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Franco-da-Silva MM, Aubin MR, de Vasconcellos AA, Sirena DH, Marchaki GB, Ruggeri LR, Hennigen AF, Muradás T, da Silveira ABT, Braganhol E, Schuh RS, Baldo G, Araújo AB, Paz AH. Effects of chorionic mesenchymal stromal cells, their conditioned medium, and membrane particles on neutrophil functionality. Cell Tissue Res 2025:10.1007/s00441-025-03970-6. [PMID: 40261417 DOI: 10.1007/s00441-025-03970-6] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/02/2024] [Accepted: 04/08/2025] [Indexed: 04/24/2025]
Abstract
Mesenchymal stromal cells (MSC) are multipotent cells that can modulate immune cells, affecting macrophages, monocytes, and lymphocytes. Neutrophils are circulating leucocytes responsible for the first line of defense and can assume different phenotypes depending on their environment: N0, the naïve form, N1 (inflammatory), N2 (anti-inflammatory). This study explores the potentially protective roles of chorionic membrane MSCs and their products-conditioned medium and pre-conditioned cMSC-derived membrane microparticles (MP-cMSC)-on neutrophils. Conditioned medium treatment reduced the rate of apoptosis and enhanced the immunosuppressive potential consistent with an anti-inflammatory profile. MP-cMSC are a noteworthy cell-free therapy, consisting of artificially generated circular lipid bilayer structures with no cargo and approximately 200 nm in size. When added to neutrophil culture, MPs increased neutral red uptake, suggesting an enhanced phagocytic activity. In the MSC co-culture group, a reduced rate of apoptosis, increased neutral red uptake, and elevated programed death-ligand 1 (PD-L1) expression were observed. These findings suggest that the distinct effects elicited by conditioned media, microparticles, and co-culture are likely influenced by the specific nature of the interactions involved-whether purely paracrine, mediated through direct cell-to-cell contact, or a combination of both.
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Affiliation(s)
- Monique Maria Franco-da-Silva
- Cells, Tissues and Genes Laboratory, Experimental Research Center, Hospital de Clínicas de Porto Alegre, Porto Alegre, Brazil
- Graduate Program in Physiology, Basic Health Sciences Institute, Universidade Federal Do Rio Grande Do Sul, Porto Alegre, Brazil
| | - Mariana Rauback Aubin
- Cells, Tissues and Genes Laboratory, Experimental Research Center, Hospital de Clínicas de Porto Alegre, Porto Alegre, Brazil
- Graduate Program in Physiology, Basic Health Sciences Institute, Universidade Federal Do Rio Grande Do Sul, Porto Alegre, Brazil
| | - Alessandra Amaral de Vasconcellos
- Cells, Tissues and Genes Laboratory, Experimental Research Center, Hospital de Clínicas de Porto Alegre, Porto Alegre, Brazil
- Graduate Program in Physiology, Basic Health Sciences Institute, Universidade Federal Do Rio Grande Do Sul, Porto Alegre, Brazil
| | - Dienifer Hermann Sirena
- Cells, Tissues and Genes Laboratory, Experimental Research Center, Hospital de Clínicas de Porto Alegre, Porto Alegre, Brazil
- Graduate Program in Physiology, Basic Health Sciences Institute, Universidade Federal Do Rio Grande Do Sul, Porto Alegre, Brazil
| | - Giovana Bangel Marchaki
- Cells, Tissues and Genes Laboratory, Experimental Research Center, Hospital de Clínicas de Porto Alegre, Porto Alegre, Brazil
| | - Laíza Rief Ruggeri
- Cells, Tissues and Genes Laboratory, Experimental Research Center, Hospital de Clínicas de Porto Alegre, Porto Alegre, Brazil
| | - André Ferreira Hennigen
- Cells, Tissues and Genes Laboratory, Experimental Research Center, Hospital de Clínicas de Porto Alegre, Porto Alegre, Brazil
| | - Thaís Muradás
