TechnologyDepartment of Anatomy and NeurobiologyUniversity of Maryland School of MedicineBaltimoreMDUSA
| | - Jessie E. Greenslade
- Center for Biomedical Engineering and TechnologyDepartment of Anatomy and NeurobiologyUniversity of Maryland School of MedicineBaltimoreMDUSA
| | - Josephine J. Wu
- Center for Biomedical Engineering and TechnologyDepartment of Anatomy and NeurobiologyUniversity of Maryland School of MedicineBaltimoreMDUSA
| | - Elena Miranda
- Department of Biology and Biotechnologies ‘Charles Darwin’Pasteur Institute – Cenci Bolognetti FoundationSapienza University of RomeRomeItaly
| | - Giovanna Galliciotti
- Institute of NeuropathologyUniversity Medical Center Hamburg‐EppendorfHamburgGermany
| | - Mervyn J. Monteiro
- Program in Molecular MedicineUniversity of Maryland School of MedicineBaltimoreMDUSA
- Center for Biomedical Engineering and TechnologyDepartment of Anatomy and NeurobiologyUniversity of Maryland School of MedicineBaltimoreMDUSA
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17
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Lim J, Lee K, Im H. Reinforcement of the Unfolded Protein Response Mitigates Cytotoxicity Induced by Human Z‐Type α
1
‐Antitrypsin. B KOREAN CHEM SOC 2021. [DOI: 10.1002/bkcs.12289] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Affiliation(s)
- Jaeyeon Lim
- Department of Integrative Bioscience and Biotechnology Sejong University Seoul 05006 South Korea
| | - Kyunghee Lee
- Department of Chemistry Sejong University Seoul 05006 South Korea
| | - Hana Im
- Department of Integrative Bioscience and Biotechnology Sejong University Seoul 05006 South Korea
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18
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McElvaney OJ, Curley GF, Rose-John S, McElvaney NG. Interleukin-6: obstacles to targeting a complex cytokine in critical illness. THE LANCET. RESPIRATORY MEDICINE 2021; 9:643-654. [PMID: 33872590 PMCID: PMC8051931 DOI: 10.1016/s2213-2600(21)00103-x] [Citation(s) in RCA: 135] [Impact Index Per Article: 33.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 11/24/2020] [Revised: 02/13/2021] [Accepted: 02/15/2021] [Indexed: 02/07/2023]
Abstract
Circulating concentrations of the pleiotropic cytokine interleukin-6 (IL-6) are known to be increased in pro-inflammatory critical care syndromes, such as sepsis and acute respiratory distress syndrome. Elevations in serum IL-6 concentrations in patients with severe COVID-19 have led to renewed interest in the cytokine as a therapeutic target. However, although the pro-inflammatory properties of IL-6 are widely known, the cytokine also has a series of important physiological and anti-inflammatory functions. An adequate understanding of the complex processes by which IL-6 signalling occurs is crucial for the correct interpretation of IL-6 concentrations in the blood or lung, the use of IL-6 as a critical care biomarker, or the design of effective anti-IL-6 strategies. Here, we outline the role of IL-6 in health and disease, explain the different types of IL-6 signalling and their contribution to the net biological effect of the cytokine, describe the approaches to IL-6 inhibition that are currently available, and discuss implications for the future use of treatments such as tocilizumab in the critical care setting.
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Affiliation(s)
- Oliver J McElvaney
- Department of Medicine, Royal College of Surgeons in Ireland, Dublin, Ireland; Beaumont Hospital, Dublin, Ireland
| | - Gerard F Curley
- Beaumont Hospital, Dublin, Ireland; Department of Anaesthesia and Critical Care, Royal College of Surgeons in Ireland, Dublin, Ireland
| | - Stefan Rose-John
- Biochemical Institute, Christian-Albrechts-Universität zu Kiel, Kiel, Germany
| | - Noel G McElvaney
- Department of Medicine, Royal College of Surgeons in Ireland, Dublin, Ireland; Beaumont Hospital, Dublin, Ireland.
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19
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Hamada M, Bhakta V, Andres SN, Sheffield WP. Stepwise Reversion of Multiply Mutated Recombinant Antitrypsin Reveals a Selective Inhibitor of Coagulation Factor XIa as Active as the M358R Variant. Front Cardiovasc Med 2021; 8:647405. [PMID: 33816577 PMCID: PMC8017132 DOI: 10.3389/fcvm.2021.647405] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/29/2020] [Accepted: 02/16/2021] [Indexed: 11/13/2022] Open
Abstract
Alpha-1 antitrypsin (AAT, also known as alpha-1 proteinase inhibitor or SERPINA1) is the most abundant member of the serpin superfamily found in human plasma. The naturally occurring variant AAT M358R, altered at the P1 position of the critical reactive center loop (RCL), is re-directed away from inhibition of AAT's chief natural target, neutrophil elastase, and toward accelerated inhibition of thrombin (FIIa), kallikrein (Kal), and other proteases such as factor XIa (FXIa). FXIa is an emerging target for the development of antithrombotic agents, since patients with FXI deficiency are protected from thromboembolic disease and do not exhibit a strong bleeding tendency. Previously, we used phage display, bacterial lysate screening, and combinatorial mutagenesis to identify AAT-RC, an engineered AAT M358R with additional changes between RCL positions P7-P3', CLEVEPR-STE [with changes bolded and the P1-P1' (R358-S359) reactive center shown as R-S]. AAT-RC was 279- and 16-fold more selective for FXIa/IIa or FXIa/Kal than AAT M358R; the increased selectivity came at a cost of a 2.3-fold decrease in the rate of FXIa inhibition and a 3.3-fold increase in the stoichiometry of inhibition (SI). Here, we asked which alterations in AAT-RC were most important for the observed increases in selectivity for FXIa inhibition. We back-mutated AAT-RC to AAT-RC-1 (P7-P3' FLEVEPRSTE), AAT-RC-2 (P7-P3' FLEAEPRSTE), and AAT RC-3 (P7-P3' FLEAIPR-STE). Proteins were expressed as cleavable, hexahistidine-tagged glutathione sulfotransferase fusion proteins in E. coli and purified by proteolytic elution from glutathione agarose, with polishing on nickel chelate agarose. Selectivity for FXIa over Kal of AAT-RC-1, −2, and −3 was 14, 21, and 2.3, respectively. AAT-RC-2 inhibited FXIa 31% more rapidly than AAT M358R, with the same SI, and enhanced selectivity for FXIa over Kal, FXa, FXIIa, activated protein C, and FIIa of 25-, 130-, 420-, 440-, and 470-fold, respectively. Structural modeling of the AAT-RC-2/FXIa encounter complex suggested that both E (Glu) substitutions at P3 and P3' may promote FXIa binding via hydrogen bonding to K192 in FXIa. AAT-RC-2 is the most selective and active AAT variant reported to date for FXIa inhibition and will be tested in animal models of thrombosis and bleeding.
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Affiliation(s)
- Mostafa Hamada
- Department of Pathology and Molecular Medicine, McMaster University, Hamilton, ON, Canada
| | - Varsha Bhakta
- Centre for Innovation, Canadian Blood Services, Hamilton, ON, Canada
| | - Sara N Andres
- Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, ON, Canada
| | - William P Sheffield
- Department of Pathology and Molecular Medicine, McMaster University, Hamilton, ON, Canada.,Centre for Innovation, Canadian Blood Services, Hamilton, ON, Canada
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Alpha-1 antitrypsin inhibits TMPRSS2 protease activity and SARS-CoV-2 infection. Nat Commun 2021; 12:1726. [PMID: 33741941 PMCID: PMC7979852 DOI: 10.1038/s41467-021-21972-0] [Citation(s) in RCA: 90] [Impact Index Per Article: 22.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/16/2020] [Accepted: 02/18/2021] [Indexed: 12/18/2022] Open
Abstract
SARS-CoV-2 is a respiratory pathogen and primarily infects the airway epithelium. As our knowledge about innate immune factors of the respiratory tract against SARS-CoV-2 is limited, we generated and screened a peptide/protein library derived from bronchoalveolar lavage for inhibitors of SARS-CoV-2 spike-driven entry. Analysis of antiviral fractions revealed the presence of α1-antitrypsin (α1AT), a highly abundant circulating serine protease inhibitor. Here, we report that α1AT inhibits SARS-CoV-2 entry at physiological concentrations and suppresses viral replication in cell lines and primary cells including human airway epithelial cultures. We further demonstrate that α1AT binds and inactivates the serine protease TMPRSS2, which enzymatically primes the SARS-CoV-2 spike protein for membrane fusion. Thus, the acute phase protein α1AT is an inhibitor of TMPRSS2 and SARS-CoV-2 entry, and may play an important role in the innate immune defense against the novel coronavirus. Our findings suggest that repurposing of α1AT-containing drugs has prospects for the therapy of COVID-19. Here, via screening of a polypeptide library from bronchoalveolar lavage, the authors identify and characterize α1-antitrypsin (α1AT) as SARS-CoV-2 inhibitor and show that α1AT binds and inactivates the serine protease TMPRSS2, which enzymatically primes the SARS-CoV-2 spike protein for membrane fusion.
