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Napolitano M, Potestio L, Nocerino M, Patruno C. Considerations for managing elderly patients with atopic dermatitis. Expert Rev Clin Immunol 2024; 20:501-511. [PMID: 38193289 DOI: 10.1080/1744666x.2024.2301967] [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: 10/21/2023] [Accepted: 12/22/2023] [Indexed: 01/10/2024]
Abstract
INTRODUCTION Atopic dermatitis (AD) diagnosis in elderly is challenging, due to its clinical polymorphism and the lack of diagnostic biomarkers. Moreover, the chronicity of the disease and the complex pathogenetic mechanism, make elderly AD management challenging. AREAS COVERED A narrative review of the current literature was performed using the PubMed, Medline, Embase, and Cochrane Skin databases, by researching the following terms: 'atopic dermatitis,' 'clinical phenotypes,' 'eczema,' 'elderly patients,' 'elderly type atopic dermatitis,' 'eczema clinical presentation.' The aim was to report the current knowledge on pathogenesis, clinical presentation, and treatment options of elderly AD. EXPERT OPINION Elderly type AD has recently been identified as a separate entity, with an increasing prevalence. With aging, both immunosenescence and barrier alterations can cause or modify AD presentation. Moreover, a chronic proinflammatory state (so-called 'inflammaging') is often present in elderly subjects. Older patients with AD may present with peculiar immunophenotypic profile, making AD diagnosis challenging. Similarly, the chronicity of the disease and the complex pathogenetic mechanism, make AD management a challenge. Indeed, systemic therapies for AD are often contraindicated or not tolerated and the management of elderly type AD is often burdened with numerous difficulties, leading to undertreated disease. Even if dupilumab and tralokinumab represent a valuable therapeutic weapon, more data on safety of JAK inhibitors are required.
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Affiliation(s)
- Maddalena Napolitano
- Section of Dermatology, Department of Clinical Medicine and Surgery, University of Naples Federico II, Naples, Italy
| | - Luca Potestio
- Section of Dermatology, Department of Clinical Medicine and Surgery, University of Naples Federico II, Naples, Italy
| | - Mariateresa Nocerino
- Section of Dermatology, Department of Clinical Medicine and Surgery, University of Naples Federico II, Naples, Italy
| | - Cataldo Patruno
- Department of Health Sciences, University Magna Graecia of Catanzaro, Catanzaro, Italy
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2
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Fang M, Tang X, Zhang J, Liao Z, Wang G, Cheng R, Zhang Z, Zhao H, Wang J, Tan Z, Kamau PM, Lu Q, Liu Q, Deng G, Lai R. An inhibitor of leukotriene-A 4 hydrolase from bat salivary glands facilitates virus infection. Proc Natl Acad Sci U S A 2022; 119:e2110647119. [PMID: 35238649 PMCID: PMC8915838 DOI: 10.1073/pnas.2110647119] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/15/2021] [Accepted: 12/20/2021] [Indexed: 12/23/2022] Open
Abstract
SignificanceAn immunosuppressant protein (MTX), which facilitates virus infection by inhibiting leukotriene A4 hydrolase (LTA4H) to produce the lipid chemoattractant leukotriene B4 (LTB4), was identified and characterized from the submandibular salivary glands of the bat Myotis pilosus. To the best of our knowledge, this is a report of an endogenous LTA4H inhibitor in animals. MTX was highly concentrated in the bat salivary glands, suggesting a mechanism for the generation of immunological privilege and immune tolerance and providing evidence of viral shedding through oral secretions. Moreover, given that the immunosuppressant MTX selectively inhibited the proinflammatory activity of LTA4H, without affecting its antiinflammatory activity, MTX might be a potential candidate for the development of antiinflammatory drugs by targeting the LTA4-LTA4H-LTB4 inflammatory axis.
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Affiliation(s)
- Mingqian Fang
- Key Laboratory of Animal Models and Human Disease Mechanisms of the Chinese Academy of Sciences/Key Laboratory of Bioactive Peptides of Yunnan Province, KIZ-CUHK Joint Laboratory of Bioresources and Molecular Research in Common Diseases, National Resource Center for Non-Human Primates, Kunming Primate Research Center, and National Research Facility for Phenotypic and Genetic Analysis of Model Animals (Primate Facility), Kunming Institute of Zoology, Kunming 650107, Yunnan, China
| | - Xiaopeng Tang
- Key Laboratory of Animal Models and Human Disease Mechanisms of the Chinese Academy of Sciences/Key Laboratory of Bioactive Peptides of Yunnan Province, KIZ-CUHK Joint Laboratory of Bioresources and Molecular Research in Common Diseases, National Resource Center for Non-Human Primates, Kunming Primate Research Center, and National Research Facility for Phenotypic and Genetic Analysis of Model Animals (Primate Facility), Kunming Institute of Zoology, Kunming 650107, Yunnan, China
| | - Juan Zhang
- Department of Infectious Diseases, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, 400038, China
| | - Zhiyi Liao
- Key Laboratory of Animal Models and Human Disease Mechanisms of the Chinese Academy of Sciences/Key Laboratory of Bioactive Peptides of Yunnan Province, KIZ-CUHK Joint Laboratory of Bioresources and Molecular Research in Common Diseases, National Resource Center for Non-Human Primates, Kunming Primate Research Center, and National Research Facility for Phenotypic and Genetic Analysis of Model Animals (Primate Facility), Kunming Institute of Zoology, Kunming 650107, Yunnan, China
- Kunming College of Life Science, University of Chinese Academy of Sciences, Beijing 100049, China
| | - Gan Wang
- Key Laboratory of Animal Models and Human Disease Mechanisms of the Chinese Academy of Sciences/Key Laboratory of Bioactive Peptides of Yunnan Province, KIZ-CUHK Joint Laboratory of Bioresources and Molecular Research in Common Diseases, National Resource Center for Non-Human Primates, Kunming Primate Research Center, and National Research Facility for Phenotypic and Genetic Analysis of Model Animals (Primate Facility), Kunming Institute of Zoology, Kunming 650107, Yunnan, China
- Institutes for Drug Discovery and Development, Chinese Academy of Sciences, Shanghai 201203, China
- Sino-African Joint Research Center, Chinese Academy of Sciences, Wuhan, Hubei 430074, China
| | - Ruomei Cheng
- Key Laboratory of Animal Models and Human Disease Mechanisms of the Chinese Academy of Sciences/Key Laboratory of Bioactive Peptides of Yunnan Province, KIZ-CUHK Joint Laboratory of Bioresources and Molecular Research in Common Diseases, National Resource Center for Non-Human Primates, Kunming Primate Research Center, and National Research Facility for Phenotypic and Genetic Analysis of Model Animals (Primate Facility), Kunming Institute of Zoology, Kunming 650107, Yunnan, China
- Kunming College of Life Science, University of Chinese Academy of Sciences, Beijing 100049, China
| | - Zhiye Zhang
- Key Laboratory of Animal Models and Human Disease Mechanisms of the Chinese Academy of Sciences/Key Laboratory of Bioactive Peptides of Yunnan Province, KIZ-CUHK Joint Laboratory of Bioresources and Molecular Research in Common Diseases, National Resource Center for Non-Human Primates, Kunming Primate Research Center, and National Research Facility for Phenotypic and Genetic Analysis of Model Animals (Primate Facility), Kunming Institute of Zoology, Kunming 650107, Yunnan, China
- Institutes for Drug Discovery and Development, Chinese Academy of Sciences, Shanghai 201203, China
- Sino-African Joint Research Center, Chinese Academy of Sciences, Wuhan, Hubei 430074, China
| | - Hongwen Zhao
- Department of Infectious Diseases, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, 400038, China
| | - Jing Wang
- Department of Laboratory Diagnosis, Chongqing Public Health Medical Center, Public Health Hospital of Southwest University, Shapingba District, Chongqing 400038, China
| | - Zhaoxia Tan
- Department of Infectious Diseases, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, 400038, China
| | - Peter Muiruri Kamau
- Key Laboratory of Animal Models and Human Disease Mechanisms of the Chinese Academy of Sciences/Key Laboratory of Bioactive Peptides of Yunnan Province, KIZ-CUHK Joint Laboratory of Bioresources and Molecular Research in Common Diseases, National Resource Center for Non-Human Primates, Kunming Primate Research Center, and National Research Facility for Phenotypic and Genetic Analysis of Model Animals (Primate Facility), Kunming Institute of Zoology, Kunming 650107, Yunnan, China
- Kunming College of Life Science, University of Chinese Academy of Sciences, Beijing 100049, China
- Sino-African Joint Research Center, Chinese Academy of Sciences, Wuhan, Hubei 430074, China
| | - Qiumin Lu
- Key Laboratory of Animal Models and Human Disease Mechanisms of the Chinese Academy of Sciences/Key Laboratory of Bioactive Peptides of Yunnan Province, KIZ-CUHK Joint Laboratory of Bioresources and Molecular Research in Common Diseases, National Resource Center for Non-Human Primates, Kunming Primate Research Center, and National Research Facility for Phenotypic and Genetic Analysis of Model Animals (Primate Facility), Kunming Institute of Zoology, Kunming 650107, Yunnan, China
- Kunming College of Life Science, University of Chinese Academy of Sciences, Beijing 100049, China
- Institutes for Drug Discovery and Development, Chinese Academy of Sciences, Shanghai 201203, China
- Sino-African Joint Research Center, Chinese Academy of Sciences, Wuhan, Hubei 430074, China
| | - Qi Liu
- Kunming College of Life Science, University of Chinese Academy of Sciences, Beijing 100049, China
| | - Guohong Deng
- Department of Infectious Diseases, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, 400038, China
| | - Ren Lai
- Key Laboratory of Animal Models and Human Disease Mechanisms of the Chinese Academy of Sciences/Key Laboratory of Bioactive Peptides of Yunnan Province, KIZ-CUHK Joint Laboratory of Bioresources and Molecular Research in Common Diseases, National Resource Center for Non-Human Primates, Kunming Primate Research Center, and National Research Facility for Phenotypic and Genetic Analysis of Model Animals (Primate Facility), Kunming Institute of Zoology, Kunming 650107, Yunnan, China
- Kunming College of Life Science, University of Chinese Academy of Sciences, Beijing 100049, China
- Institutes for Drug Discovery and Development, Chinese Academy of Sciences, Shanghai 201203, China
- Sino-African Joint Research Center, Chinese Academy of Sciences, Wuhan, Hubei 430074, China
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Review of the Mechanisms of Snake Venom Induced Pain: It's All about Location, Location, Location. Int J Mol Sci 2022; 23:ijms23042128. [PMID: 35216244 PMCID: PMC8879488 DOI: 10.3390/ijms23042128] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/07/2022] [Revised: 02/12/2022] [Accepted: 02/14/2022] [Indexed: 02/06/2023] Open
Abstract
Pain—acute, chronic and debilitating—is the most feared neurotoxicity resulting from a survivable venomous snake bite. The purpose of this review is to present in a novel paradigm what we know about the molecular mechanisms responsible for pain after envenomation. Progressing from known pain modulating peptides and enzymes, to tissue level interactions with venom resulting in pain, to organ system level pain syndromes, to geographical level distribution of pain syndromes, the present work demonstrates that understanding the mechanisms responsible for pain is dependent on “location, location, location”. It is our hope that this work can serve to inspire the molecular and epidemiologic investigations needed to better understand the neurotoxic mechanisms responsible for these snake venom mediated diverse pain syndromes and ultimately lead to agent specific treatments beyond anti-venom alone.