- Pharmaceutical Sciences - Graduate Program in Pharmaceutical Sciences, Pharmacy Faculty, Universidade Federal Do Rio Grande Do Sul, Porto Alegre, Brazil
| | - Ana Beatriz Tittoni da Silveira
- Cells, Tissues and Genes Laboratory, Experimental Research Center, Hospital de Clínicas de Porto Alegre, Porto Alegre, Brazil
| | - Elizandra Braganhol
- Graduate Program in Biosciences, Federal University of Health Sciences of Porto Alegre, Porto Alegre, Brazil
| | - Roselena Silvestri Schuh
- Cells, Tissues and Genes Laboratory, Experimental Research Center, Hospital de Clínicas de Porto Alegre, Porto Alegre, Brazil
- Pharmaceutical Sciences - Graduate Program in Pharmaceutical Sciences, Pharmacy Faculty, Universidade Federal Do Rio Grande Do Sul, Porto Alegre, Brazil
| | - Guilherme Baldo
- Cells, Tissues and Genes Laboratory, Experimental Research Center, Hospital de Clínicas de Porto Alegre, Porto Alegre, Brazil
- Graduate Program in Physiology, Basic Health Sciences Institute, Universidade Federal Do Rio Grande Do Sul, Porto Alegre, Brazil
| | - Anelise Bergmann Araújo
- Cell Processing Center, Hemotherapy Service, Hospital de Clínicas de Porto Alegre, Porto Alegre, Brazil
| | - Ana Helena Paz
- Cells, Tissues and Genes Laboratory, Experimental Research Center, Hospital de Clínicas de Porto Alegre, Porto Alegre, Brazil.
- Graduate Program in Physiology, Basic Health Sciences Institute, Universidade Federal Do Rio Grande Do Sul, Porto Alegre, Brazil.
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Božić Nedeljković B, Ćilerdžić J, Zmijanjac D, Marković M, Džopalić T, Vasilijić S, Stajić M, Vučević D. Immunomodulatory effects of extract of Lingzhi or Reishi medicinal Mushroom Ganoderma lucidum (Agaricomycetes) basidiocarps cultivated on alternative substrate. Int J Med Mushrooms 2022; 24:45-59. [DOI: 10.1615/intjmedmushrooms.2022044452] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022]
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Tucovic D, Mirkov I, Kulas J, Zeljkovic M, Popovic D, Zolotarevski L, Djurdjic S, Mutic J, Kataranovski M, Popov Aleksandrov A. Dermatotoxicity of oral cadmium is strain-dependent and related to differences in skin stress response and inflammatory/immune activity. ENVIRONMENTAL TOXICOLOGY AND PHARMACOLOGY 2020; 75:103326. [PMID: 31924569 DOI: 10.1016/j.etap.2020.103326] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/09/2019] [Revised: 12/13/2019] [Accepted: 01/02/2020] [Indexed: 06/10/2023]
Abstract
Adverse effects of non-occupational exposure to cadmium (Cd) are increasingly acknowledged. Since our previous study has showed that orally acquired Cd affects skin, the contribution of genetic background to dermatotoxicity of oral cadmium was examined in two rat strains, Albino Oxford (AO) and Dark Agouti (DA), which differed in response to chemicals. While similar accumulation of Cd in the skin of both strains was noted, the skin response to the metal differed. DA rat individuals mounted antioxidant enzyme defense in the skin already at lower Cd dose, in contrast to AO rats which reacted to higher metal dose solely (and less pronounced), implying higher susceptibility of DA strain to Cd dermatotoxicity. Epidermal cells from both strains developed stress response, but higher intensity of antioxidant response in AO rats implied this strain`s better ability to defend against Cd insult. Cd induced epidermal cells' proinflammatory cytokine response only in DA rats. Increased IL-10 seems responsible for the lack of response in AO rats. Differences in the pattern of skin/epidermal cell responsiveness to cadmium give a new insight into repercussion of genetic variability to dermatotoxicity of orally acquired cadmium, bearing relevance for variations in the link between dietary cadmium and inflammation-based skin pathologies.