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21
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The Discovery of Endoplasmic Reticulum Storage Disease. The Connection between an H&E Slide and the Brain. Int J Mol Sci 2021; 22:ijms22062899. [PMID: 33809321 PMCID: PMC8001541 DOI: 10.3390/ijms22062899] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/27/2021] [Revised: 03/06/2021] [Accepted: 03/09/2021] [Indexed: 12/31/2022] Open
Abstract
The revolutionary evolution in science and technology over the last few decades has made it possible to face more adequately three main challenges of modern medicine: changes in old diseases, the appearance of new diseases, and diseases that are unknown (mostly genetic), despite research efforts. In this paper we review the road travelled by pathologists in search of a method based upon the use of routine instruments and techniques which once were available for research only. The application to tissue studies of techniques from immunology, molecular biology, and genetics has allowed dynamic interpretations of biological phenomena with special regard to gene regulation and expression. That implies stepwise investigations, including light microscopy, immunohistochemistry, in situ hybridization, electron microscopy, molecular histopathology, protein crystallography, and gene sequencing, in order to progress from suggestive features detectable in routinely stained preparations to more characteristic, specific, and finally, pathognomonic features. Hematoxylin and Eosin (H&E)-stained preparations and appropriate immunohistochemical stains have enabled the recognition of phenotypic changes which may reflect genotypic alterations. That has been the case with hepatocytic inclusions detected in H&E-stained preparations, which appeared to correspond to secretory proteins that, due to genetic mutations, were retained within the rough endoplasmic reticulum (RER) and were deficient in plasma. The identification of this phenomenon affecting the molecules alpha-1-antitrypsin and fibrinogen has led to the discovery of a new field of cell organelle pathology, endoplasmic reticulum storage disease(s) (ERSD). Over fifty years, pathologists have wandered through a dark forest of complicated molecules with strange conformations, and by detailed observations in simple histopathological sections, accompanied by a growing background of molecular techniques and revelations, have been able to recognize and identify arrays of grotesque polypeptide arrangements.
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22
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Loureiro C, Buzalaf MAR, Moraes FRN, Ventura TMO, Pelá VT, Pessan JP, Jacinto RC. Quantitative proteomic analysis in symptomatic and asymptomatic apical periodontitis. Int Endod J 2021; 54:834-847. [PMID: 33480079 DOI: 10.1111/iej.13480] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/11/2020] [Accepted: 01/19/2021] [Indexed: 12/31/2022]
Abstract
AIM To quantitatively and qualitatively compare the host proteomic profile in samples of symptomatic and asymptomatic apical periodontitis (AP) using nano-liquid chromatography-electron spray tandem mass spectrometry. METHODOLOGY Samples were obtained from 18 patients with radiographically evident AP, divided into symptomatic and asymptomatic groups (nine per group) according to clinical characteristics. After sample collection, protein extraction, purification and quantification of the samples were performed, which were analysed by reverse-phase liquid chromatography coupled to mass spectrometry. Label-free quantitative proteomic analysis was performed by Protein Lynx Global Service software. Differences in expression of proteins between the groups were calculated using the Monte Carlo algorithm, considering P < 0.05 for down-regulated proteins and 1 - P > 0.95 for up-regulated proteins. Proteins were identified with the embedded ion accounting algorithm in the software and a search of the Homo sapiens UniProt database. RESULTS A total of 853 individual human proteins were identified. In the quantitative analysis, common proteins to both groups accounted for 143 proteins. Differences in expression between groups resulted in 51 up-regulated proteins (1 - P > 0.95) in the symptomatic group, including alpha-1-antitrypsin, protein S100-A8, myeloperoxidase, peroxiredoxin and lactotransferrin. This group also had 43 down-regulated proteins (P < 0.05), comprising immunoglobulin, neutrophil defensin, pyruvate kinase and alpha-enolase. The qualitative analysis considered only the exclusive proteins of each group. For the symptomatic group, 318 complete proteins and 29 fragments were identified, such as dedicator of cytokinesis protein, intersectin, prostaglandin, phospholipase DDHD2 and superoxide dismutase. For the asymptomatic group, 326 complete proteins and 37 fragments were identified, including azurocidin, C-reactive protein, collagen alpha, cathepsin, heat shock and laminin. CONCLUSIONS Quantitative differences in the expression of common proteins in cases of symptomatic and asymptomatic AP were found, which were mostly related to host immune response in both groups. Exclusive proteins in the symptomatic group were mainly related to the host response to the presence of viruses in endodontic infections, oxidative stress and proteolytic enzymes. The results provide a basis for a better understanding of cellular and molecular pathways involved in AP, establishing specific proteomic profiles for symptomatic and asymptomatic conditions.
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Affiliation(s)
- C Loureiro
- Department of Preventive and Restorative Dentistry, Aracatuba School of Dentistry, São Paulo State University (UNESP), Araçatuba, Brazil
| | - M A R Buzalaf
- Department of Biological Sciences, Bauru School of Dentistry, University of São Paulo, Bauru, Brazil
| | - F R N Moraes
- Department of Preventive and Restorative Dentistry, Aracatuba School of Dentistry, São Paulo State University (UNESP), Araçatuba, Brazil
| | - T M O Ventura
- Department of Biological Sciences, Bauru School of Dentistry, University of São Paulo, Bauru, Brazil
| | - V T Pelá
- Department of Genetics and Evolution, Federal University of Sao Carlos, São Carlos, Brazil
| | - J P Pessan
- Department of Preventive and Restorative Dentistry, Aracatuba School of Dentistry, São Paulo State University (UNESP), Araçatuba, Brazil
| | - R C Jacinto
- Department of Preventive and Restorative Dentistry, Aracatuba School of Dentistry, São Paulo State University (UNESP), Araçatuba, Brazil
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23
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Ulfig A, Leichert LI. The effects of neutrophil-generated hypochlorous acid and other hypohalous acids on host and pathogens. Cell Mol Life Sci 2021; 78:385-414. [PMID: 32661559 PMCID: PMC7873122 DOI: 10.1007/s00018-020-03591-y] [Citation(s) in RCA: 137] [Impact Index Per Article: 34.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/20/2020] [Revised: 06/21/2020] [Accepted: 07/01/2020] [Indexed: 12/15/2022]
Abstract
Neutrophils are predominant immune cells that protect the human body against infections by deploying sophisticated antimicrobial strategies including phagocytosis of bacteria and neutrophil extracellular trap (NET) formation. Here, we provide an overview of the mechanisms by which neutrophils kill exogenous pathogens before we focus on one particular weapon in their arsenal: the generation of the oxidizing hypohalous acids HOCl, HOBr and HOSCN during the so-called oxidative burst by the enzyme myeloperoxidase. We look at the effects of these hypohalous acids on biological systems in general and proteins in particular and turn our attention to bacterial strategies to survive HOCl stress. HOCl is a strong inducer of protein aggregation, which bacteria can counteract by chaperone-like holdases that bind unfolding proteins without the need for energy in the form of ATP. These chaperones are activated by HOCl through thiol oxidation (Hsp33) or N-chlorination of basic amino acid side-chains (RidA and CnoX) and contribute to bacterial survival during HOCl stress. However, neutrophil-generated hypohalous acids also affect the host system. Recent studies have shown that plasma proteins act not only as sinks for HOCl, but get actively transformed into modulators of the cellular immune response through N-chlorination. N-chlorinated serum albumin can prevent aggregation of proteins, stimulate immune cells, and act as a pro-survival factor for immune cells in the presence of cytotoxic antigens. Finally, we take a look at the emerging role of HOCl as a potential signaling molecule, particularly its role in neutrophil extracellular trap formation.
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Affiliation(s)
- Agnes Ulfig
- Ruhr University Bochum, Institute for Biochemistry and Pathobiochemistry-Microbial Biochemistry, Universitätsstrasse 150, 44780, Bochum, Germany
| | - Lars I Leichert
- Ruhr University Bochum, Institute for Biochemistry and Pathobiochemistry-Microbial Biochemistry, Universitätsstrasse 150, 44780, Bochum, Germany.
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24
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Zheng W, Wang Z, Jiang X, Zhao Q, Shen J. Targeted Drugs for Treatment of Pulmonary Arterial Hypertension: Past, Present, and Future Perspectives. J Med Chem 2020; 63:15153-15186. [PMID: 33314936 DOI: 10.1021/acs.jmedchem.0c01093] [Citation(s) in RCA: 21] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
Pulmonary arterial hypertension (PAH) is a devastating disease that can lead to right ventricular failure and premature death. Although approved drugs have been shown to be safe and effective, PAH remains a severe clinical condition, and the long-term survival of patients with PAH is still suboptimal. Thus, potential therapeutic targets and new agents to treat PAH are urgently needed. In recent years, a variety of related pathways and potential therapeutic targets have been found, which brings new hope for PAH therapy. In this perspective, not only are the marketed drugs used to treat PAH summarized but also the recently developed novel pharmaceutical therapies currently in clinical trials are discussed. Furthermore, the advances in natural products as potential treatment for PAH are also updated.