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Kim YS, Go G, Yun CW, Yea JH, Yoon S, Han SY, Lee G, Lee MY, Lee SH. Topical Administration of Melatonin-Loaded Extracellular Vesicle-Mimetic Nanovesicles Improves 2,4-Dinitrofluorobenzene-Induced Atopic Dermatitis. Biomolecules 2021; 11:1450. [PMID: 34680082 PMCID: PMC8533309 DOI: 10.3390/biom11101450] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/06/2021] [Revised: 09/25/2021] [Accepted: 10/01/2021] [Indexed: 12/18/2022] Open
Abstract
Atopic dermatitis (AD) is caused by multiple factors that trigger chronic skin inflammation, including a defective skin barrier, immune cell activation, and microbial exposure. Although melatonin has an excellent biosafety profile and a potential to treat AD, there is limited clinical evidence from controlled trials that support the use of melatonin as an AD treatment. The delivery of melatonin via the transdermal delivery system is also a challenge in designing melatonin-based AD treatments. In this study, we generated melatonin-loaded extracellular vesicle-mimetic nanoparticles (MelaNVs) to improve the transdermal delivery of melatonin and to evaluate their therapeutic potential in AD. The MelaNVs were spherical nanoparticles with an average size of 100 nm, which is the optimal size for the transdermal delivery of drugs. MelaNVs showed anti-inflammatory effects by suppressing the release of TNF-α and β-hexosaminidase in LPS-treated RAW264.7 cells and compound 48/80-treated RBL-2H3 cells, respectively. MelaNVs showed a superior suppressive effect compared to an equivalent concentration of free melatonin. Treating a 2,4-dinitrofluorobenzene (DNCB)-induced AD-like mouse model with MelaNVs improved AD by suppressing local inflammation, mast cell infiltration, and fibrosis. In addition, MelaNVs effectively suppressed serum IgE levels and regulated serum IFN-γ and IL-4 levels. Taken together, these results suggest that MelaNVs are novel and efficient transdermal delivery systems of melatonin and that MelaNVs can be used as a treatment to improve AD.
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Affiliation(s)
- Yoon Seon Kim
- Department of Medical Science, Soonchunhyang University, Asan-si 31538, Korea;
| | - Gyeongyun Go
- Department of Biochemistry, College of Medicine, Soonchunhyang University, Cheonan 31151, Korea; (G.G.); (J.-H.Y.); (G.L.)
- Department of Biochemistry, BK21FOUR Project2, College of Medicine, Soonchunhyang University, Cheonan 31151, Korea
| | - Chul-Won Yun
- Medical Science Research Institute, Soonchunhyang University Seoul Hospital, Seoul 04401, Korea;
| | - Ji-Hye Yea
- Department of Biochemistry, College of Medicine, Soonchunhyang University, Cheonan 31151, Korea; (G.G.); (J.-H.Y.); (G.L.)
| | - Sungtae Yoon
- Stembio Ltd., Entrepreneur 306, Asan-si 31538, Korea; (S.Y.); (S.-Y.H.)
| | - Su-Yeon Han
- Stembio Ltd., Entrepreneur 306, Asan-si 31538, Korea; (S.Y.); (S.-Y.H.)
| | - Gaeun Lee
- Department of Biochemistry, College of Medicine, Soonchunhyang University, Cheonan 31151, Korea; (G.G.); (J.-H.Y.); (G.L.)
- Department of Biochemistry, BK21FOUR Project2, College of Medicine, Soonchunhyang University, Cheonan 31151, Korea
| | - Mi-Young Lee
- Department of Medical Science, Soonchunhyang University, Asan-si 31538, Korea;
- Department of Medical Biotechnology, Soonchunhyang University, Asan-si 31538, Korea
| | - Sang Hun Lee
- Department of Biochemistry, College of Medicine, Soonchunhyang University, Cheonan 31151, Korea; (G.G.); (J.-H.Y.); (G.L.)
- Department of Biochemistry, BK21FOUR Project2, College of Medicine, Soonchunhyang University, Cheonan 31151, Korea
- Medical Science Research Institute, Soonchunhyang University Seoul Hospital, Seoul 04401, Korea;
- Stembio Ltd., Entrepreneur 306, Asan-si 31538, Korea; (S.Y.); (S.-Y.H.)
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Bocheva GS, Slominski RM, Slominski AT. Immunological Aspects of Skin Aging in Atopic Dermatitis. Int J Mol Sci 2021; 22:ijms22115729. [PMID: 34072076 PMCID: PMC8198400 DOI: 10.3390/ijms22115729] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/10/2021] [Revised: 05/22/2021] [Accepted: 05/25/2021] [Indexed: 12/13/2022] Open
Abstract
The cutaneous immune response is important for the regulation of skin aging well as for the development of immune-mediated skin diseases. Aging of the human skin undergoes immunosenescence with immunological alterations and can be affected by environmental stressors and internal factors, thus leading to various epidermal barrier abnormalities. The dysfunctional epidermal barrier, immune dysregulation, and skin dysbiosis in the advanced age, together with the genetic factors, facilitate the late onset of atopic dermatitis (AD) in the elderly, whose cases have recently been on the rise. Controversial to the healthy aged skin, where overproduction of many cytokines is found, the levels of Th2/Th22 related cytokines inversely correlated with age in the skin of older AD patients. As opposed to an endogenously aged skin, the expression of the terminal differentiation markers significantly increases with age in AD. Despite the atenuated barrier disturbances in older AD patients, the aged skin carries an impairment associated with the aging process, which reflects the persistence of AD. The chronicity of AD in older patients might not directly affect skin aging but does not allow spontaneous remission. Thus, adult- and elderly subtypes of AD are considered as a lifelong disease.
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Affiliation(s)
- Georgeta St. Bocheva
- Department of Pharmacology and Toxicology, Medical University of Sofia, 1431 Sofia, Bulgaria
- Correspondence: (G.S.B.); (A.T.S.)
| | - Radomir M. Slominski
- Division of Rheumatology, Department of Medicine, University of Alabama at Birmingham, Birmingham, AL 35294, USA;
| | - Andrzej T. Slominski
- Department of Dermatology, Comprehensive Cancer Center, Cancer Chemoprevention Program, University of Alabama at Birmingham, Birmingham, AL 35294, USA
- Veteran Administration Medical Center, Birmingham, AL 35294, USA
- Correspondence: (G.S.B.); (A.T.S.)
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6
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Zhan MX, Tang L, Lu YF, Wu HH, Guo ZB, Shi ZM, Yang CL, Zou YQ, Yang F, Chen GZ. Ulinastatin Exhibits Antinociception in Rat Models of Acute Somatic and Visceral Pain Through Inhibiting the Local and Central Inflammation. J Pain Res 2021; 14:1201-1214. [PMID: 33976570 PMCID: PMC8106509 DOI: 10.2147/jpr.s303595] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/28/2021] [Accepted: 03/31/2021] [Indexed: 12/28/2022] Open
Abstract
Introduction Ulinastatin, a broad-spectrum serine protease inhibitor, has been widely used to treat various diseases clinically. However, so far, the antinociceptive effect of ulinastatin remains less studied experimentally and the underlying mechanisms of ulinastatin for pain relief remain unclear. This study aimed to find evidence of the analgesic effect of ulinastatin on acute somatic and visceral pain. Methods The analgesic effect of ulinastatin on acute somatic and visceral pain was evaluated by using formalin and acetic acid-induced writhing test. The analgesic mechanism of ulinastatin was verified by detecting the peripheral inflammatory cell infiltration and spinal glial activation with hematoxylin-eosin (H&E) and immunohistochemistry staining. Results We found that both of intraperitoneal (i.p.) pre-administration and post-administration of ulinastatin could reduce the total number of flinching and the licking duration following intraplantar formalin injection in a dose-related manner. However, the inhibitory effect of ulinastatin existed only in the second phase (Phase 2) of formalin-induced spontaneous pain response, with no effect in the first phase (Phase 1). The formalin-induced edema and ulcer were also improved by i.p. administration of ulinastatin. Moreover, i.p. administration of ulinastatin was also able to delay the occurrence of acetic acid-induced writhing and reduced the total number of writhes dose-dependently. We further demonstrated that ulinastatin significantly decreased the local inflammatory cell infiltration in injured paw and peritoneum tissue under formalin and acetic acid test separately. The microglial and astrocytic activation in the spinal dorsal horn induced by intraplantar formalin and i.p. acetic acid injection were also dramatically inhibited by i.p. administration of ulinastatin. Conclusion Our results for the first time provided a new line of evidence showing that ulinastatin could attenuate acute somatic and visceral pain by inhibiting the peripheral and spinal inflammatory reaction.