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Affiliation(s)
- Dina Tucovic
- Immunotoxicology Group, Department of Ecology, Institute for Biological Research "Siniša Stanković"- National Institute of Republic of Serbia, University of Belgrade, 142 Bulevar Despota Stefana, 11000, Belgrade, Serbia
| | - Ivana Mirkov
- Immunotoxicology Group, Department of Ecology, Institute for Biological Research "Siniša Stanković"- National Institute of Republic of Serbia, University of Belgrade, 142 Bulevar Despota Stefana, 11000, Belgrade, Serbia
| | - Jelena Kulas
- Immunotoxicology Group, Department of Ecology, Institute for Biological Research "Siniša Stanković"- National Institute of Republic of Serbia, University of Belgrade, 142 Bulevar Despota Stefana, 11000, Belgrade, Serbia
| | - Milica Zeljkovic
- Immunotoxicology Group, Department of Ecology, Institute for Biological Research "Siniša Stanković"- National Institute of Republic of Serbia, University of Belgrade, 142 Bulevar Despota Stefana, 11000, Belgrade, Serbia
| | - Dusanka Popovic
- Immunotoxicology Group, Department of Ecology, Institute for Biological Research "Siniša Stanković"- National Institute of Republic of Serbia, University of Belgrade, 142 Bulevar Despota Stefana, 11000, Belgrade, Serbia
| | - Lidija Zolotarevski
- Immunotoxicology Group, Department of Ecology, Institute for Biological Research "Siniša Stanković"- National Institute of Republic of Serbia, University of Belgrade, 142 Bulevar Despota Stefana, 11000, Belgrade, Serbia
| | - Sladjana Djurdjic
- Innovation Center of the Faculty of Chemistry, University of Belgrade, 12-16 Studentski Trg, 11000, Belgrade, Serbia
| | - Jelena Mutic
- Innovation Center of the Faculty of Chemistry, University of Belgrade, 12-16 Studentski Trg, 11000, Belgrade, Serbia
| | - Milena Kataranovski
- Immunotoxicology Group, Department of Ecology, Institute for Biological Research "Siniša Stanković"- National Institute of Republic of Serbia, University of Belgrade, 142 Bulevar Despota Stefana, 11000, Belgrade, Serbia
| | - Aleksandra Popov Aleksandrov
- Immunotoxicology Group, Department of Ecology, Institute for Biological Research "Siniša Stanković"- National Institute of Republic of Serbia, University of Belgrade, 142 Bulevar Despota Stefana, 11000, Belgrade, Serbia.
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Tucovic D, Popov Aleksandrov A, Mirkov I, Ninkov M, Kulas J, Zolotarevski L, Vukojevic V, Mutic J, Tatalovic N, Kataranovski M. Oral cadmium exposure affects skin immune reactivity in rats. ECOTOXICOLOGY AND ENVIRONMENTAL SAFETY 2018; 164:12-20. [PMID: 30092388 DOI: 10.1016/j.ecoenv.2018.07.117] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/04/2018] [Revised: 07/11/2018] [Accepted: 07/28/2018] [Indexed: 06/08/2023]
Abstract
Skin can acquire cadmium (Cd) by oral route, but there is paucity of data concerning cutaneous effects of this metal. Cd acquired by oral route can affect skin wound healing, but the effect of Cd on other activities involved in skin homeostasis, including skin immunity, are not explored. Using the rat model of 30-day oral administration of Cd (5 ppm and 50 ppm) in drinking water, basic aspects of immune-relevant activity of epidermal cells were examined. Dose-dependent Cd deposition in the the skin was observed (0.035 ± 0.02 µg/g and 0.127 ± 0.04 µg/g at 5 ppm and 50 ppm, respectively, compared to 0.012 ± 0.009 µg/g at 0 ppm of Cd). This resulted in skin inflammation (oxidative stress at both Cd doses and dose-dependent structural changes in the skin and the presence/activation of innate immunity cells). At low Cd dose inflammatory response (nitric oxide and IL-1β) was observed. Other inflammatory cytokines (IL-6 and TNF) response occurred at 50 ppm, which was increased further following skin sensitization with contact allergen dinitro-chlorobenzene (DNCB). Epidermal cells exposed to both Cd doses enhanced concanavalin A (ConA)-stimulated lymphocyte production of IL-17. This study showed for the first time the effect of the metal which gained access to the skin via gut on immune reactivity of epidermal cells. Presented data might be relevant for the link between dietary Cd and the risk of skin pathologies.