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Affiliation(s)
- Wei Zheng
- CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.,School of Pharmacy, University of the Chinese Academy of Sciences, Beijing 100049, China
| | - Zhen Wang
- CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
| | - Xiangrui Jiang
- CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
| | - Qingjie Zhao
- CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
| | - Jingshan Shen
- CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.,School of Pharmacy, University of the Chinese Academy of Sciences, Beijing 100049, China
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25
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McElvaney OJ, Hobbs BD, Qiao D, McElvaney OF, Moll M, McEvoy NL, Clarke J, O'Connor E, Walsh S, Cho MH, Curley GF, McElvaney NG. A linear prognostic score based on the ratio of interleukin-6 to interleukin-10 predicts outcomes in COVID-19. EBioMedicine 2020; 61:103026. [PMID: 33039714 PMCID: PMC7543971 DOI: 10.1016/j.ebiom.2020.103026] [Citation(s) in RCA: 65] [Impact Index Per Article: 13.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/15/2020] [Revised: 09/10/2020] [Accepted: 09/10/2020] [Indexed: 02/07/2023] Open
Abstract
BACKGROUND Prognostic tools are required to guide clinical decision-making in COVID-19. METHODS We studied the relationship between the ratio of interleukin (IL)-6 to IL-10 and clinical outcome in 80 patients hospitalized for COVID-19, and created a simple 5-point linear score predictor of clinical outcome, the Dublin-Boston score. Clinical outcome was analysed as a three-level ordinal variable ("Improved", "Unchanged", or "Declined"). For both IL-6:IL-10 ratio and IL-6 alone, we associated clinical outcome with a) baseline biomarker levels, b) change in biomarker level from day 0 to day 2, c) change in biomarker from day 0 to day 4, and d) slope of biomarker change throughout the study. The associations between ordinal clinical outcome and each of the different predictors were performed with proportional odds logistic regression. Associations were run both "unadjusted" and adjusted for age and sex. Nested cross-validation was used to identify the model for incorporation into the Dublin-Boston score. FINDINGS The 4-day change in IL-6:IL-10 ratio was chosen to derive the Dublin-Boston score. Each 1 point increase in the score was associated with a 5.6 times increased odds for a more severe outcome (OR 5.62, 95% CI -3.22-9.81, P = 1.2 × 10-9). Both the Dublin-Boston score and the 4-day change in IL-6:IL-10 significantly outperformed IL-6 alone in predicting clinical outcome at day 7. INTERPRETATION The Dublin-Boston score is easily calculated and can be applied to a spectrum of hospitalized COVID-19 patients. More informed prognosis could help determine when to escalate care, institute or remove mechanical ventilation, or drive considerations for therapies. FUNDING Funding was received from the Elaine Galwey Research Fellowship, American Thoracic Society, National Institutes of Health and the Parker B Francis Research Opportunity Award.
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Affiliation(s)
- Oliver J McElvaney
- Department of Medicine, Royal College of Surgeons in Ireland, Dublin, Ireland; Beaumont Hospital, Dublin, Ireland.
| | - Brian D Hobbs
- Channing Division of Network Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, United States; Division of Pulmonary and Critical Care Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, United States
| | - Dandi Qiao
- Channing Division of Network Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, United States
| | - Oisín F McElvaney
- Department of Medicine, Royal College of Surgeons in Ireland, Dublin, Ireland; Beaumont Hospital, Dublin, Ireland
| | - Matthew Moll
- Channing Division of Network Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, United States; Division of Pulmonary and Critical Care Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, United States
| | - Natalie L McEvoy
- Department of Anaesthesia and Critical Care Medicine, Royal College of Surgeons in Ireland, Dublin, Ireland
| | - Jennifer Clarke
- Beaumont Hospital, Dublin, Ireland; Department of Anaesthesia and Critical Care Medicine, Royal College of Surgeons in Ireland, Dublin, Ireland
| | | | | | - Michael H Cho
- Channing Division of Network Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, United States; Division of Pulmonary and Critical Care Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, United States
| | - Gerard F Curley
- Beaumont Hospital, Dublin, Ireland; Department of Anaesthesia and Critical Care Medicine, Royal College of Surgeons in Ireland, Dublin, Ireland
| | - Noel G McElvaney
- Department of Medicine, Royal College of Surgeons in Ireland, Dublin, Ireland; Beaumont Hospital, Dublin, Ireland.
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26
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27
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Yoosefian M, Mirhaji E, Moghani MZ, Ebrahimpour Y, Fouladi M. The effect of water/ethanol solvent mixtures on interactions of an antibody selective for wild-type alpha-1-antitrypsin in complex with its antigen. J Mol Liq 2020. [DOI: 10.1016/j.molliq.2020.113437] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
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28
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Pye A, Turner AM. Experimental and investigational drugs for the treatment of alpha-1 antitrypsin deficiency. Expert Opin Investig Drugs 2019; 28:891-902. [PMID: 31550938 DOI: 10.1080/13543784.2019.1672656] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/21/2023]
Abstract
Introduction: Alpha-1 antitrypsin deficiency (AATD) is most often associated with chronic lung disease, early onset emphysema, and liver disease. The standard of care in lung disease due to AATD is alpha-1 antitrypsin augmentation but there are several new and emerging treatment options under investigation for both lung and liver manifestations. Areas covered: We review therapeutic approaches to lung and liver disease in alpha-1 antitrypsin deficiency (AATD) and the agents in clinical development according to their mode of action. The focus is on products in clinical trials, but data from pre-clinical studies are described where relevant, particularly where progression to trials appears likely. Expert opinion: Clinical trials directed at lung and liver disease separately are now taking place. Multimodality treatment may be the future, but this could be limited by treatment costs. The next 5-10 years may reveal new guidance on when to use therapeutics for slowing disease progression with personalized treatment regimes coming to the forefront.
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Affiliation(s)
- Anita Pye
- Institute of Applied Health Research, University of Birmingham , Birmingham , UK
| | - Alice M Turner
- Institute of Applied Health Research, University of Birmingham , Birmingham , UK
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29
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McElvaney OJ, Wade P, Murphy M, Reeves EP, McElvaney NG. Targeting airway inflammation in cystic fibrosis. Expert Rev Respir Med 2019; 13:1041-1055. [PMID: 31530195 DOI: 10.1080/17476348.2019.1666715] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
Introduction: The major cause of morbidity and mortality in patients with cystic fibrosis (CF) is lung disease. Inflammation in the CF airways occurs from a young age and contributes significantly to disease progression and shortened life expectancy. Areas covered: In this review, we discuss the key immune cells involved in airway inflammation in CF, the contribution of the intrinsic genetic defect to the CF inflammatory phenotype, and anti-inflammatory strategies designed to overcome what is a critical factor in the pathogenesis of CF lung disease. Review of the literature was carried out using the MEDLINE (from 1975 to 2018), Google Scholar and The Cochrane Library databases. Expert opinion: Therapeutic interventions specifically targeting the defective CF transmembrane conductance regulator (CFTR) protein have changed the clinical landscape and significantly improved the outlook for CF. As survival estimates for people with CF increase, long-term management has become an important focus, with an increased need for therapies targeted at specific elements of inflammation, to complement CFTR modulator therapies.
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Affiliation(s)
- Oliver J McElvaney
- Irish Centre for Genetic Lung Disease, Royal College of Surgeons in Ireland, Beaumont Hospital , Dublin , Ireland
| | - Patricia Wade
- Irish Centre for Genetic Lung Disease, Royal College of Surgeons in Ireland, Beaumont Hospital , Dublin , Ireland
| | - Mark Murphy
- Irish Centre for Genetic Lung Disease, Royal College of Surgeons in Ireland, Beaumont Hospital , Dublin , Ireland
| | - Emer P Reeves
- Irish Centre for Genetic Lung Disease, Royal College of Surgeons in Ireland, Beaumont Hospital , Dublin , Ireland
| | - Noel G McElvaney
- Irish Centre for Genetic Lung Disease, Royal College of Surgeons in Ireland, Beaumont Hospital , Dublin , Ireland
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30
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Arora V, Cruz M, Lang J, Klos AM, Merritt WK, Price J, Taylor G, Vandeberg P, Wee K, Willis T. Comparison of the liquid and lyophilized formulations of Prolastin®-C for Alpha 1-Antitrypsin deficiency: Biochemical characteristics, pharmacokinetics, safety and neoantigenicity in rabbits. Biologicals 2019; 62:77-84. [PMID: 31522909 DOI: 10.1016/j.biologicals.2019.09.002] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/26/2019] [Revised: 08/22/2019] [Accepted: 09/04/2019] [Indexed: 11/25/2022] Open
Abstract
Multiple analytical and preclinical studies were performed to compare the biochemical characteristics, pharmacokinetics (PK), safety and neoantigenicity of a new 5% liquid formulation of Alpha-1 Proteinase Inhibitor (Liquid A1PI, Prolastin®-C Liquid) with the lyophilized version (Lyophilized A1PI, Prolastin®-C). Liquid A1PI and Lyophilized A1PI had similar average mass (~52 kDa), and both forms exhibited glycoform patterns consistent with the known banding pattern of A1PI (dominated by the M6 and M4 bands, including deconvoluted masses). Both Liquid A1PI and Lyophilized A1PI yielded average percent purity values ranging from 96% to 99% and had active content ranging from 53 mg/mL to 59 mg/mL. The PK profile of Liquid A1PI was similar to Lyophilized A1PI. Safety assessments in rabbits showed good tolerability and no test article-related changes in mortality, clinical signs, clinical pathology, body weight, food consumption, or urinalysis parameters. Following immunodepletion of antibodies that recognize Lyophilized A1PI, there were no significant differences in the anti-drug titers among animals immunized with Lyophilized A1PI and Liquid A1PI (p > 0.05), indicating that no antibodies to neoantigens were generated. Liquid A1PI and Lyophilized A1PI have similar profiles with respect to biochemical characteristics, PK, safety and neoantigenicity.