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Affiliation(s)
- Mei-Xiang Zhan
- Department of Anesthesiology and Perioperative Medicine, Clinical Medical College, (900 Hospital of the Joint Logistic Support Force), Fujian Medical University, Fuzhou, Fujian, 350025, People's Republic of China.,Department of Anesthesiology and Perioperative Medicine, Dongfang Hospital, Xiamen University, Fuzhou, Fujian, 350025, People's Republic of China
| | - Li Tang
- Department of Stomatology, Beijing Friendship Hospital, Capital Medical University, Beijing, 100050, People's Republic of China
| | - Yun-Fei Lu
- Department of Anesthesiology, Fifth Medical Center of Chinese PLA General Hospital, Beijing, 100039, People's Republic of China
| | - Huang-Hui Wu
- Department of Anesthesiology and Perioperative Medicine, Clinical Medical College, (900 Hospital of the Joint Logistic Support Force), Fujian Medical University, Fuzhou, Fujian, 350025, People's Republic of China
| | - Zhi-Bin Guo
- Department of Anesthesiology and Perioperative Medicine, Clinical Medical College, (900 Hospital of the Joint Logistic Support Force), Fujian Medical University, Fuzhou, Fujian, 350025, People's Republic of China
| | - Zhong-Mou Shi
- Department of Anesthesiology and Perioperative Medicine, Clinical Medical College, (900 Hospital of the Joint Logistic Support Force), Fujian Medical University, Fuzhou, Fujian, 350025, People's Republic of China
| | - Chen-Long Yang
- Department of Anesthesiology and Perioperative Medicine, Clinical Medical College, (900 Hospital of the Joint Logistic Support Force), Fujian Medical University, Fuzhou, Fujian, 350025, People's Republic of China
| | - Yi-Qing Zou
- Department of Anesthesiology and Perioperative Medicine, Clinical Medical College, (900 Hospital of the Joint Logistic Support Force), Fujian Medical University, Fuzhou, Fujian, 350025, People's Republic of China.,Department of Anesthesiology and Perioperative Medicine, Dongfang Hospital, Xiamen University, Fuzhou, Fujian, 350025, People's Republic of China
| | - Fei Yang
- Department of Anesthesiology and Perioperative Medicine, Clinical Medical College, (900 Hospital of the Joint Logistic Support Force), Fujian Medical University, Fuzhou, Fujian, 350025, People's Republic of China.,Department of Anesthesiology and Perioperative Medicine, Dongfang Hospital, Xiamen University, Fuzhou, Fujian, 350025, People's Republic of China.,Laboratory of Pain Research, School of Basic Medical Sciences, Fujian Medical University, Fuzhou, Fujian, 350122, People's Republic of China
| | - Guo-Zhong Chen
- Department of Anesthesiology and Perioperative Medicine, Clinical Medical College, (900 Hospital of the Joint Logistic Support Force), Fujian Medical University, Fuzhou, Fujian, 350025, People's Republic of China.,Department of Anesthesiology and Perioperative Medicine, Dongfang Hospital, Xiamen University, Fuzhou, Fujian, 350025, People's Republic of China
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Stein R, Berger M, Santana de Cecco B, Mallmann LP, Terraciano PB, Driemeier D, Rodrigues E, Beys-da-Silva WO, Konrath EL. Chymase inhibition: A key factor in the anti-inflammatory activity of ethanolic extracts and spilanthol isolated from Acmella oleracea. JOURNAL OF ETHNOPHARMACOLOGY 2021; 270:113610. [PMID: 33246121 DOI: 10.1016/j.jep.2020.113610] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/13/2020] [Revised: 11/03/2020] [Accepted: 11/18/2020] [Indexed: 06/11/2023]
Abstract
ETHNOPHARMACOLOGICAL RELEVANCE Acmella oleracea (L.) R. K. Jansen (Asteraceae), known as jambú in Brazil, is used in traditional medicine as analgesic and for inflammatory conditions, characterized by the presence of N-alkylamides, mainly spilanthol. This bioactive compound is responsible for the above-described pharmacological properties, including sialagogue and anesthetic. AIM OF THE STUDY This study aimed to characterize the anti-inflammatory effects of A. oleracea leaves (AOEE-L) and flowers (AOEE-F) extracts, including an isolated alkylamide (spilanthol), using in vitro and in vivo models. The mechanism underlying this effect was also investigated. MATERIALS AND METHODS Extracts were analyzed by HPLC-ESI-MS/MS in order to characterize the N-alkylamides content. AOEE-L, AOEE-F (25-100 μg/mL) and spilanthol (50-200 μM) were tested in vitro on VSMC after stimulation with hyperglycemic medium (25 mM glucose). Their effects over nitric oxide (NO) generation, chymase inhibition and expression, catalase (CAT), superoxide anion (SOD) radical activity were evaluated. After an acute administration of extracts (10-100 mg/mL) and spilanthol (6.2 mg/mL), the anti-inflammatory effects were evaluated by applying the formalin test in rats. Blood was collected to measure serum aminotransferases activities, NO activity, creatinine and urea. RESULTS A number of distinct N-alkylamides were detected and quantified in AOEE-L and AOEE-F. Spilanthol was identified in both extracts and selected for experimental tests. Hyperglycemic stimulation in VSMC promoted the expression of inflammatory parameters, including chymase, NO, CAT and SOD activity and chymase expression, all of them attenuated by the presence of the extracts and spilanthol. The administration of extracts or spilanthol significantly inhibited edema formation, NO production and cell tissue infiltration in the formalin test, without causing kidney and liver toxicity. CONCLUSION Taken together, these results provide evidence for the anti-inflammatory activity of leaves and flowers extracts of jambú associated distinctly with their chemical profile. The effects appear to be associated with the inhibition of chymase activity, suppression of the proinflammatory cytokine NO and antioxidant activities.
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Affiliation(s)
- Renan Stein
- Programa de Pós-Graduação em Ciências Farmacêuticas, Faculdade de Farmácia, Universidade Federal do Rio Grande do Sul (UFRGS), CEP, 90610-000, Porto Alegre, RS, Brazil
| | - Markus Berger
- Laboratório de Bioquímica Farmacológica, Centro de Pesquisa Experimental (CPE), Hospital de Clínicas de Porto Alegre (HCPA-UFRGS), CEP, 90035-007, Porto Alegre, RS, Brazil
| | - Bianca Santana de Cecco
- Departamento de Patologia Clínica Veterinária, Faculdade de Medicina Veterinária, Universidade Federal do Rio Grande do Sul (UFRGS), CEP, 91540-000, Porto Alegre, RS, Brazil
| | - Luana Peixoto Mallmann
- Instituto de Ciência e Tecnologia de Alimentos, Universidade Federal do Rio Grande do Sul (UFRGS), CEP, 91501-970, Porto Alegre, RS, Brazil
| | - Paula Barros Terraciano
- Laboratório de Embriologia e Diferenciação Celular, Centro de Pesquisa Experimental (CPE), Hospital de Clínicas de Porto Alegre (HCPA-UFRGS), CEP, 90035-007, Porto Alegre, RS, Brazil
| | - David Driemeier
- Departamento de Patologia Clínica Veterinária, Faculdade de Medicina Veterinária, Universidade Federal do Rio Grande do Sul (UFRGS), CEP, 91540-000, Porto Alegre, RS, Brazil
| | - Eliseu Rodrigues
- Instituto de Ciência e Tecnologia de Alimentos, Universidade Federal do Rio Grande do Sul (UFRGS), CEP, 91501-970, Porto Alegre, RS, Brazil
| | - Walter Orlando Beys-da-Silva
- Faculdade de Farmácia, Universidade Federal do Rio Grande do Sul- UFRGS, CEP, 90610-000, Porto Alegre, RS, Brazil
| | - Eduardo Luis Konrath
- Programa de Pós-Graduação em Ciências Farmacêuticas, Faculdade de Farmácia, Universidade Federal do Rio Grande do Sul (UFRGS), CEP, 90610-000, Porto Alegre, RS, Brazil.