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Affiliation(s)
- Dina Tucovic
- Immunotoxicology Group, Department of Ecology, Institute for Biological Research "Sinisa Stankovic", University of Belgrade, 142 Bulevar despota Stefana, 11000 Belgrade, Serbia
| | - Aleksandra Popov Aleksandrov
- Immunotoxicology Group, Department of Ecology, Institute for Biological Research "Sinisa Stankovic", University of Belgrade, 142 Bulevar despota Stefana, 11000 Belgrade, Serbia
| | - Ivana Mirkov
- Immunotoxicology Group, Department of Ecology, Institute for Biological Research "Sinisa Stankovic", University of Belgrade, 142 Bulevar despota Stefana, 11000 Belgrade, Serbia
| | - Marina Ninkov
- Immunotoxicology Group, Department of Ecology, Institute for Biological Research "Sinisa Stankovic", University of Belgrade, 142 Bulevar despota Stefana, 11000 Belgrade, Serbia
| | - Jelena Kulas
- Immunotoxicology Group, Department of Ecology, Institute for Biological Research "Sinisa Stankovic", University of Belgrade, 142 Bulevar despota Stefana, 11000 Belgrade, Serbia
| | - Lidija Zolotarevski
- Immunotoxicology Group, Department of Ecology, Institute for Biological Research "Sinisa Stankovic", University of Belgrade, 142 Bulevar despota Stefana, 11000 Belgrade, Serbia
| | - Vesna Vukojevic
- Innovation Center of the Faculty of Chemistry, University of Belgrade, 12-16 Studentski trg, 11000 Belgrade, Serbia
| | - Jelena Mutic
- Innovation Center of the Faculty of Chemistry, University of Belgrade, 12-16 Studentski trg, 11000 Belgrade, Serbia
| | - Nikola Tatalovic
- Department of Physiology, Institute for Biological Research "Siniša Stanković", University of Belgrade, 142 Bulevar despota Stefana, 11000 Belgrade, Serbia
| | - Milena Kataranovski
- Immunotoxicology Group, Department of Ecology, Institute for Biological Research "Sinisa Stankovic", University of Belgrade, 142 Bulevar despota Stefana, 11000 Belgrade, Serbia; Institute of Physiology and Biochemistry, Faculty of Biology, University of Belgrade, 16 Studentski trg, 11000 Belgrade, Serbia.
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Aleksandrov AP, Mirkov I, Zolotarevski L, Ninkov M, Mileusnic D, Kataranovski D, Kataranovski M. Oral warfarin intake affects skin inflammatory cytokine responses in rats. ENVIRONMENTAL TOXICOLOGY AND PHARMACOLOGY 2017; 54:93-98. [PMID: 28704755 DOI: 10.1016/j.etap.2017.06.027] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/16/2016] [Revised: 05/15/2017] [Accepted: 06/26/2017] [Indexed: 06/07/2023]
Abstract
Warfarin is an anticoagulant used in prevention/prophylaxis of thromboembolism. Besides the effects on coagulation, non-hemorrhagic reactions have also been documented. Although cutaneous reactions were reported in some patients, the impact on skin immunity was not explored. In the present paper, the effect of 30-day oral warfarin intake on skin cytokine responses in rats was analyzed. Increased release of inflammatory cytokines (TNF, IL-1β and IL-10) was noted by skin explants from rats which received warfarin, but without effect on IL-6. No impact on epidermal cell cytokine secretion was seen, except a tendency of an increase of IL-6 response to stimulation with microbial product lipopolysaccharide (LPS). Topical application of contact allergen dinitrochlorobenzene (DNCB) resulted in slight (numerical solely) increase of TNF release by skin explants of warfarin-treated animals, while epidermal cells responded by increased secretion of all four cytokines examined. The data presented provide new information on the potential of oral warfarin to modulate skin innate immune activity.