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Affiliation(s)
- Vikram Arora
- Grifols Bioscience Research Group, 85 TW Alexander Drive, Research Triangle Park, NC, 27709, USA.
| | - Maria Cruz
- Grifols Bioscience Research Group, 85 TW Alexander Drive, Research Triangle Park, NC, 27709, USA
| | - John Lang
- Grifols Bioscience Research Group, 85 TW Alexander Drive, Research Triangle Park, NC, 27709, USA
| | - Anthony M Klos
- Grifols Bioscience Research Group, 85 TW Alexander Drive, Research Triangle Park, NC, 27709, USA
| | - W Keither Merritt
- Grifols Bioscience Research Group, 85 TW Alexander Drive, Research Triangle Park, NC, 27709, USA
| | - Jeffrey Price
- Grifols Bioscience Research Group, 85 TW Alexander Drive, Research Triangle Park, NC, 27709, USA
| | - George Taylor
- Grifols Bioscience Research Group, 85 TW Alexander Drive, Research Triangle Park, NC, 27709, USA
| | - Pete Vandeberg
- Grifols Bioscience Research Group, 85 TW Alexander Drive, Research Triangle Park, NC, 27709, USA
| | - Kevin Wee
- Grifols Bioscience Research Group, 85 TW Alexander Drive, Research Triangle Park, NC, 27709, USA
| | - Todd Willis
- Grifols Bioscience Research Group, 85 TW Alexander Drive, Research Triangle Park, NC, 27709, USA
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31
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Shan D, Wang H, Khatri P, Niu Y, Song W, Zhao S, Jiang Y, Ma Q, Liu X, Zhang R, Wang W, Yin C. The Urinary Peptidome as a Noninvasive Biomarker Development Strategy for Prenatal Screening of Down's Syndrome. OMICS-A JOURNAL OF INTEGRATIVE BIOLOGY 2019; 23:439-447. [PMID: 31381471 DOI: 10.1089/omi.2019.0098] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
Prenatal screening for Down's syndrome based on maternal age, ultrasound measures, and maternal serum biomarkers is recommended worldwide, but the false-positive rate and poor diagnostic performance of these screening tests remain problematic. Genetic analysis of cell-free DNA in maternal blood has been developed as a new prenatal screening for Down's syndrome, but it has a number of limitations, including turnaround time and cost. Prenatal screening diagnostic innovation calls for new tests that are noninvasive, accurate, and affordable. We report original observations on potential peptide biomarkers in maternal urine for screening of fetal Down's syndrome. The peptidome of urine samples from 23 pregnant women carrying Down's syndrome fetuses and 30 pregnant women carrying fetuses with normal karyotype was fractionated by weak cation exchange magnetic beads and analyzed by MALDI-TOF mass spectrometry. Levels of six peptides (m/z 1022.1, 1032.1, 1099.5, 1155.9, 1306.6, and 2365.6) were significantly altered between the case and control groups after controlling for maternal and gestational age. A classification model was constructed based on these candidate peptides that could differentiate fetuses with Down's syndrome from controls with a sensitivity of 95.7%, a specificity of 70.0%, and an area under receiver operating characteristic curves of 0.909 (95% confidence interval, 0.835-0.984). Peptide peaks at m/z 1099.5 and 1155.9 were identified as the partial sequences of alpha-1-antitrypsin and heat shock protein beta-1, respectively. These new findings support the new idea that maternal urinary peptidome offers prospects for noninvasive biomarker discovery and development for the prenatal screening of fetal Down's syndrome.
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Affiliation(s)
- Dan Shan
- Department of Perinatal Medicine, Beijing Obstetrics and Gynecology Hospital, Capital Medical University, Beijing, China
| | - Hao Wang
- Beijing Key Laboratory of Clinical Epidemiology, School of Public Health, Capital Medical University, Beijing, China.,School of Medical and Health Sciences, Edith Cowan University, Perth, Australia
| | - Prekshya Khatri
- Department of Perinatal Medicine, Beijing Obstetrics and Gynecology Hospital, Capital Medical University, Beijing, China
| | - Yue Niu
- Department of Perinatal Medicine, Beijing Obstetrics and Gynecology Hospital, Capital Medical University, Beijing, China
| | - Wei Song
- Department of Perinatal Medicine, Beijing Obstetrics and Gynecology Hospital, Capital Medical University, Beijing, China
| | - Shenglong Zhao
- Department of Perinatal Medicine, Beijing Obstetrics and Gynecology Hospital, Capital Medical University, Beijing, China
| | - Yan Jiang
- Department of Perinatal Medicine, Beijing Obstetrics and Gynecology Hospital, Capital Medical University, Beijing, China
| | - Qingwei Ma
- Bioyong Technology Co., Ltd., Beijing, China
| | - Xinchao Liu
- Bioyong Technology Co., Ltd., Beijing, China
| | - Rong Zhang
- Bioyong Technology Co., Ltd., Beijing, China
| | - Wei Wang
- Beijing Key Laboratory of Clinical Epidemiology, School of Public Health, Capital Medical University, Beijing, China.,School of Medical and Health Sciences, Edith Cowan University, Perth, Australia.,School of Public Health, Shandong First Medical University, Taian, China
| | - Chenghong Yin
- Department of Perinatal Medicine, Beijing Obstetrics and Gynecology Hospital, Capital Medical University, Beijing, China
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Guo J, Lodge K, Newnham M, Bunclark K, Toshner M, Morrell NW, Li W. Increased Antielastase Activity in Idiopathic Pulmonary Arterial Hypertension and Chronic Thromboembolic Pulmonary Hypertension. Am J Respir Cell Mol Biol 2018; 59:652-655. [PMID: 30382774 PMCID: PMC6236689 DOI: 10.1165/rcmb.2018-0060le] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022] Open
Affiliation(s)
- Jingxu Guo
- University of Cambridge School of Clinical MedicineCambridge, United Kingdomand
| | - Katharine Lodge
- University of Cambridge School of Clinical MedicineCambridge, United Kingdomand
| | - Michael Newnham
- University of Cambridge School of Clinical MedicineCambridge, United Kingdomand
- Royal Papworth HospitalCambridge, United Kingdom
| | | | - Mark Toshner
- University of Cambridge School of Clinical MedicineCambridge, United Kingdomand
- Royal Papworth HospitalCambridge, United Kingdom
| | - Nicholas W. Morrell
- University of Cambridge School of Clinical MedicineCambridge, United Kingdomand
| | - Wei Li
- University of Cambridge School of Clinical MedicineCambridge, United Kingdomand
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McElvaney OJ, Gunaratnam C, McElvaney OF, Bagwe I, Reeves EP, McElvaney NG. Emerging pharmacotherapies in cystic fibrosis. Expert Rev Respir Med 2018; 12:843-855. [DOI: 10.1080/17476348.2018.1512409] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/22/2023]
Affiliation(s)
- Oliver J McElvaney
- Irish Centre for Genetic Lung Disease, Royal College of Surgeons in Ireland, Beaumont Hospital, Dublin, Ireland
| | - Cedric Gunaratnam
- Irish Centre for Genetic Lung Disease, Royal College of Surgeons in Ireland, Beaumont Hospital, Dublin, Ireland
| | - Oisin Fiachra McElvaney
- Irish Centre for Genetic Lung Disease, Royal College of Surgeons in Ireland, Beaumont Hospital, Dublin, Ireland
| | - Isha Bagwe
- Irish Centre for Genetic Lung Disease, Royal College of Surgeons in Ireland, Beaumont Hospital, Dublin, Ireland
| | - Emer P Reeves
- Irish Centre for Genetic Lung Disease, Royal College of Surgeons in Ireland, Beaumont Hospital, Dublin, Ireland
| | - Noel G McElvaney
- Irish Centre for Genetic Lung Disease, Royal College of Surgeons in Ireland, Beaumont Hospital, Dublin, Ireland
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McElvaney OJ, Gunaratnam C, Reeves EP, McElvaney NG. A specialized method of sputum collection and processing for therapeutic interventions in cystic fibrosis. J Cyst Fibros 2018; 18:203-211. [PMID: 29960875 DOI: 10.1016/j.jcf.2018.06.001] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/12/2018] [Revised: 05/12/2018] [Accepted: 06/04/2018] [Indexed: 11/30/2022]
Abstract
Cystic fibrosis (CF) lung disease is characterized by aggressive neutrophil-dominated inflammation mediated in large part by neutrophil elastase (NE), an omnivorous protease released by activated or disintegrating neutrophils and a key therapeutic target. To date, several short-term studies have shown that anti-NE compounds can inhibit NE and have anti-inflammatory effects. However, progression to large-scale or multicenter clinical trials has been hampered by the fact that the current gold standard methodology of evaluating airway NE inhibition, bronchoalveolar lavage (BAL), is invasive, difficult to standardize across sites and excludes those with severe lung disease. Attempts to utilize sputum that is either spontaneously expectorated (SS) or induced (IS) have been hindered by poor reproducibility, often due to the various processing methods employed. In this study, we evaluate TEmperature-controlled Two-step Rapid Isolation of Sputum (TETRIS), a specialized method for the acquisition and processing of SS and IS. Using TETRIS, we show for the first time that NE activity and cytokine levels are comparable in BAL, SS and IS samples taken from the same people with CF (PWCF) on the same day once this protocol is used. We correlate biomarkers in TETRIS-processed IS and clinical outcome measures including FEV1, and show stability and reproducible inhibition of NE over time in IS processed by TETRIS. The data offer a tremendous opportunity to evaluate prognosis and therapeutic interventions in CF and to study the full spectrum of people with PWCF, many of whom have been excluded from previous studies due to being unfit for BAL or unable to expectorate sputum.