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8
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Carco C, Young W, Gearry RB, Talley NJ, McNabb WC, Roy NC. Increasing Evidence That Irritable Bowel Syndrome and Functional Gastrointestinal Disorders Have a Microbial Pathogenesis. Front Cell Infect Microbiol 2020; 10:468. [PMID: 33014892 PMCID: PMC7509092 DOI: 10.3389/fcimb.2020.00468] [Citation(s) in RCA: 49] [Impact Index Per Article: 9.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/04/2020] [Accepted: 07/29/2020] [Indexed: 12/12/2022] Open
Abstract
The human gastrointestinal tract harbors most of the microbial cells inhabiting the body, collectively known as the microbiota. These microbes have several implications for the maintenance of structural integrity of the gastrointestinal mucosal barrier, immunomodulation, metabolism of nutrients, and protection against pathogens. Dysfunctions in these mechanisms are linked to a range of conditions in the gastrointestinal tract, including functional gastrointestinal disorders, ranging from irritable bowel syndrome, to functional constipation and functional diarrhea. Irritable bowel syndrome is characterized by chronic abdominal pain with changes in bowel habit in the absence of morphological changes. Despite the high prevalence of irritable bowel syndrome in the global population, the mechanisms responsible for this condition are poorly understood. Although alterations in the gastrointestinal microbiota, low-grade inflammation and immune activation have been implicated in the pathophysiology of functional gastrointestinal disorders, there is inconsistency between studies and a lack of consensus on what the exact role of the microbiota is, and how changes to it relate to these conditions. The complex interplay between host factors, such as microbial dysbiosis, immune activation, impaired epithelial barrier function and motility, and environmental factors, including diet, will be considered in this narrative review of the pathophysiology of functional gastrointestinal disorders.
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Affiliation(s)
- Caterina Carco
- School of Food and Advanced Technology, Massey University, Palmerston North, New Zealand.,Riddet Institute, Massey University, Palmerston North, New Zealand.,Food Nutrition and Health Team, AgResearch Grasslands, Palmerston North, New Zealand.,The High-Value Nutrition National Science Challenge, Auckland, New Zealand
| | - Wayne Young
- Riddet Institute, Massey University, Palmerston North, New Zealand.,Food Nutrition and Health Team, AgResearch Grasslands, Palmerston North, New Zealand.,The High-Value Nutrition National Science Challenge, Auckland, New Zealand
| | - Richard B Gearry
- The High-Value Nutrition National Science Challenge, Auckland, New Zealand.,Department of Medicine, University of Otago, Christchurch, New Zealand
| | - Nicholas J Talley
- Faculty of Health and Medicine, University of Newcastle, Callaghan, NSW, Australia
| | - Warren C McNabb
- Riddet Institute, Massey University, Palmerston North, New Zealand.,The High-Value Nutrition National Science Challenge, Auckland, New Zealand
| | - Nicole C Roy
- Riddet Institute, Massey University, Palmerston North, New Zealand.,The High-Value Nutrition National Science Challenge, Auckland, New Zealand.,Liggins Institute, University of Auckland, Auckland, New Zealand.,Department of Human Nutrition, University of Otago, Dunedin, New Zealand
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9
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Buhl T, Ikoma A, Kempkes C, Cevikbas F, Sulk M, Buddenkotte J, Akiyama T, Crumrine D, Camerer E, Carstens E, Schön MP, Elias P, Coughlin SR, Steinhoff M. Protease-Activated Receptor-2 Regulates Neuro-Epidermal Communication in Atopic Dermatitis. Front Immunol 2020; 11:1740. [PMID: 32903402 PMCID: PMC7435019 DOI: 10.3389/fimmu.2020.01740] [Citation(s) in RCA: 48] [Impact Index Per Article: 9.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/20/2019] [Accepted: 06/29/2020] [Indexed: 01/01/2023] Open
Abstract
Background: Activation of protease-activated receptor-2 (PAR2) has been implicated in inflammation, pruritus, and skin barrier regulation, all characteristics of atopic dermatitis (AD), as well as Netherton syndrome which has similar characteristics. However, understanding the precise role of PAR2 on neuro-immune communication in AD has been hampered by the lack of appropriate animal models. Methods: We used a recently established mouse model with epidermal overexpression of PAR2 (PAR2OE) and littermate WT mice to study the impact of increased PAR2 expression in epidermal cells on spontaneous and house dust mite (HDM)-induced skin inflammation, itch, and barrier dysfunction in AD, in vivo and ex vivo. Results: PAR2OE newborns displayed no overt abnormalities, but spontaneously developed dry skin, severe pruritus, and eczema. Dermatological, neurophysiological, and immunological analyses revealed the hallmarks of AD-like skin disease. Skin barrier defects were observed before onset of skin lesions. Application of HDM onto PAR2OE mice triggered pruritus and the skin phenotype. PAR2OE mice displayed an increased density of nerve fibers, increased nerve growth factor and endothelin-1 expression levels, alloknesis, enhanced scratching (hyperknesis), and responses of dorsal root ganglion cells to non-histaminergic pruritogens. Conclusion: PAR2 in keratinocytes, activated by exogenous and endogenous proteases, is sufficient to drive barrier dysfunction, inflammation, and pruritus and sensitize skin to the effects of HDM in a mouse model that mimics human AD. PAR2 signaling in keratinocytes appears to be sufficient to drive several levels of neuro-epidermal communication, another feature of human AD.
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Affiliation(s)
- Timo Buhl
- Department of Dermatology and Surgery, University of California, San Francisco, San Francisco, CA, United States.,Department of Dermatology, Venereology and Allergology, University Medical Center Göttingen, Göttingen, Germany
| | - Akihiko Ikoma
- Department of Dermatology and Surgery, University of California, San Francisco, San Francisco, CA, United States.,Department of Dermatology and UCD Charles Institute for Translational Dermatology, University College Dublin, Dublin, Ireland
| | - Cordula Kempkes
- Department of Dermatology and Surgery, University of California, San Francisco, San Francisco, CA, United States
| | - Ferda Cevikbas
- Department of Dermatology and Surgery, University of California, San Francisco, San Francisco, CA, United States
| | - Mathias Sulk
- Department of Dermatology and Surgery, University of California, San Francisco, San Francisco, CA, United States.,Department of Dermatology, University Hospital Münster, Münster, Germany
| | - Joerg Buddenkotte
- Department of Dermatology and Venerology, Hamad Medical Corporation, Doha, Qatar.,Translational Research Institute, Academic Health System, Hamad Medical Corporation, Doha, Qatar
| | - Tasuku Akiyama
- Department of Dermatology, Anatomy and Cell Biology, Temple Itch Center, Temple University, Philadelphia, PA, United States.,Department of Neurobiology, Physiology and Behavior, University of California, Davis, Davis, CA, United States
| | - Debbie Crumrine
- Department of Dermatology and Surgery, University of California, San Francisco, San Francisco, CA, United States
| | - Eric Camerer
- INSERM U970, Paris Cardiovascular Research Centre, Paris, France
| | - Earl Carstens
- Department of Neurobiology, Physiology and Behavior, University of California, Davis, Davis, CA, United States
| | - Michael P Schön
- Department of Dermatology, Venereology and Allergology, University Medical Center Göttingen, Göttingen, Germany
| | - Peter Elias
- Department of Dermatology and Surgery, University of California, San Francisco, San Francisco, CA, United States
| | - Shaun R Coughlin
- Cardiovascular Research Institute, University of California, San Francisco, San Francisco, CA, United States
| | - Martin Steinhoff
- Department of Dermatology and Surgery, University of California, San Francisco, San Francisco, CA, United States.,Department of Dermatology and UCD Charles Institute for Translational Dermatology, University College Dublin, Dublin, Ireland.,Department of Dermatology and Venerology, Hamad Medical Corporation, Doha, Qatar.,Translational Research Institute, Academic Health System, Hamad Medical Corporation, Doha, Qatar.,Department of Dermatology, Medical School, University of Qatar, Doha, Qatar.,School of Medicine, Weill Cornell Medicine-Qatar, Doha, Qatar.,Department of Dermatology, Weill Cornell Medicine, New York, NY, United States
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10
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Sánchez Di Maggio L, Tirloni L, Uhl M, Carmona C, Logullo C, Mulenga A, da Silva Vaz I, Berasain P. Serpins in Fasciola hepatica: insights into host-parasite interactions. Int J Parasitol 2020; 50:931-943. [PMID: 32668271 DOI: 10.1016/j.ijpara.2020.05.010] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/16/2020] [Revised: 05/14/2020] [Accepted: 05/20/2020] [Indexed: 12/28/2022]
Abstract
Protease inhibitors play crucial roles in parasite development and survival, modulating the immune responses of their vertebrate hosts. Members of the serpin family are irreversible inhibitors of serine proteases and regulate systems related to defence against parasites. Limited information is currently available on protease inhibitors from the liver fluke Fasciola hepatica. In this study, we characterised four serpins from F. hepatica (FhS-1-FhS-4). Biochemical characterisation revealed that recombinant FhS-2 (rFhS) inhibits the activity of human neutrophil cathepsin G, while rFhS-4 inhibits the activity of bovine pancreatic chymotrypsin and cathepsin G. Consistent with inhibitor function profiling data, rFhS-4 inhibited cathepsin G-activated platelet aggregation in a dose-responsive manner.Similar to other serpins, rFhS2 and rFhS-4 bind to heparin with high affinity. Tissue localisation demonstrated that these serpins have different spatial distributions. FhS-2 is localised in the ovary, while FhS-4 was found in gut cells. Both of them co-localised in the spines within the tegument. These findings provide the basis for study of functional roles of these proteins as part of an immune evasion mechanism in the adult fluke, and in protection of eggs to ensure parasite life cycle continuity. Further understanding of serpins from the liver fluke may lead to the discovery of novel anti-parasitic interventions.