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Affiliation(s)
- Aleksandra Popov Aleksandrov
- Immunotoxicology Group, Department of Ecology, Institute for Biological Research "Sinisa Stankovic", University of Belgrade, Belgrade, Serbia
| | - Ivana Mirkov
- Immunotoxicology Group, Department of Ecology, Institute for Biological Research "Sinisa Stankovic", University of Belgrade, Belgrade, Serbia
| | | | - Marina Ninkov
- Immunotoxicology Group, Department of Ecology, Institute for Biological Research "Sinisa Stankovic", University of Belgrade, Belgrade, Serbia
| | - Dina Mileusnic
- Immunotoxicology Group, Department of Ecology, Institute for Biological Research "Sinisa Stankovic", University of Belgrade, Belgrade, Serbia
| | - Dragan Kataranovski
- Immunotoxicology Group, Department of Ecology, Institute for Biological Research "Sinisa Stankovic", University of Belgrade, Belgrade, Serbia; Institute of Zoology, Faculty of Biology, University of Belgrade, Belgrade, Serbia
| | - Milena Kataranovski
- Immunotoxicology Group, Department of Ecology, Institute for Biological Research "Sinisa Stankovic", University of Belgrade, Belgrade, Serbia; Institute of Physiology and Biochemistry, Faculty of Biology, University of Belgrade, Belgrade, Serbia.
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Popov Aleksandrov A, Belij-Rammerstorfer S, Mirkov I, Subota V, Kulas J, Kataranovski D, Kataranovski M. Oral warfarin affects some aspects of systemic immunomodulation with topical dinitrochlorobenzene (DNCB) in rats. Cutan Ocul Toxicol 2017; 37:29-35. [PMID: 28486821 DOI: 10.1080/15569527.2017.1328690] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/19/2022]
Abstract
PURPOSE The efficacy of topical dinitrochlorobenzene (DNCB) in the treatment of some skin dermatoses is based both on local and systemic effects. It is not known, however, whether it can be applied to patients receiving some other therapy associated with systemic immunomodulation. The aim of the present paper using a rat model was to examine whether oral warfarin (WF) intake, as shown by others and by us, had an immunomodulatory potential to interfere with effects of topical DNCB as systemic immunotherapy. MATERIALS AND METHODS Rats received 3.5 mg/l of WF sodium in drinking water for 30 days and were thereafter skin-sensitized with 0.4% DNCB. Changes in the oxidative activity (myeloperoxidase/MPO, reduction of nitroblue tetrazolium/NBT and nitric oxide/NO production) as well as tumor necrosis factor (TNF) production by peripheral blood polymorphonuclear cells (PMN) were measured and compared with PMN from sensitized unexposed to WF rats. RESULTS WF intake enhanced some aspects of PMN activity (intracellular MPO activity and unstimulated NO production) as well as their responsiveness to exogenous stimulation (NBT reduction and TNF production from sensitized animals). However, WF also decreased PMN responsiveness of NO production to stimulation. WF affected NO and TNF production solely by PMN, as no effect on these activities of peripheral blood mononuclear cells was seen. CONCLUSION Having in mind that polymorphonuclear leukocytes are the most abundant cell type in peripheral blood in humans, increase of basic aspects of PMN activity described in the present paper might be relevant for consideration of using WF as therapeutic modality in patients topically treated with DNCB.