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Affiliation(s)
- O J McElvaney
- Irish Centre for Genetic Lung Disease, Royal College of Surgeons in Ireland, Beaumont Hospital, Dublin, Ireland
| | - C Gunaratnam
- Irish Centre for Genetic Lung Disease, Royal College of Surgeons in Ireland, Beaumont Hospital, Dublin, Ireland
| | - E P Reeves
- Irish Centre for Genetic Lung Disease, Royal College of Surgeons in Ireland, Beaumont Hospital, Dublin, Ireland
| | - N G McElvaney
- Irish Centre for Genetic Lung Disease, Royal College of Surgeons in Ireland, Beaumont Hospital, Dublin, Ireland.
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Pérez-Ibave DC, Burciaga-Flores CH, Elizondo-Riojas MÁ. Prostate-specific antigen (PSA) as a possible biomarker in non-prostatic cancer: A review. Cancer Epidemiol 2018; 54:48-55. [DOI: 10.1016/j.canep.2018.03.009] [Citation(s) in RCA: 30] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/31/2017] [Revised: 03/13/2018] [Accepted: 03/14/2018] [Indexed: 12/26/2022]
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Cabezas-Llobet N, Camprubí S, García B, Alberch J, Xifró X. Human alpha 1-antitrypsin protects neurons and glial cells against oxygen and glucose deprivation through inhibition of interleukins expression. Biochim Biophys Acta Gen Subj 2018; 1862:1852-1861. [PMID: 29857082 DOI: 10.1016/j.bbagen.2018.05.017] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/30/2018] [Revised: 05/09/2018] [Accepted: 05/23/2018] [Indexed: 12/11/2022]
Abstract
BACKGROUND Death due to cerebral stroke afflicts a large number of neuronal populations, including glial cells depending on the brain region affected. Drugs with a wide cellular range of protection are needed to develop effective therapies for stroke. Human alpha 1-antitrypsin (hAAT) is a serine proteinase inhibitor with potent anti-inflammatory, anti-apoptotic and immunoregulatory activities. This study aimed to test whether hAAT can protect different kind of neurons and glial cells after the oxygen and glucose deprivation (OGD). METHODS Addition of hAAT to mouse neuronal cortical, hippocampal and striatal cultures, as well as glial cultures, was performed 30 min after OGD induction and cell viability was assessed 24 h later. The expression of different apoptotic markers and several inflammatory parameters were assessed by immunoblotting and RT-PCR. RESULTS hAAT had a concentration-dependent survival effect in all neuronal cultures exposed to OGD, with a maximal effect at 1-2 mg/mL. The addition of hAAT at 1 mg/mL reduced the OGD-mediated necrotic and apoptotic death in all neuronal cultures. This neuroprotective activity of hAAT was associated with a decrease of cleaved caspase-3 and an increase of MAP2 levels. It was also associated with a reduction of pro-inflammatory cytokines protein levels and expression, increase of IL-10 protein levels and decrease of nuclear localization of nuclear factor-kappaB. Similar to neurons, addition of hAAT protected astrocytes and oligodendrocytes against OGD-induced cell death. CONCLUSIONS Human AAT protects neuronal and glial cells against OGD through interaction with cytokines. GENERAL SIGNIFICANCE Human AAT could be a good therapeutic neuroprotective candidate to treat ischemic stroke.
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Affiliation(s)
- Núria Cabezas-Llobet
- New Therapeutic Targets Group (TargetsLab), Departament de Ciències Mèdiques, Facultat de Medicina, Universitat de Girona, E-17071 Girona, Spain
| | | | | | - Jordi Alberch
- Departament de Biomedicina, Institut de Neurociències, Facultat de Medicina, Universitat de Barcelona, E-08036 Barcelona, Spain; Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), E-08036 Barcelona, Spain; Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Spain
| | - Xavier Xifró
- New Therapeutic Targets Group (TargetsLab), Departament de Ciències Mèdiques, Facultat de Medicina, Universitat de Girona, E-17071 Girona, Spain; Departament de Biomedicina, Institut de Neurociències, Facultat de Medicina, Universitat de Barcelona, E-08036 Barcelona, Spain; Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), E-08036 Barcelona, Spain; Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Spain.
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Callea F, Giovannoni I, Francalanci P, Boldrini R, Faa G, Medicina D, Nobili V, Desmet VJ, Ishak K, Seyama K, Bellacchio E. Mineralization of alpha-1-antitrypsin inclusion bodies in Mmalton alpha-1-antitrypsin deficiency. Orphanet J Rare Dis 2018; 13:79. [PMID: 29769092 PMCID: PMC5956786 DOI: 10.1186/s13023-018-0821-7] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/11/2017] [Accepted: 05/08/2018] [Indexed: 02/07/2023] Open
Abstract
Background Alpha-1-antitrypsin (AAT) deficiency (AATD) of Z, Mmalton, Siiyama type is associated with liver storage of the mutant proteins and liver disease. The Z variant can be diagnosed on isoelectric focusing (IEF) while Mmalton and Siiyama may be missed or misdiagnosed with this technique. Therefore, molecular analysis is mandatory for their characterization. In particular, that holds true for the Mmalton variant as on IEF profile it resembles the wild M2 subtype. Methods This is a retrospective analysis involving review of medical records and of liver biopsy specimens from a series of Mmalton, Z and Siiyama Alpha-1-antitrypsin deficiency patients. The review has been implemented by additional histological stains, electron microscopic observations and 3-D modeling studies of the sites of the mutations. Results Z, Mmalton and Siiyama liver specimen contained characteristic intrahepatocytic PAS-D globules. The globules differed in the three variants as only Mmalton cases showed dark basophilic precipitates within the AAT inclusions. The precipitates were visualized in haematoxylin-eosin (H.E.) stained preparations and corresponded to calcium precipitates as demonstrated by von Kossa staining. On immunohistochemistry, ZAAT inclusions were stained by polyclonal as well as monoclonal noncommercial anti-AAT antibody (AZT11), whilst Mmalton and Siiyama inclusion bodies remained negative with the monoclonal anti-Z antibody. 3-D protein analysis allowed to predict more severe misfolding of the Mmalton molecule as compared to Z and Siiyama that could trigger anomalous interaction with endoplasmic reticulum chaperon proteins, namely calcium binding proteins. Conclusions Mmalton AAT inclusion bodies contain calcium precipitates inside them that allow the differential diagnosis with Siiyama and ZAAT inclusions in routine histological sections. The study has confirmed the specificity of the monoclonal AZT11 for the Z mutant. Thus, the combination of these two features is crucial for the distinction between the three variants and for predicting the genotype, whose confirmation would definitely require molecular analysis. Our study provides new data on the pathomorphogenesis of Mmalton inclusion bodies whose mineralization could play a central role in disease pathogenesis of Mmalton that is distinct from the Z and Siiyama variants. Calcium is known to be a major effector of cell death either via the increased intracellular concentration or the alteration of homeostasis.
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Affiliation(s)
- Francesco Callea
- Department of Pathology, Bambino Gesù Children's Hospital, IRCCS, Piazza S. Onofrio 4, 00165, Rome, Italy.
| | - Isabella Giovannoni
- Department of Pathology, Bambino Gesù Children's Hospital, IRCCS, Piazza S. Onofrio 4, 00165, Rome, Italy
| | - Paola Francalanci
- Department of Pathology, Bambino Gesù Children's Hospital, IRCCS, Piazza S. Onofrio 4, 00165, Rome, Italy
| | - Renata Boldrini
- Department of Pathology, Bambino Gesù Children's Hospital, IRCCS, Piazza S. Onofrio 4, 00165, Rome, Italy
| | - Gavino Faa
- Department of Cytomorphology, University of Cagliari, Cagliari, Italy
| | - Daniela Medicina
- Department of Pathology Spedali Civili, University of Brescia, Brescia, Italy
| | - Valerio Nobili
- Hepato-metabolic Unit, Bambino Gesù Children's Hospital, IRCCS, Rome, Italy
| | | | - Kamal Ishak
- Armed Forces Institute of Pathology, Washington, USA
| | - Kuniaki Seyama
- Division of Respiratory Medicine, Juntendo University Faculty of Medicine and Graduate School of Medicine, 2-1-1 Hongo, Bunkyo-Ku, Tokyo, 113-8421, Japan
| | - Emanuele Bellacchio
- Genetic and Rare Diseases, Research Division, Bambino Gesù Children's Hospital, IRCCS, Rome, Italy
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Dunlea DM, Fee LT, McEnery T, McElvaney NG, Reeves EP. The impact of alpha-1 antitrypsin augmentation therapy on neutrophil-driven respiratory disease in deficient individuals. J Inflamm Res 2018; 11:123-134. [PMID: 29618937 PMCID: PMC5875399 DOI: 10.2147/jir.s156405] [Citation(s) in RCA: 21] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
Alpha-1 antitrypsin (AAT) is the most abundant serine protease inhibitor circulating in the blood. AAT deficiency (AATD) is an autosomal codominant condition affecting an estimated 3.4 million individuals worldwide. The clinical disease associated with AATD can present in a number of ways including COPD, liver disease, panniculitis and antineutrophil cytoplasmic antibody vasculitis. AATD is the only proven genetic risk factor for the development of COPD, and deficient individuals who smoke are disposed to more aggressive disease. Principally, AAT is a serine protease inhibitor; however, over the past number of years, the assessment of AAT as simply an antiprotease has evolved, and it is now recognized that AAT has significant anti-inflammatory properties affecting a wide range of cells, including the circulating neutrophil.