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Affiliation(s)
- Lucía Sánchez Di Maggio
- Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil; Unidad de Biología Parasitaria, Facultad de Ciencias, Universidad de la República Oriental del Uruguay, Montevideo, Uruguay
| | - Lucas Tirloni
- Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil; Department of Veterinary Pathobiology, College of Veterinary Medicine, Texas A&M University, College Station, USA
| | - Marcelle Uhl
- Laboratory of Chemistry and Function of Proteins and Peptides, Animal Experimentation Unit, Universidade Estadual do Norte Fluminence Darcy Ribeiro, Campos dos Goytacazes, RJ, Brazil
| | - Carlos Carmona
- Unidad de Biología Parasitaria, Facultad de Ciencias, Universidad de la República Oriental del Uruguay, Montevideo, Uruguay
| | - Carlos Logullo
- Laboratory of Chemistry and Function of Proteins and Peptides, Animal Experimentation Unit, Universidade Estadual do Norte Fluminence Darcy Ribeiro, Campos dos Goytacazes, RJ, Brazil
| | - Albert Mulenga
- Department of Veterinary Pathobiology, College of Veterinary Medicine, Texas A&M University, College Station, USA
| | - Itabajara da Silva Vaz
- Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil; Faculdade de Veterinária, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil.
| | - Patrícia Berasain
- Unidad de Biología Parasitaria, Facultad de Ciencias, Universidad de la República Oriental del Uruguay, Montevideo, Uruguay.
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11
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Coutinho ML, Bizzarro B, Tirloni L, Berger M, Freire Oliveira CJ, Sá-Nunes A, Silva Vaz I. Rhipicephalus microplus serpins interfere with host immune responses by specifically modulating mast cells and lymphocytes. Ticks Tick Borne Dis 2020; 11:101425. [PMID: 32335011 PMCID: PMC11000276 DOI: 10.1016/j.ttbdis.2020.101425] [Citation(s) in RCA: 17] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/05/2019] [Revised: 03/10/2020] [Accepted: 03/22/2020] [Indexed: 01/09/2023]
Abstract
Rhipicephalus microplus ticks feed on a bovine host for three weeks. At the attachment site, inflammatory and immune responses are triggered resulting in the recruitment of cells and production of a set of immunological mediators. To oppose the host's immune responses, ticks inoculate bioactive salivary molecules capable of interfering with these defense mechanisms. Serpins are among the most frequent molecules present in tick saliva and have been shown to negatively affect the host's anti-tick immunity. R. microplus has at least eighteen full-length serpins (RmS) and eleven are transcribed during blood feeding. Among them, RmS-3, RmS-6, and RmS-17 are present in the saliva of engorged females. Here, the effect of these serpins on the immune responses was evaluated in cells involved in innate/inflammatory (mast cells and macrophages) and adaptive (T cells) immunity. RmS-3 modulated mast cells due to its inhibitory activity on peritoneal rat chymase and on vascular permeability in acute inflammation. In addition, both RmS-6 and RmS-17 inhibited vascular permeability. Of the three serpins studied, neither affected activation nor inflammatory cytokine production by murine macrophages. On the other hand, RmS-3 and RmS-17 presented an inhibitory effect on the metabolic activity of lymphocytes, with the latter being the most potent, while RmS-6 had no effect on it. This activity was associated with a decrease in lymphocyte proliferation, but not with induction of cell death. The present study highlights the powerful modulatory role of tick salivary serpins in the host's immune system and inspire the discovery of targets for the treatment of inflammatory/immune disorders.
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Affiliation(s)
- Mariana L Coutinho
- Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, 91501-970, Brazil.
| | - Bruna Bizzarro
- Departamento de Imunologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, SP, 05508-000, Brazil.
| | - Lucas Tirloni
- Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, 91501-970, Brazil.
| | - Markus Berger
- Laboratório de Bioquímica Farmacológica, Centro de Pesquisa Experimental, Hospital de Clínicas de Porto Alegre, Porto Alegre, RS, 90035-007, Brazil.
| | - Carlo Jose Freire Oliveira
- Instituto de Ciências Biológicas e Naturais, Universidade Federal do Triângulo Mineiro, Uberaba, MG, 38025-180, Brazil; Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular (INCT-EM), Rio de Janeiro, RJ, 21941-902, Brazil.
| | - Anderson Sá-Nunes
- Departamento de Imunologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, SP, 05508-000, Brazil; Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular (INCT-EM), Rio de Janeiro, RJ, 21941-902, Brazil.
| | - Itabajara Silva Vaz
- Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, 91501-970, Brazil; Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular (INCT-EM), Rio de Janeiro, RJ, 21941-902, Brazil; Faculdade de Veterinária, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, 91501-970, Brazil.
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12
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Kim TK, Tirloni L, Pinto AFM, Diedrich JK, Moresco JJ, Yates JR, da Silva Vaz I, Mulenga A. Time-resolved proteomic profile of Amblyomma americanum tick saliva during feeding. PLoS Negl Trop Dis 2020; 14:e0007758. [PMID: 32049966 PMCID: PMC7041860 DOI: 10.1371/journal.pntd.0007758] [Citation(s) in RCA: 40] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/03/2019] [Revised: 02/25/2020] [Accepted: 01/03/2020] [Indexed: 12/26/2022] Open
Abstract
Amblyomma americanum ticks transmit more than a third of human tick-borne disease (TBD) agents in the United States. Tick saliva proteins are critical to success of ticks as vectors of TBD agents, and thus might serve as targets in tick antigen-based vaccines to prevent TBD infections. We describe a systems biology approach to identify, by LC-MS/MS, saliva proteins (tick = 1182, rabbit = 335) that A. americanum ticks likely inject into the host every 24 h during the first 8 days of feeding, and towards the end of feeding. Searching against entries in GenBank grouped tick and rabbit proteins into 27 and 25 functional categories. Aside from housekeeping-like proteins, majority of tick saliva proteins belong to the tick-specific (no homology to non-tick organisms: 32%), protease inhibitors (13%), proteases (8%), glycine-rich proteins (6%) and lipocalins (4%) categories. Global secretion dynamics analysis suggests that majority (74%) of proteins in this study are associated with regulating initial tick feeding functions and transmission of pathogens as they are secreted within 24–48 h of tick attachment. Comparative analysis of the A. americanum tick saliva proteome to five other tick saliva proteomes identified 284 conserved tick saliva proteins: we speculate that these regulate critical tick feeding functions and might serve as tick vaccine antigens. We discuss our findings in the context of understanding A. americanum tick feeding physiology as a means through which we can find effective targets for a vaccine against tick feeding. The lone star tick, Amblyomma americanum, is a medically important species in US that transmits 5 of the 16 reported tick-borne disease agents. Most recently, bites of this tick were associated with red meat allergies in humans. Vaccination of animals against tick feeding has been shown to be a sustainable and an effective alternative to current acaricide based tick control method which has several limitations. The pre-requisite to tick vaccine development is to understand the molecular basis of tick feeding physiology. Toward this goal, this study has identified proteins that A. americanum ticks inject into the host at different phases of its feeding cycle. This data set has identified proteins that A. americanum inject into the host within 24–48 h of feeding before it starts to transmit pathogens. Of high importance, we identified 284 proteins that are present in saliva of other tick species, which we suspect regulate important role(s) in tick feeding success and might represent rich source target antigens for a tick vaccine. Overall, this study provides a foundation to understand the molecular mechanisms regulating tick feeding physiology.
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Affiliation(s)
- Tae Kwon Kim
- Department of Veterinary Pathobiology, College of Veterinary Medicine, Texas A&M University, College Station, Texas, United States of America
| | - Lucas Tirloni
- Department of Veterinary Pathobiology, College of Veterinary Medicine, Texas A&M University, College Station, Texas, United States of America
- Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil
| | - Antônio F. M. Pinto
- Foundation Peptide Biology Lab, Salk Institute for Biological Studies, La Jolla, Californai, United States of America
- Department of Molecular Medicine, The Scripps Research Institute, La Jolla, California, United States of America
| | - Jolene K. Diedrich
- Department of Molecular Medicine, The Scripps Research Institute, La Jolla, California, United States of America
- Mass Spectrometry Core, Salk Institute for Biological Studies, La Jolla, California, United States of America
| | - James J. Moresco
- Department of Molecular Medicine, The Scripps Research Institute, La Jolla, California, United States of America
- Mass Spectrometry Core, Salk Institute for Biological Studies, La Jolla, California, United States of America
| | - John R. Yates
- Department of Molecular Medicine, The Scripps Research Institute, La Jolla, California, United States of America
| | - Itabajara da Silva Vaz
- Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil
- Faculdade de Veterinária, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil
| | - Albert Mulenga
- Department of Veterinary Pathobiology, College of Veterinary Medicine, Texas A&M University, College Station, Texas, United States of America
- * E-mail:
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13
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Amblyomma americanum serpin 27 (AAS27) is a tick salivary anti-inflammatory protein secreted into the host during feeding. PLoS Negl Trop Dis 2019; 13:e0007660. [PMID: 31449524 PMCID: PMC6730956 DOI: 10.1371/journal.pntd.0007660] [Citation(s) in RCA: 24] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/03/2019] [Revised: 09/06/2019] [Accepted: 07/24/2019] [Indexed: 11/20/2022] Open
Abstract
Ticks successfully feed and transmit pathogens by injecting pharmacological compounds in saliva to thwart host defenses. We have previously used LC-MS/MS to identify proteins that are present in saliva of unfed Amblyomma americanum ticks that were exposed to different hosts. Here we show that A. americanum serine protease inhibitor (serpin) 27 (AAS27) is an immunogenic saliva protein that is injected into the host within the first day of tick feeding and is an anti-inflammatory protein that might act by blocking plasmin and trypsin functions. Although AAS27 is injected into the host throughout tick feeding, qRT-PCR and western blotting analyses indicate that the respective transcript and protein are present in high amounts within the first 24 h of tick feeding. Biochemical screening of Pichia pastoris-expressed recombinant (r) AAS27 against mammalian proteases related to host defense shows it is an inhibitor of trypsin and plasmin, with stoichiometry of inhibition indices of 3.5 and 3.8, respectively. Consistent with typical inhibitory serpins, rAAS27 formed heat- and SDS-stable irreversible complexes with both proteases. We further demonstrate that rAAS27 inhibits trypsin with ka of 6.46 ± 1.24 x 104 M-1 s-1, comparable to serpins of other tick species. We show that native AAS27 is part of the repertoire of proteins responsible for the inhibitory activity against trypsin in crude tick saliva. AAS27 is likely utilized by the tick to evade the hosts inflammation defense since rAAS27 blocks both formalin and compound 48/80-induced inflammation in rats. Tick immune sera of rabbits that had acquired resistance against tick feeding following repeated infestations with A. americanum or Ixodes scapularis ticks reacts with rAAS27. Of significant interest, antibody to rAAS27 blocks this serpin inhibitory functions. Taken together, we conclude that AAS27 is an anti-inflammatory protein secreted into the host during feeding and may represent a potential candidate for development of an anti-tick vaccine.