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Affiliation(s)
- Aleksandra Popov Aleksandrov
- a Immunotoxicology Group, Department of Ecology , Institute for Biological Research "Sinisa Stankovic", University of Belgrade , Belgrade , Serbia
| | - Sandra Belij-Rammerstorfer
- a Immunotoxicology Group, Department of Ecology , Institute for Biological Research "Sinisa Stankovic", University of Belgrade , Belgrade , Serbia
| | - Ivana Mirkov
- a Immunotoxicology Group, Department of Ecology , Institute for Biological Research "Sinisa Stankovic", University of Belgrade , Belgrade , Serbia
| | - Vesna Subota
- b Institute for Biochemistry, Military Medical Academy , Belgrade , Serbia
| | - Jelena Kulas
- a Immunotoxicology Group, Department of Ecology , Institute for Biological Research "Sinisa Stankovic", University of Belgrade , Belgrade , Serbia
| | - Dragan Kataranovski
- a Immunotoxicology Group, Department of Ecology , Institute for Biological Research "Sinisa Stankovic", University of Belgrade , Belgrade , Serbia.,c Institute of Zoology, Faculty of Biology, University of Belgrade , Belgrade , Serbia , and
| | - Milena Kataranovski
- a Immunotoxicology Group, Department of Ecology , Institute for Biological Research "Sinisa Stankovic", University of Belgrade , Belgrade , Serbia.,d Institute of Physiology and Biochemistry, Faculty of Biology, University of Belgrade , Belgrade , Serbia
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Mirkov I, Popov Aleksandrov A, Demenesku J, Ninkov M, Mileusnic D, Kataranovski D, Kataranovski M. Warfarin affects acute inflammatory response induced by subcutaneous polyvinyl sponge implantation in rats. Cutan Ocul Toxicol 2017; 36:283-288. [DOI: 10.1080/15569527.2016.1275664] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/20/2022]
Affiliation(s)
- Ivana Mirkov
- Ecology Department, Institute for Biological Research “Sinisa Stankovic”, University of Belgrade, Belgrade, Serbia,
| | - Aleksandra Popov Aleksandrov
- Ecology Department, Institute for Biological Research “Sinisa Stankovic”, University of Belgrade, Belgrade, Serbia,
| | - Jelena Demenesku
- Ecology Department, Institute for Biological Research “Sinisa Stankovic”, University of Belgrade, Belgrade, Serbia,
| | - Marina Ninkov
- Ecology Department, Institute for Biological Research “Sinisa Stankovic”, University of Belgrade, Belgrade, Serbia,
| | - Dina Mileusnic
- Ecology Department, Institute for Biological Research “Sinisa Stankovic”, University of Belgrade, Belgrade, Serbia,
| | - Dragan Kataranovski
- Ecology Department, Institute for Biological Research “Sinisa Stankovic”, University of Belgrade, Belgrade, Serbia,
- Institute of Zoology, Faculty of Biology, University of Belgrade, Belgrade, Serbia, and
| | - Milena Kataranovski
- Ecology Department, Institute for Biological Research “Sinisa Stankovic”, University of Belgrade, Belgrade, Serbia,
- Institute of Physiology and Biochemistry, Faculty of Biology, University of Belgrade, Belgrade, Serbia
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Mirkov I, Popov Aleksandrov A, Ninkov M, Mileusnic D, Demenesku J, Zolotarevski L, Subota V, Stefik D, Kataranovski D, Kataranovski M. Strain differences in intestinal toxicity of warfarin in rats. ENVIRONMENTAL TOXICOLOGY AND PHARMACOLOGY 2016; 48:175-182. [PMID: 27816002 DOI: 10.1016/j.etap.2016.10.019] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/15/2016] [Revised: 10/18/2016] [Accepted: 10/21/2016] [Indexed: 06/06/2023]
Abstract
Intestinal hemorrhage characterizes effectiveness of warfarin (WF) as rodenticide and is among adverse effects of therapy in humans. Having in mind genetic variations in the effectiveness of WF in wild rats and in the doses required for therapeutic effect, strain differences in the intestinal toxicity of oral warfarin in rats were examined in this study. High WF dose (3.5mg/l) led to mortality in Albino Oxford (AO) rats, with no lethality in Dark Agouti (DA) rats. Higher values of prothrombin time were noted at low WF dose (0.35mg/l) in the former strain. Leukocyte infiltration in intestine noted at this dose in both strains was associated with oxidative injury and more pronounced anti-oxidative response in AO rats. Suppression of mesenteric lymph node cell proliferation and IFN-γ and IL-10 production in AO rats and lack of these effects in DA rats, represent different strategies to protect vulnerable intestine from harmful immune responses.