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Affiliation(s)
- Danielle M Dunlea
- Irish Centre for Genetic Lung Disease, Department of Medicine, Royal College of Surgeons in Ireland, Beaumont Hospital, Dublin, Ireland
| | - Laura T Fee
- Irish Centre for Genetic Lung Disease, Department of Medicine, Royal College of Surgeons in Ireland, Beaumont Hospital, Dublin, Ireland
| | - Thomas McEnery
- Irish Centre for Genetic Lung Disease, Department of Medicine, Royal College of Surgeons in Ireland, Beaumont Hospital, Dublin, Ireland
| | - Noel G McElvaney
- Irish Centre for Genetic Lung Disease, Department of Medicine, Royal College of Surgeons in Ireland, Beaumont Hospital, Dublin, Ireland
| | - Emer P Reeves
- Irish Centre for Genetic Lung Disease, Department of Medicine, Royal College of Surgeons in Ireland, Beaumont Hospital, Dublin, Ireland
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Giese M, Turiello N, Molenda N, Palesch D, Meid A, Schroeder R, Basilico P, Benarafa C, Halatsch ME, Zimecki M, Westhoff MA, Wirtz CR, Burster T. Exogenous cathepsin G upregulates cell surface MHC class I molecules on immune and glioblastoma cells. Oncotarget 2018; 7:74602-74611. [PMID: 27806341 PMCID: PMC5342690 DOI: 10.18632/oncotarget.12980] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/28/2016] [Accepted: 10/25/2016] [Indexed: 11/25/2022] Open
Abstract
Major histocompatibility complex (MHC) class I molecules present antigenic peptides to cytotoxic T cells. During an adaptive immune response, MHC molecules are regulated by several mechanisms including lipopolysaccharide (LPS) and interferon gamma (IFN-g). However, it is unclear whether the serine protease cathepsin G (CatG), which is generally secreted by neutrophils at the site of inflammation, might regulate MHC I molecules. We identified CatG, and to a higher extend CatG and lactoferrin (LF), as an exogenous regulator of cell surface MHC I expression of immune cells and glioblastoma stem cells. In addition, levels of MHC I molecules are reduced on dendritic cells from CatG deficient mice compared to their wild type counterparts. Furthermore, cell surface CatG on immune cells, including T cells, B cells, and NK cells triggers MHC I on THP-1 monocytes suggesting a novel mechanism for CatG to facilitate intercellular communication between infiltrating cells and the respective target cell. Subsequently, our findings highlight the pivotal role of CatG as a checkpoint protease which might force target cells to display their intracellular MHC I:antigen repertoire.
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Affiliation(s)
- Madleen Giese
- Department of Neurosurgery, Ulm University Medical Center, Ulm, Germany
| | - Nadine Turiello
- Department of Neurosurgery, Ulm University Medical Center, Ulm, Germany
| | - Nicole Molenda
- Department of Neurosurgery, Ulm University Medical Center, Ulm, Germany
| | - David Palesch
- Institute of Molecular Virology, Ulm University Medical Center, Ulm, Germany
| | - Annika Meid
- Department of Neurosurgery, Ulm University Medical Center, Ulm, Germany
| | - Roman Schroeder
- Department of Neurosurgery, Ulm University Medical Center, Ulm, Germany
| | - Paola Basilico
- Theodor Kocher Institute, University of Bern, Bern, Switzerland.,Graduate School for Cellular and Biomedical Sciences, University of Bern, Bern, Switzerland
| | - Charaf Benarafa
- Theodor Kocher Institute, University of Bern, Bern, Switzerland
| | | | - Michal Zimecki
- Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland
| | - Mike-Andrew Westhoff
- Department Pediatrics and Adolescent Medicine, Ulm University Medical Center, Ulm, Germany
| | | | - Timo Burster
- Department of Neurosurgery, Ulm University Medical Center, Ulm, Germany
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40
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Mahon BP, Ambadapadi S, Yaron JR, Lomelino CL, Pinard MA, Keinan S, Kurnikov I, Macaulay C, Zhang L, Reeves W, McFadden G, Tibbetts S, McKenna R, Lucas AR. Crystal Structure of Cleaved Serp-1, a Myxomavirus-Derived Immune Modulating Serpin: Structural Design of Serpin Reactive Center Loop Peptides with Improved Therapeutic Function. Biochemistry 2018; 57:1096-1107. [PMID: 29227673 DOI: 10.1021/acs.biochem.7b01171] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
The Myxomavirus-derived protein Serp-1 has potent anti-inflammatory activity in models of vasculitis, lupus, viral sepsis, and transplant. Serp-1 has also been tested successfully in a Phase IIa clinical trial in unstable angina, representing a "first-in-class" therapeutic. Recently, peptides derived from the reactive center loop (RCL) have been developed as stand-alone therapeutics for reducing vasculitis and improving survival in MHV68-infected mice. However, both Serp-1 and the RCL peptides lose activity in MHV68-infected mice after antibiotic suppression of intestinal microbiota. Here, we utilize a structure-guided approach to design and test a series of next-generation RCL peptides with improved therapeutic potential that is not reduced when the peptides are combined with antibiotic treatments. The crystal structure of cleaved Serp-1 was determined to 2.5 Å resolution and reveals a classical serpin structure with potential for serpin-derived RCL peptides to bind and inhibit mammalian serpins, plasminogen activator inhibitor 1 (PAI-1), anti-thrombin III (ATIII), and α-1 antitrypsin (A1AT), and target proteases. Using in silico modeling of the Serp-1 RCL peptide, S-7, we designed several modified RCL peptides that were predicted to have stronger interactions with human serpins because of the larger number of stabilizing hydrogen bonds. Two of these peptides (MPS7-8 and -9) displayed extended activity, improving survival where activity was previously lost in antibiotic-treated MHV68-infected mice (P < 0.0001). Mass spectrometry and kinetic assays suggest interaction of the peptides with ATIII, A1AT, and target proteases in mouse and human plasma. In summary, we present the next step toward the development of a promising new class of anti-inflammatory serpin-based therapeutics.
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Affiliation(s)
- Brian P Mahon
- Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health , Bethesda, Maryland 20892, United States.,Department of Biochemistry and Molecular Biology, University of Florida College of Medicine , Gainesville, Florida 32610-0277, United States
| | - Sriram Ambadapadi
- Department of Medicine, Divisions of Cardiovascular Medicine and Rheumatology, University of Florida , Gainesville, Florida 32610-0277, United States
| | | | - Carrie L Lomelino
- Department of Biochemistry and Molecular Biology, University of Florida College of Medicine , Gainesville, Florida 32610-0277, United States
| | - Melissa A Pinard
- Department of Biochemistry and Molecular Biology, University of Florida College of Medicine , Gainesville, Florida 32610-0277, United States
| | - Shahar Keinan
- Cloud Pharmaceuticals , 6 Davis Drive, Research Triangle Park, North Carolina 27709, United States
| | - Igor Kurnikov
- Cloud Pharmaceuticals , 6 Davis Drive, Research Triangle Park, North Carolina 27709, United States
| | | | | | - Westley Reeves
- Department of Medicine, Divisions of Cardiovascular Medicine and Rheumatology, University of Florida , Gainesville, Florida 32610-0277, United States
| | | | | | - Robert McKenna
- Department of Biochemistry and Molecular Biology, University of Florida College of Medicine , Gainesville, Florida 32610-0277, United States
| | - Alexandra R Lucas
- Department of Medicine, Divisions of Cardiovascular Medicine and Rheumatology, University of Florida , Gainesville, Florida 32610-0277, United States.,Saint Joseph's Hospital, Dignity Health , Phoenix, Arizona 85013, United States
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Quaglia A, Roberts EA, Torbenson M. Developmental and Inherited Liver Disease. MACSWEEN'S PATHOLOGY OF THE LIVER 2018:111-274. [DOI: 10.1016/b978-0-7020-6697-9.00003-0] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/02/2025]
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Recombinant Human Alpha-1 Antitrypsin-Fc Fusion Protein Reduces Mouse Myocardial Inflammatory Injury After Ischemia-Reperfusion Independent of Elastase Inhibition. J Cardiovasc Pharmacol 2017; 68:27-32. [PMID: 26945157 DOI: 10.1097/fjc.0000000000000383] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
Abstract
BACKGROUND Alpha-1-antitrypsin (AAT) is an abundant plasma protein with neutrophil elastase-inhibiting activity, and AAT is available as a plasma-derived therapeutic (pAAT). In experimental myocardial infarction, pAAT reduced acute inflammatory injury because of ischemia-reperfusion. The aim of the present study was to assess the properties of a recombinant protein composed of human AAT fused to the human immunoglobulin (Ig) G1 Fc fragment (rhAAT-Fc) in experimental myocardial infarction. METHODS Ten-week-old CD1 male mice underwent transient occlusion (30 minutes) of the left anterior coronary artery. rhAAT-Fc (2 mg/kg) or pAAT (60 mg/kg) were administered upon reperfusion. We used human plasma-derived Ig (2 mg/kg) or a matching volume of NaCl 0.9% as control solutions. After 24 hours, infarct size and caspase-1 activity were quantified. The left ventricular ejection fraction (LVEF) was measured by echocardiography at 24 hours and 7 days. A variant of rhAAT-Fc lacking elastase inhibition activity, rhAAT-Fc, was also tested. RESULTS The rhAAT-Fc induced a significant reduction in infarct size (P < 0.01 vs. all controls, P > 0.05 vs. pAAT). Caspase-1 activity was reduced to the same degree with rhAAT-Fc and pAAT (-70%; P < 0.05; P > 0.05 rhAAT-Fc vs. pAAT). The effects on infarct size after a single administration were reflected by preservation of LVEF at 24 hours and 7 days (all P < 0.05). rhAAT-Fc without elastase inhibiting activity, rhAAT-Fc, conferred comparable effects on infarct size, caspase-1 activity, and LVEF (P > 0.2 vs. rhAAT-Fc). CONCLUSIONS The pAAT and recombinant human AAT-Fc reduce the acute myocardial inflammatory injury after ischemia-reperfusion in the mouse leading to preservation of viable myocardium and systolic function, independent on the effects on neutrophil elastase.