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14
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Henehan M, De Benedetto A. Update on protease‐activated receptor 2 in cutaneous barrier, differentiation, tumorigenesis and pigmentation, and its role in related dermatologic diseases. Exp Dermatol 2019; 28:877-885. [DOI: 10.1111/exd.13936] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/27/2018] [Revised: 03/03/2019] [Accepted: 03/18/2019] [Indexed: 12/20/2022]
Affiliation(s)
- Mason Henehan
- Department of Dermatology College of Medicine University of Florida Gainesville Florida
| | - Anna De Benedetto
- Department of Dermatology College of Medicine University of Florida Gainesville Florida
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15
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Di Maggio LS, Tirloni L, Pinto AFM, Diedrich JK, Yates JR, Carmona C, Berasain P, da Silva Vaz I. A proteomic comparison of excretion/secretion products in Fasciola hepatica newly excysted juveniles (NEJ) derived from Lymnaea viatrix or Pseudosuccinea columella. Exp Parasitol 2019; 201:11-20. [PMID: 31022392 DOI: 10.1016/j.exppara.2019.04.004] [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] [Received: 12/19/2018] [Revised: 03/19/2019] [Accepted: 04/12/2019] [Indexed: 12/13/2022]
Abstract
The characteristics of parasitic infections are often tied to host behavior. Although most studies have investigated definitive hosts, intermediate hosts can also play a role in shaping the distribution and accumulation of parasites. This is particularly relevant in larval stages, where intermediate host's behavior could potentially interfere in the molecules secreted by the parasite into the next host during infection. To investigate this hypothesis, we used a proteomic approach to analyze excretion/secretion products (ESP) from Fasciola hepatica newly excysted juveniles (NEJ) derived from two intermediate host species, Lymnaea viatrix and Pseudosuccinea columella. The two analyzed proteomes showed differences in identity, abundance, and functional classification of the proteins. This observation could be due to differences in the biological cycle of the parasite in the host, environmental aspects, and/or host-dependent factors. Categories such as protein modification machinery, protease inhibitors, signal transduction, and cysteine-rich proteins showed different abundance between samples. More specifically, differences in abundance of individual proteins such as peptidyl-prolyl cis-trans isomerase, thioredoxin, cathepsin B, cathepsin L, and Kunitz-type inhibitors were identified. Based on the differences identified between NEJ ESP samples, we can conclude that the intermediate host is a factor influencing the proteomic profile of ESP in F. hepatica.
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Affiliation(s)
- Lucía Sánchez Di Maggio
- Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil; Unidad de Biología Parasitaria, Facultad de Ciencias, Universidad de la República Oriental del Uruguay, Montevideo, Uruguay
| | - Lucas Tirloni
- Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil; College of Veterinary Medicine, Department of Veterinary Pathobiology, Texas A&M University, College Station, TX, USA
| | - Antônio F M Pinto
- Department of Molecular Medicine, The Scripps Research Institute, CA, USA
| | - Jolene K Diedrich
- Department of Molecular Medicine, The Scripps Research Institute, CA, USA
| | - John R Yates
- Department of Molecular Medicine, The Scripps Research Institute, CA, USA
| | - Carlos Carmona
- Unidad de Biología Parasitaria, Facultad de Ciencias, Universidad de la República Oriental del Uruguay, Montevideo, Uruguay
| | - Patricia Berasain
- Unidad de Biología Parasitaria, Facultad de Ciencias, Universidad de la República Oriental del Uruguay, Montevideo, Uruguay.
| | - Itabajara da Silva Vaz
- Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil; Faculdade de Veterinária, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil.
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16
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Sun L, Li PB, Yao YF, Xiu AY, Peng Z, Bai YH, Gao YJ. Proteinase-activated receptor 2 promotes tumor cell proliferation and metastasis by inducing epithelial-mesenchymal transition and predicts poor prognosis in hepatocellular carcinoma. World J Gastroenterol 2018; 24:1120-1133. [PMID: 29563756 PMCID: PMC5850131 DOI: 10.3748/wjg.v24.i10.1120] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/08/2017] [Revised: 12/29/2017] [Accepted: 01/23/2018] [Indexed: 02/06/2023] Open
Abstract
AIM To clarify the role of proteinase-activated receptor 2 (PAR2) in hepatocellular carcinoma, especially in the process of metastasis.
METHODS PAR2 expression levels were assessed by qRT-PCR and immunohistochemistry (IHC) in patient tissues and in hepatocellular carcinoma cell lines SMMC-7721 and HepG2. Cell proliferation and metastasis were assessed both in vitro and in vitro. Immunoblotting was carried out to monitor the levels of mitogen-activated protein kinase (MAPK) and epithelial-mesenchymal transition markers.
RESULTS The prognosis was significantly poorer in patients with high PAR2 levels than in those with low PAR2 levels. Patients with high PAR2 levels had advanced tumor stage (P = 0.001, chi-square test), larger tumor size (P = 0.032, chi-square test), and high microvascular invasion rate (P = 0.037, chi-square test). The proliferation and metastasis ability of SMMC-7721 and HepG2 cells was increased after PAR2 overexpression, while knockdown of PAR2 decreased the proliferation and metastasis ability of SMMC-7721 and HepG2 cells. Knockdown of PAR2 also inhibited hepatocellular carcinoma tumor cell growth and liver metastasis in nude mice. Mechanistically, PAR2 increased the proliferation ability of SMMC-7721 and HepG2 cells via ERK activation. Activated ERK further promoted the epithelial-mesenchymal transition of these cells, which endowed them with enhanced migration and invasion ability.
CONCLUSION These data suggest that PAR2 plays an important role in the proliferation and metastasis of hepatocellular carcinoma. Therefore, targeting PAR2 may present a favorable target for treatment of this malignancy.
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Affiliation(s)
- Liang Sun
- Qilu Hospital of Shandong University, Jinan 250012, Shandong Province, China
- Department of Critical Care Medicine, Shandong Traffic Hospital, Jinan 250000, Shandong Province, China
| | - Pi-Bao Li
- Department of Critical Care Medicine, Shandong Traffic Hospital, Jinan 250000, Shandong Province, China
| | - Yan-Fen Yao
- Department of Critical Care Medicine, Shandong Traffic Hospital, Jinan 250000, Shandong Province, China
| | - Ai-Yuan Xiu
- Department of Gastroenterology, Qilu Hospital of Shandong University, Jinan 250012, Shandong Province, China
| | - Zhi Peng
- Department of Gastroenterology, Qilu Hospital of Shandong University, Jinan 250012, Shandong Province, China
| | - Yu-Huan Bai
- Department of Gastroenterology, Qilu Hospital of Shandong University, Jinan 250012, Shandong Province, China
| | - Yan-Jing Gao
- Department of Gastroenterology, Qilu Hospital of Shandong University, Jinan 250012, Shandong Province, China
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17
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da Silva Barth C, Tolentino de Souza HG, Rocha LW, da Silva GF, Dos Anjos MF, Pastor VD, Belle Bresolin TM, Garcia Couto A, Roberto Santin J, Meira Quintão NL. Ipomoea pes-caprae (L.) R. Br (Convolvulaceae) relieved nociception and inflammation in mice - A topical herbal medicine against effects due to cnidarian venom-skin contact. JOURNAL OF ETHNOPHARMACOLOGY 2017; 200:156-164. [PMID: 28196712 DOI: 10.1016/j.jep.2017.02.014] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/17/2016] [Revised: 02/09/2017] [Accepted: 02/10/2017] [Indexed: 06/06/2023]
Abstract
ETHNOPHARMACOLOGICAL RELEVANCE Ipomoea pes-caprae is known as bayhops, beach morning glory or goat's foot, and in Brazil as salsa-de-praia. Its leaves are used in worldwide folk medicine for the relief of jellyfish-stings symptoms. The literature only reports the neutralizing effects of nonpolar plant derived over jellyfish venoms, without validating the popular use or exploring the mechanism of action. AIM OF THE STUDY This study aimed to evaluate and validate the topical effects of a semisolid containing hydroethanolic extract obtained from the aerial parts of I. pes-caprae using different models of paw- and ear-oedema and spontaneous nociception in mice, attempting to better understand the mechanism involved in its effect. MATERIALS AND METHODS Leaf and stem of I. pes-caprae were extracted by ethanol 50% (v/v) and the soft-extract was incorporated in Hostacerin® cream base at 0.5%, 1.0% and 2% (w/w). The anti-hypersensitivity effects were evaluated by injecting the Physalia physalis venom into the right mice's hindpaw pre-treated either with the semisolid containing the I. pes-caprae extract or with the isolated majority compound 3,5-Di-O-caffeoylquinic acid (ISA). The topical anti-inflammatory activity was investigated using both preclinical models: paw oedema induced by trypsin, bradykinin (BK), histamine and carrageenan, and ear oedema induced by capsaicin. Additionally, the model of spontaneous nociception induced by trypsin and capsaicin were used to verify the topical antinociceptive activity. RESULTS The animals pre-treated with the semisolid containing I. pes-caprae extract or with the intraplantar injection of the major component (ISA) had the mechanical hypersensitivity induced by P. physalis venom significantly reduced. Significant inhibition was also observed in paw-oedema induced by trypsin, histamine and BK, and in a less extent in carrageenan-induced paw oedema. Similar effect was observed in mice challenged to the capsaicin-induced ear-oedema. Besides the vascular effects, the extract interfered with leukocyte migration induced by histamine injection. Finally, the semisolid presented significant inhibition in trypsin- and capsaicin-induced spontaneous nociception. CONCLUSIONS The hydroethanolic extract of I. pes-caprae showed compliance with the topical popular use of the herbal product to relieve the symptoms evoked by the cnidarian venom-skin contact, such as neurogenic oedema and nociception. The extract components seem to interfere with the effects resulting from the TRPV1, B2R and PAR-2 activation, once it interfered with painful-behaviour and oedema induced by capsaicin, BK and trypsin, pointing the histaminergic system as the main target, once it is an important mediator in the signalling pathway of the aforementioned receptors.
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Affiliation(s)
| | - Hugo Guilherme Tolentino de Souza
- Núcleo de Investigações Químico-Farmacêuticas (NIQFAR), Itajaí, SC, Brazil; Pharmacy Course, Universidade do Vale de Itajaí (UNIVALI), Itajaí, SC, Brazil
| | - Lilian W Rocha
- Department of Structural and Functional Biology, State University of Campinas (UNICAMP), São Paulo, Brazil
| | | | | | | | - Tania Mari Belle Bresolin
- Postgraduate Program in Pharmaceutical Sciences, Itajaí, SC, Brazil; Núcleo de Investigações Químico-Farmacêuticas (NIQFAR), Itajaí, SC, Brazil
| | - Angelica Garcia Couto
- Postgraduate Program in Pharmaceutical Sciences, Itajaí, SC, Brazil; Núcleo de Investigações Químico-Farmacêuticas (NIQFAR), Itajaí, SC, Brazil
| | - José Roberto Santin
- Postgraduate Program in Pharmaceutical Sciences, Itajaí, SC, Brazil; Núcleo de Investigações Químico-Farmacêuticas (NIQFAR), Itajaí, SC, Brazil
| | - Nara Lins Meira Quintão
- Postgraduate Program in Pharmaceutical Sciences, Itajaí, SC, Brazil; Núcleo de Investigações Químico-Farmacêuticas (NIQFAR), Itajaí, SC, Brazil.
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Tirloni L, Kim TK, Coutinho ML, Ali A, Seixas A, Termignoni C, Mulenga A, da Silva Vaz I. The putative role of Rhipicephalus microplus salivary serpins in the tick-host relationship. INSECT BIOCHEMISTRY AND MOLECULAR BIOLOGY 2016; 71:12-28. [PMID: 26844868 PMCID: PMC4808628 DOI: 10.1016/j.ibmb.2016.01.004] [Citation(s) in RCA: 44] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/23/2015] [Revised: 01/28/2016] [Accepted: 01/29/2016] [Indexed: 05/13/2023]
Abstract
Inflammation and hemostasis are part of the host's first line of defense to tick feeding. These systems are in part serine protease mediated and are tightly controlled by their endogenous inhibitors, in the serpin superfamily (serine protease inhibitors). From this perspective ticks are thought to use serpins to evade host defenses during feeding. The cattle tick Rhipicephalus microplus encodes at least 24 serpins, of which RmS-3, RmS-6, and RmS-17 were previously identified in saliva of this tick. In this study, we screened inhibitor functions of these three saliva serpins against a panel of 16 proteases across the mammalian defense pathway. Our data confirm that Pichia pastoris-expressed rRmS-3, rRmS-6, and rRmS-17 are likely inhibitors of pro-inflammatory and pro-coagulant proteases. We show that rRmS-3 inhibited chymotrypsin and cathepsin G with stoichiometry of inhibition (SI) indices of 1.8 and 2.0, and pancreatic elastase with SI higher than 10. Likewise, rRmS-6 inhibited trypsin with SI of 2.6, chymotrypsin, factor Xa, factor XIa, and plasmin with SI higher than 10, while rRmS-17 inhibited trypsin, cathepsin G, chymotrypsin, plasmin, and factor XIa with SI of 1.6, 2.6, 2.7, 3.4, and 9.0, respectively. Additionally, we observed the formation of irreversible complexes between rRmS-3 and chymotrypsin, rRmS-6/rRmS-17 and trypsin, and rRmS-3/rRmS-17 and cathepsin G, which is consistent with typical mechanism of inhibitory serpins. In blood clotting assays, rRmS-17 delayed plasma clotting by 60 s in recalcification time assay, while rRmS-3 and rRmS-6 did not have any effect. Consistent with inhibitor function profiling data, 2.0 μM rRmS-3 and rRmS-17 inhibited cathepsin G-activated platelet aggregation in a dose-responsive manner by up to 96% and 95% respectively. Of significant interest, polyclonal antibodies blocked inhibitory functions of the three serpins. Also notable, antibodies to Amblyomma americanum, Ixodes scapularis, and Rhipicephalus sanguineus tick saliva proteins cross-reacted with the three R. microplus saliva serpins, suggesting the potential of these proteins as candidates for universal anti-tick vaccines.
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Affiliation(s)
- Lucas Tirloni
- Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil; Department of Veterinary Pathobiology, College of Veterinary Medicine, Texas A&M University, College Station, TX, USA
| | - Tae Kwon Kim
- Department of Veterinary Pathobiology, College of Veterinary Medicine, Texas A&M University, College Station, TX, USA
| | - Mariana Loner Coutinho
- Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil; Faculdade de Veterinária, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil
| | - Abid Ali
- Institute of Biotechnology Genetic Engineering, The University of Agriculture, Peshawar, Khyber Pakhtunkhwa, Pakistan
| | - Adriana Seixas
- Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil; Departamento de Farmacociências, Universidade Federal de Ciências da Saúde de Porto Alegre, Porto Alegre, RS, Brazil
| | - Carlos Termignoni
- Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil; Departamento de Bioquímica, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil
| | - Albert Mulenga
- Department of Veterinary Pathobiology, College of Veterinary Medicine, Texas A&M University, College Station, TX, USA
| | - Itabajara da Silva Vaz
- Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil; Faculdade de Veterinária, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil.
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Ruiz-Laguna J, Vélez JM, Pueyo C, Abril N. Global gene expression profiling using heterologous DNA microarrays to analyze alterations in the transcriptome of Mus spretus mice living in a heavily polluted environment. ENVIRONMENTAL SCIENCE AND POLLUTION RESEARCH INTERNATIONAL 2016; 23:5853-5867. [PMID: 26590064 DOI: 10.1007/s11356-015-5824-5] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/23/2015] [Accepted: 11/16/2015] [Indexed: 06/05/2023]
Abstract
Microarray platforms are a good approach for assessing biological responses to pollution as they enable the simultaneous analyses of changes in the expression of thousands of genes. As an omic and non-targeted methodology, this technique is open to unforeseen responses under particular environmental conditions. In this study, we successfully apply a commercial oligonucleotide microarray containing Mus musculus whole-genome probes to compare and assess the biological effects of living in a heavily polluted settlement, the Domingo Rubio stream (DRS), at the Huelva Estuary (SW Spain), on inhabitant free-living Mus spretus mice. Our microarray results show that mice living in DRS suffer dramatic changes in gene and protein expression compared with reference specimens. DRS mice showed alteration in the oxidative status of hepatocytes, with activation of both the innate and the acquired immune responses and the induction of chronic inflammation, accompanied by metabolic alterations that imply the accumulation of lipids in the liver (hepatic steatosis). The identified deregulated genes may be useful as biomarkers of environmental pollution.
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Affiliation(s)
- Julia Ruiz-Laguna
- Department of Biochemistry and Molecular Biology and Agrifood Campus of International Excellence (ceiA3-UCO), University of Córdoba, Severo Ochoa Building, Rabanales Campus, 14071, Córdoba, Spain
| | - José M Vélez
- Department of Biochemistry and Molecular Biology and Agrifood Campus of International Excellence (ceiA3-UCO), University of Córdoba, Severo Ochoa Building, Rabanales Campus, 14071, Córdoba, Spain
| | - Carmen Pueyo
- Department of Biochemistry and Molecular Biology and Agrifood Campus of International Excellence (ceiA3-UCO), University of Córdoba, Severo Ochoa Building, Rabanales Campus, 14071, Córdoba, Spain
| | - Nieves Abril
- Department of Biochemistry and Molecular Biology and Agrifood Campus of International Excellence (ceiA3-UCO), University of Córdoba, Severo Ochoa Building, Rabanales Campus, 14071, Córdoba, Spain.
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20
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Luo J, Feng J, Liu S, Walters ET, Hu H. Molecular and cellular mechanisms that initiate pain and itch. Cell Mol Life Sci 2015; 72:3201-23. [PMID: 25894692 PMCID: PMC4534341 DOI: 10.1007/s00018-015-1904-4] [Citation(s) in RCA: 69] [Impact Index Per Article: 6.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/30/2014] [Revised: 03/20/2015] [Accepted: 04/07/2015] [Indexed: 12/17/2022]
Abstract
Somatosensory neurons mediate our sense of touch. They are critically involved in transducing pain and itch sensations under physiological and pathological conditions, along with other skin-resident cells. Tissue damage and inflammation can produce a localized or systemic sensitization of our senses of pain and itch, which can facilitate our detection of threats in the environment. Although acute pain and itch protect us from further damage, persistent pain and itch are debilitating. Recent exciting discoveries have significantly advanced our knowledge of the roles of membrane-bound G protein-coupled receptors and ion channels in the encoding of information leading to pain and itch sensations. This review focuses on molecular and cellular events that are important in early stages of the biological processing that culminates in our senses of pain and itch.