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Busch R, Hobbs BD, Zhou J, Castaldi PJ, McGeachie MJ, Hardin ME, Hawrylkiewicz I, Sliwinski P, Yim JJ, Kim WJ, Kim DK, Agusti A, Make BJ, Crapo JD, Calverley PM, Donner CF, Lomas DA, Wouters EF, Vestbo J, Tal-Singer R, Bakke P, Gulsvik A, Litonjua AA, Sparrow D, Paré PD, Levy RD, Rennard SI, Beaty TH, Hokanson J, Silverman EK, Cho MH. Genetic Association and Risk Scores in a Chronic Obstructive Pulmonary Disease Meta-analysis of 16,707 Subjects. Am J Respir Cell Mol Biol 2017; 57:35-46. [PMID: 28170284 PMCID: PMC5516277 DOI: 10.1165/rcmb.2016-0331oc] [Citation(s) in RCA: 47] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/04/2023] Open
Abstract
The heritability of chronic obstructive pulmonary disease (COPD) cannot be fully explained by recognized genetic risk factors identified as achieving genome-wide significance. In addition, the combined contribution of genetic variation to COPD risk has not been fully explored. We sought to determine: (1) whether studies of variants from previous studies of COPD or lung function in a larger sample could identify additional associated variants, particularly for severe COPD; and (2) the impact of genetic risk scores on COPD. We genotyped 3,346 single-nucleotide polymorphisms (SNPs) in 2,588 cases (1,803 severe COPD) and 1,782 control subjects from four cohorts, and performed association testing with COPD, combining these results with existing genotyping data from 6,633 cases (3,497 severe COPD) and 5,704 control subjects. In addition, we developed genetic risk scores from SNPs associated with lung function and COPD and tested their discriminatory power for COPD-related measures. We identified significant associations between SNPs near PPIC (P = 1.28 × 10-8) and PPP4R4/SERPINA1 (P = 1.01 × 10-8) and severe COPD; the latter association may be driven by recognized variants in SERPINA1. Genetic risk scores based on SNPs previously associated with COPD and lung function had a modest ability to discriminate COPD (area under the curve, ∼0.6), and accounted for a mean 0.9-1.9% lower forced expiratory volume in 1 second percent predicted for each additional risk allele. In a large genetic association analysis, we identified associations with severe COPD near PPIC and SERPINA1. A risk score based on combining genetic variants had modest, but significant, effects on risk of COPD and lung function.
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Affiliation(s)
- Robert Busch
- Channing Division of Network Medicine, Brigham and Women’s Hospital, Boston, Massachusetts
| | - Brian D. Hobbs
- Channing Division of Network Medicine, Brigham and Women’s Hospital, Boston, Massachusetts
| | - Jin Zhou
- University of Arizona, Tucson, Arizona
| | - Peter J. Castaldi
- Channing Division of Network Medicine, Brigham and Women’s Hospital, Boston, Massachusetts
| | - Michael J. McGeachie
- Channing Division of Network Medicine, Brigham and Women’s Hospital, Boston, Massachusetts
| | - Megan E. Hardin
- Channing Division of Network Medicine, Brigham and Women’s Hospital, Boston, Massachusetts
| | | | - Pawel Sliwinski
- National Tuberculosis and Lung Disease Research Institute, Warsaw, Poland
| | - Jae-Joon Yim
- Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, Seoul National University College of Medicine, Seoul, Korea
| | - Woo Jin Kim
- Kangwon National University, Chuncheon, Korea
| | - Deog K. Kim
- Seoul National University College of Medicine Boramae Medical Center, Seoul, Korea
| | - Alvar Agusti
- Thorax Institute, Hospital Clinic, IDIBAPS, University of Barcelona, CIBERES, Barcelona, Spain
| | | | | | | | - Claudio F. Donner
- Mondo Medico di I.F.I.M. srl, Multidisciplinary and Rehabilitation Outpatient Clinic, Borgomanero, Novara, Italy
| | | | | | - Jørgen Vestbo
- University of Manchester, Manchester, United Kingdom
| | - Ruth Tal-Singer
- GlaxoSmithKline Research and Development, King of Prussia, Pennsylvania
| | - Per Bakke
- University of Bergen, Bergen, Norway
| | | | - Augusto A. Litonjua
- Channing Division of Network Medicine, Brigham and Women’s Hospital, Boston, Massachusetts
| | - David Sparrow
- Brigham and Women’s Hospital and the Veterans Administration Medical Center–Jamaica Plain, Jamaica Plain, Massachusetts
| | - Peter D. Paré
- Respiratory Division, Department of Medicine, University of British Columbia, Vancouver, British Columbia, Canada
| | - Robert D. Levy
- Respiratory Division, Department of Medicine, University of British Columbia, Vancouver, British Columbia, Canada
| | | | - Terri H. Beaty
- Department of Epidemiology, Bloomberg School of Public Health, the Johns Hopkins University, Baltimore, Maryland; and
| | - John Hokanson
- Department of Epidemiology, University of Colorado Anschutz Medical Campus, Aurora, Colorado
| | - Edwin K. Silverman
- Channing Division of Network Medicine, Brigham and Women’s Hospital, Boston, Massachusetts
| | - Michael H. Cho
- Channing Division of Network Medicine, Brigham and Women’s Hospital, Boston, Massachusetts
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Giełdoń A, Witt MM, Gajewicz A, Puzyn T. Rapid insight into C60 influence on biological functions of proteins. Struct Chem 2017. [DOI: 10.1007/s11224-017-0957-4] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/19/2022]
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Bengtson P, Valtonen-André C, Jonsson M. Phenotyping of α-1-Antitrypsin by liquid chromatography-high resolution mass spectrometry. CLINICAL MASS SPECTROMETRY (DEL MAR, CALIF.) 2016; 2:34-40. [PMID: 39192843 PMCID: PMC11324609 DOI: 10.1016/j.clinms.2017.02.002] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/28/2016] [Revised: 02/16/2017] [Accepted: 02/16/2017] [Indexed: 11/19/2022]
Abstract
More than seventy-five isotypes of α-1-antitrypsin (AAT) have been described. To assess risks associated with AAT deficiency, isotype identification is necessary. Isoelectric focusing (IEF) is traditionally used for isotype differentiation, however, IEF has limited scope since it is a manual procedure that is not suitable for automation, and antitrypsin variants must differ in net charge in order to be resolved. In comparison, mass spectrometric assays are easily automated and offer a more complete solution for characterization of proteins. To capitalize on these advantages, we have developed a qualitative top-down liquid chromatography-mass spectrometry (LC-MS) method for selective phenotyping of AAT. This technique requires no sample pretreatment, and has the potential for use in routine clinical diagnostics. We have validated our LC-MS results against both DNA sequencing and IEF. Thus far, this method has identified the AAT variants PLowell, S and Z, as well as unique fragments shared by different M alleles. Its high selectivity is indirectly illustrated by the detection of a variant carrying the amino acid substitution p.Ala308Ser, which cannot be visualized by IEF.
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Affiliation(s)
- Per Bengtson
- Department of Clinical Chemistry, University Health Care in Region Skåne, Lund, Sweden
| | | | - Magnus Jonsson
- Department of Clinical Chemistry, University Health Care in Region Skåne, Malmö, Sweden
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Busch R, Qiu W, Lasky-Su J, Morrow J, Criner G, DeMeo D. Differential DNA methylation marks and gene comethylation of COPD in African-Americans with COPD exacerbations. Respir Res 2016; 17:143. [PMID: 27814717 PMCID: PMC5097392 DOI: 10.1186/s12931-016-0459-8] [Citation(s) in RCA: 45] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/16/2016] [Accepted: 10/27/2016] [Indexed: 12/18/2022] Open
Abstract
BACKGROUND Chronic obstructive pulmonary disease (COPD) is the third-leading cause of death worldwide. Identifying COPD-associated DNA methylation marks in African-Americans may contribute to our understanding of racial disparities in COPD susceptibility. We determined differentially methylated genes and co-methylation network modules associated with COPD in African-Americans recruited during exacerbations of COPD and smoking controls from the Pennsylvania Study of Chronic Obstructive Pulmonary Exacerbations (PA-SCOPE) cohort. METHODS We assessed DNA methylation from whole blood samples in 362 African-American smokers in the PA-SCOPE cohort using the Illumina Infinium HumanMethylation27 BeadChip Array. Final analysis included 19302 CpG probes annotated to the nearest gene transcript after quality control. We tested methylation associations with COPD case-control status using mixed linear models. Weighted gene comethylation networks were constructed using weighted gene coexpression network analysis (WGCNA) and network modules were analyzed for association with COPD. RESULTS There were five differentially methylated CpG probes significantly associated with COPD among African-Americans at an FDR less than 5 %, and seven additional probes that approached significance at an FDR less than 10 %. The top ranked gene association was MAML1, which has been shown to affect NOTCH-dependent angiogenesis in murine lung. Network modeling yielded the "yellow" and "blue" comethylation modules which were significantly associated with COPD (p-value 4 × 10-10 and 4 × 10-9, respectively). The yellow module was enriched for gene sets related to inflammatory pathways known to be relevant to COPD. The blue module contained the top ranked genes in the concurrent differential methylation analysis (FXYD1/LGI4, gene significance p-value 1.2 × 10-26; MAML1, p-value 2.0 × 10-26; CD72, p-value 2.1 × 10-25; and LPO, p-value 7.2 × 10-25), and was significantly associated with lung development processes in Gene Ontology gene-set enrichment analysis. CONCLUSION We identified 12 differentially methylated CpG sites associated with COPD that mapped to biologically plausible genes. Network module comethylation patterns have identified candidate genes that may be contributing to racial differences in COPD susceptibility and severity. COPD-associated comethylation modules contained genes previously associated with lung disease and inflammation and recapitulated known COPD-associated genes. The genes implicated by differential methylation and WGCNA analysis may provide mechanistic targets contributing to COPD susceptibility, exacerbations, and outcomes among African-Americans. TRIAL REGISTRATION Trial Registration: NCT00774176 , Registry: ClinicalTrials.gov, URL: www.clinicaltrials.gov , Date of Enrollment of First Participant: June 2004, Date Registered: 04 January 2008 (retrospectively registered).
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Affiliation(s)
- Robert Busch
- Channing Division of Network Medicine, Department of Medicine, Brigham and Women’s Hospital, 181 Longwood Ave, Room 449, Boston, 02111 MA USA
| | - Weiliang Qiu
- Channing Division of Network Medicine, Department of Medicine, Brigham and Women’s Hospital, 181 Longwood Ave, Room 449, Boston, 02111 MA USA
| | - Jessica Lasky-Su
- Channing Division of Network Medicine, Department of Medicine, Brigham and Women’s Hospital, 181 Longwood Ave, Room 449, Boston, 02111 MA USA
| | - Jarrett Morrow
- Channing Division of Network Medicine, Department of Medicine, Brigham and Women’s Hospital, 181 Longwood Ave, Room 449, Boston, 02111 MA USA
| | - Gerard Criner
- Temple Lung Center, Temple University Health System, Philadelphia, PA USA
| | - Dawn DeMeo
- Channing Division of Network Medicine, Department of Medicine, Brigham and Women’s Hospital, 181 Longwood Ave, Room 449, Boston, 02111 MA USA
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Quantitation of human milk proteins and their glycoforms using multiple reaction monitoring (MRM). Anal Bioanal Chem 2016; 409:589-606. [PMID: 27796459 DOI: 10.1007/s00216-016-0029-4] [Citation(s) in RCA: 37] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/28/2016] [Revised: 09/26/2016] [Accepted: 10/11/2016] [Indexed: 01/11/2023]
Abstract
Human milk plays a substantial role in the child growth, development and determines their nutritional and health status. Despite the importance of the proteins and glycoproteins in human milk, very little quantitative information especially on their site-specific glycosylation is known. As more functions of milk proteins and other components continue to emerge, their fine-detailed quantitative information is becoming a key factor in milk research efforts. The present work utilizes a sensitive label-free MRM method to quantify seven milk proteins (α-lactalbumin, lactoferrin, secretory immunoglobulin A, immunoglobulin G, immunoglobulin M, α1-antitrypsin, and lysozyme) using their unique peptides while at the same time, quantifying their site-specific N-glycosylation relative to the protein abundance. The method is highly reproducible, has low limit of quantitation, and accounts for differences in glycosylation due to variations in protein amounts. The method described here expands our knowledge about human milk proteins and provides vital details that could be used in monitoring the health of the infant and even the mother. Graphical Abstract The glycopeptides EICs generated from QQQ.
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Anzueto A. Alpha-1 Antitrypsin Deficiency-Associated Chronic Obstructive Pulmonary Disease: A Family Perspective. COPD 2016; 12:462-7. [PMID: 25474273 DOI: 10.3109/15412555.2014.974746] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023]
Abstract
Alpha-1 antitrypsin (AAT) deficiency (AATD) is a genetic condition that can lead to the early onset of chronic obstructive pulmonary disease (COPD), a disorder that comprises elements of chronic bronchitis and emphysema. AATD is characterized by reduced levels of the AAT protease inhibitor, leading to unrestricted protease activity in the lung, which promotes destruction of lung tissue. In severe cases, patients with AATD have an increased mortality risk and, potentially, a poor quality of life due to more frequent COPD exacerbations and/or limitations on daily activity. However, the burden of AATD on members of the patient's immediate family who may serve as caregivers has not been described. Because the age range at which most patients are diagnosed with AATD may affect the economic status of an individual and/or of a family, it is likely that a diagnosis of AATD may have negative effects that extend beyond those on the diagnosed person to include immediate family members. Here, we review the literature to investigate the impact of the caregiver role of family members in disease states that affect an age group similar to AATD. Furthermore, we provide a case study showing the effect of AATD on immediate family members.
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Affiliation(s)
- Antonio Anzueto
- a South Texas Veterans Health Care System, Audie L. Murphy Hospital, and University of Texas Health Science Center , San Antonio , Texas , USA
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Identification and characterization of alpha-I-proteinase inhibitor from common carp sarcoplasmic proteins. Food Chem 2016; 192:1090-7. [DOI: 10.1016/j.foodchem.2015.07.099] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/03/2015] [Revised: 06/26/2015] [Accepted: 07/22/2015] [Indexed: 11/22/2022]
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Cavalcante MDS, Torres-Romero JC, Lobo MDP, Moreno FBMB, Bezerra LP, Lima DS, Matos JC, Moreira RDA, Monteiro-Moreira ACDO. A panel of glycoproteins as candidate biomarkers for early diagnosis and treatment evaluation of B-cell acute lymphoblastic leukemia. Biomark Res 2016; 4:1. [PMID: 26823978 PMCID: PMC4730630 DOI: 10.1186/s40364-016-0055-6] [Citation(s) in RCA: 34] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/30/2015] [Accepted: 01/04/2016] [Indexed: 12/21/2022] Open
Abstract
BACKGROUND Acute lymphoblastic leukemia is the most common malignant cancer in childhood. The signs and symptoms of childhood cancer are difficult to recognize, as it is not the first diagnosis to be considered for nonspecific complaints, leading to potential uncertainty in diagnosis. The aim of this study was to perform proteomic analysis of serum from pediatric patients with B-cell acute lymphoblastic leukemia (B-ALL) to identify candidate biomarker proteins, for use in early diagnosis and evaluation of treatment. METHODS Serum samples were obtained from ten patients at the time of diagnosis (B-ALL group) and after induction therapy (AIT group). Sera from healthy children were used as controls (Control group). The samples were subjected to immunodepletion, affinity chromatography with α-d-galactose-binding lectin (from Artocarpus incisa seeds) immobilized on a Sepharose(TM) 4B gel, concentration, and digestion for subsequent analysis with nano-UPLC tandem nano-ESI-MS(E). The program Expression (E) was used to quantify differences in protein expression between groups. RESULTS A total of 96 proteins were identified. Leucine-rich alpha-2-glycoprotein 1 (LRG1), Clusterin (CLU), thrombin (F2), heparin cofactor II (SERPIND1), alpha-2-macroglobulin (A2M), alpha-2-antiplasmin (SERPINF2), Alpha-1 antitrypsin (SERPINA1), Complement factor B (CFB) and Complement C3 (C3) were identified as candidate biomarkers for early diagnosis of B-ALL, as they were upregulated in the B-ALL group relative to the control and AIT groups. Expression levels of the candidate biomarkers did not differ significantly between the AIT and control groups, providing further evidence that the candidate biomarkers are present only in the disease state, as all patients achieved complete remission after treatment. CONCLUSION A panel of protein biomarker candidates has been developed for pre-diagnosis of B-ALL and also provided information that would indicate a favorable response to treatment after induction therapy.
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Affiliation(s)
| | - José Camilo Torres-Romero
- />Center of Experimental Biology (NUBEX), University of Fortaleza (UNIFOR), Fortaleza, Ceará Brazil
- />Department of Biochemistry and Molecular Biology, Federal University of Ceará, Fortaleza, Ceará Brazil
| | - Marina Duarte Pinto Lobo
- />Center of Experimental Biology (NUBEX), University of Fortaleza (UNIFOR), Fortaleza, Ceará Brazil
- />Department of Biochemistry and Molecular Biology, Federal University of Ceará, Fortaleza, Ceará Brazil
| | | | - Leonardo Primo Bezerra
- />Center of Experimental Biology (NUBEX), University of Fortaleza (UNIFOR), Fortaleza, Ceará Brazil
| | - Diego Silva Lima
- />Development and Technological Innovation in Drug Program, Federal University of Ceará, Fortaleza, Ceará Brazil
| | - Jesamar Correia Matos
- />Reference Center at Children’s Cancer Diagnosis and Adolescents Dr. Murilo Martins, Albert Sabin Hospital, Fortaleza, Ceará Brazil
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