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Affiliation(s)
- Jialie Luo
- Department of Anesthesiology, The Center for the Study of Itch, Washington University School of Medicine, 660 S. Euclid Ave., St. Louis, MO, 63110, USA
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21
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Alloy AP, Kayode O, Wang R, Hockla A, Soares AS, Radisky ES. Mesotrypsin Has Evolved Four Unique Residues to Cleave Trypsin Inhibitors as Substrates. J Biol Chem 2015; 290:21523-35. [PMID: 26175157 DOI: 10.1074/jbc.m115.662429] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/30/2015] [Indexed: 01/18/2023] Open
Abstract
Human mesotrypsin is highly homologous to other mammalian trypsins, and yet it is functionally unique in possessing resistance to inhibition by canonical serine protease inhibitors and in cleaving these inhibitors as preferred substrates. Arg-193 and Ser-39 have been identified as contributors to the inhibitor resistance and cleavage capability of mesotrypsin, but it is not known whether these residues fully account for the unusual properties of mesotrypsin. Here, we use human cationic trypsin as a template for engineering a gain of catalytic function, assessing mutants containing mesotrypsin-like mutations for resistance to inhibition by bovine pancreatic trypsin inhibitor (BPTI) and amyloid precursor protein Kunitz protease inhibitor (APPI), and for the ability to hydrolyze these inhibitors as substrates. We find that Arg-193 and Ser-39 are sufficient to confer mesotrypsin-like resistance to inhibition; however, compared with mesotrypsin, the trypsin-Y39S/G193R double mutant remains 10-fold slower at hydrolyzing BPTI and 2.5-fold slower at hydrolyzing APPI. We identify two additional residues in mesotrypsin, Lys-74 and Asp-97, which in concert with Arg-193 and Ser-39 confer the full catalytic capability of mesotrypsin for proteolysis of BPTI and APPI. Novel crystal structures of trypsin mutants in complex with BPTI suggest that these four residues function cooperatively to favor conformational dynamics that assist in dissociation of cleaved inhibitors. Our results reveal that efficient inhibitor cleavage is a complex capability to which at least four spatially separated residues of mesotrypsin contribute. These findings suggest that inhibitor cleavage represents a functional adaptation of mesotrypsin that may have evolved in response to positive selection pressure.
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Affiliation(s)
- Alexandre P Alloy
- From the Department of Cancer Biology, Mayo Clinic Comprehensive Cancer Center, Jacksonville, Florida 32224 and
| | - Olumide Kayode
- From the Department of Cancer Biology, Mayo Clinic Comprehensive Cancer Center, Jacksonville, Florida 32224 and
| | - Ruiying Wang
- From the Department of Cancer Biology, Mayo Clinic Comprehensive Cancer Center, Jacksonville, Florida 32224 and
| | - Alexandra Hockla
- From the Department of Cancer Biology, Mayo Clinic Comprehensive Cancer Center, Jacksonville, Florida 32224 and
| | - Alexei S Soares
- the Biology Department, Brookhaven National Laboratory, Upton, New York 11973
| | - Evette S Radisky
- From the Department of Cancer Biology, Mayo Clinic Comprehensive Cancer Center, Jacksonville, Florida 32224 and
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22
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Conserved Amblyomma americanum tick Serpin19, an inhibitor of blood clotting factors Xa and XIa, trypsin and plasmin, has anti-haemostatic functions. Int J Parasitol 2015; 45:613-27. [PMID: 25957161 DOI: 10.1016/j.ijpara.2015.03.009] [Citation(s) in RCA: 43] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/28/2015] [Revised: 03/21/2015] [Accepted: 03/23/2015] [Indexed: 12/21/2022]
Abstract
Tick saliva serine protease inhibitors (serpins) facilitate tick blood meal feeding through inhibition of protease mediators of host defense pathways. We previously identified a highly conserved Amblyomma americanum serpin 19 that is characterised by its reactive center loop being 100% conserved in ixodid ticks. In this study, biochemical characterisation reveals that the ubiquitously transcribed A. americanum serpin 19 is an anti-coagulant protein, inhibiting the activity of five of the eight serine protease blood clotting factors. Pichia pastoris-expressed recombinant (r) A. americanum serpin 19 inhibits the enzyme activity of trypsin, plasmin and blood clotting factors (f) Xa and XIa, with stoichiometry of inhibition estimated at 5.1, 9.4, 23.8 and 28, respectively. Similar to typical inhibitory serpins, recombinant A. americanum serpin 19 forms irreversible complexes with trypsin, fXa and fXIa. At a higher molar excess of recombinant A. americanum serpin 19, fXIIa is inhibited by 82.5%, and thrombin (fIIa), fIXa, chymotrypsin and tryptase are inhibited moderately by 14-29%. In anti-hemostatic functional assays, recombinant A. americanum serpin 19 inhibits thrombin but not ADP and cathepsin G activated platelet aggregation, delays clotting in recalcification and thrombin time assays by up to 250s, and up to 40s in the activated partial thromboplastin time assay. Given A. americanum serpin 19 high cross-tick species conservation, and specific reactivity of recombinant A. americanum serpin 19 with antibodies to A. americanum tick saliva proteins, we conclude that recombinant A. americanum serpin 19 is a potential candidate for development of a universal tick vaccine.
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Haerteis S, Krappitz A, Krappitz M, Murphy JE, Bertog M, Krueger B, Nacken R, Chung H, Hollenberg MD, Knecht W, Bunnett NW, Korbmacher C. Proteolytic activation of the human epithelial sodium channel by trypsin IV and trypsin I involves distinct cleavage sites. J Biol Chem 2014; 289:19067-78. [PMID: 24841206 DOI: 10.1074/jbc.m113.538470] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022] Open
Abstract
Proteolytic activation is a unique feature of the epithelial sodium channel (ENaC). However, the underlying molecular mechanisms and the physiologically relevant proteases remain to be identified. The serine protease trypsin I can activate ENaC in vitro but is unlikely to be the physiologically relevant activating protease in ENaC-expressing tissues in vivo. Herein, we investigated whether human trypsin IV, a form of trypsin that is co-expressed in several extrapancreatic epithelial cells with ENaC, can activate human ENaC. In Xenopus laevis oocytes, we monitored proteolytic activation of ENaC currents and the appearance of γENaC cleavage products at the cell surface. We demonstrated that trypsin IV and trypsin I can stimulate ENaC heterologously expressed in oocytes. ENaC cleavage and activation by trypsin IV but not by trypsin I required a critical cleavage site (Lys-189) in the extracellular domain of the γ-subunit. In contrast, channel activation by trypsin I was prevented by mutating three putative cleavage sites (Lys-168, Lys-170, and Arg-172) in addition to mutating previously described prostasin (RKRK(178)), plasmin (Lys-189), and neutrophil elastase (Val-182 and Val-193) sites. Moreover, we found that trypsin IV is expressed in human renal epithelial cells and can increase ENaC-mediated sodium transport in cultured human airway epithelial cells. Thus, trypsin IV may regulate ENaC function in epithelial tissues. Our results show, for the first time, that trypsin IV can stimulate ENaC and that trypsin IV and trypsin I activate ENaC by cleavage at distinct sites. The presence of distinct cleavage sites may be important for ENaC regulation by tissue-specific proteases.
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Affiliation(s)
- Silke Haerteis
- From the Institut für Zelluläre und Molekulare Physiologie, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), Waldstrasse 6, 91054 Erlangen, Germany
| | - Annabel Krappitz
- From the Institut für Zelluläre und Molekulare Physiologie, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), Waldstrasse 6, 91054 Erlangen, Germany
| | - Matteus Krappitz
- From the Institut für Zelluläre und Molekulare Physiologie, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), Waldstrasse 6, 91054 Erlangen, Germany
| | - Jane E Murphy
- the UCSF Center for the Neurobiology of Digestive Diseases, Department of Surgery, University of California, San Francisco, California
| | - Marko Bertog
- From the Institut für Zelluläre und Molekulare Physiologie, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), Waldstrasse 6, 91054 Erlangen, Germany
| | - Bettina Krueger
- From the Institut für Zelluläre und Molekulare Physiologie, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), Waldstrasse 6, 91054 Erlangen, Germany
| | - Regina Nacken
- From the Institut für Zelluläre und Molekulare Physiologie, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), Waldstrasse 6, 91054 Erlangen, Germany
| | - Hyunjae Chung
- the Department of Physiology and Pharmacology, University of Calgary, Calgary, Alberta T2N 4N1, Canada
| | - Morley D Hollenberg
- the Department of Physiology and Pharmacology, University of Calgary, Calgary, Alberta T2N 4N1, Canada
| | - Wolfgang Knecht
- Bioscience, CVGI iMed, AstraZeneca Research and Development, 43181 Mölndal, Sweden
| | - Nigel W Bunnett
- the Monash Institute of Pharmaceutical Sciences, 381 Royal Parade, Parkville, Victoria 3052, Australia, and the Department of Pharmacology, University of Melbourne, Melbourne, Victoria 3010, Australia
| | - Christoph Korbmacher
- From the Institut für Zelluläre und Molekulare Physiologie, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), Waldstrasse 6, 91054 Erlangen, Germany,
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