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Affiliation(s)
- Ivana Mirkov
- Immunotoxicology group, Department of Ecology, Institute for Biological Research "Sinisa Stankovic", University of Belgrade, 142 Bulevar despota Stefana, 11000 Belgrade, Serbia
| | - Aleksandra Popov Aleksandrov
- Immunotoxicology group, Department of Ecology, Institute for Biological Research "Sinisa Stankovic", University of Belgrade, 142 Bulevar despota Stefana, 11000 Belgrade, Serbia
| | - Marina Ninkov
- Immunotoxicology group, Department of Ecology, Institute for Biological Research "Sinisa Stankovic", University of Belgrade, 142 Bulevar despota Stefana, 11000 Belgrade, Serbia
| | - Dina Mileusnic
- Immunotoxicology group, Department of Ecology, Institute for Biological Research "Sinisa Stankovic", University of Belgrade, 142 Bulevar despota Stefana, 11000 Belgrade, Serbia
| | - Jelena Demenesku
- Immunotoxicology group, Department of Ecology, Institute for Biological Research "Sinisa Stankovic", University of Belgrade, 142 Bulevar despota Stefana, 11000 Belgrade, Serbia
| | - Lidija Zolotarevski
- Institute for Pathology, Military Medical Academy, 17 Crnotravska, 11000 Belgrade, Serbia
| | - Vesna Subota
- Institute for Medical Biochemistry, Military Medical Academy, 17 Crnotravska, 11000 Belgrade, Serbia
| | - Debora Stefik
- Immunotoxicology group, Department of Ecology, Institute for Biological Research "Sinisa Stankovic", University of Belgrade, 142 Bulevar despota Stefana, 11000 Belgrade, Serbia
| | - Dragan Kataranovski
- Immunotoxicology group, Department of Ecology, Institute for Biological Research "Sinisa Stankovic", University of Belgrade, 142 Bulevar despota Stefana, 11000 Belgrade, Serbia; Institute of Zoology, Faculty of Biology, University of Belgrade,16 Studentski trg, 11000 Belgrade, Serbia
| | - Milena Kataranovski
- Immunotoxicology group, Department of Ecology, Institute for Biological Research "Sinisa Stankovic", University of Belgrade, 142 Bulevar despota Stefana, 11000 Belgrade, Serbia; Institute of Physiology and Biochemistry, Faculty of Biology, University of Belgrade, 16 Studentski trg, 11000 Belgrade, Serbia.
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Mirkov I, Popov Aleksandrov A, Demenesku J, Ninkov M, Mileusnic D, Zolotarevski L, Subota V, Kataranovski D, Kataranovski M. Intestinal toxicity of oral warfarin intake in rats. Food Chem Toxicol 2016; 94:11-8. [PMID: 27181730 DOI: 10.1016/j.fct.2016.05.007] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/25/2016] [Revised: 05/09/2016] [Accepted: 05/10/2016] [Indexed: 11/18/2022]
Abstract
Though warfarin is extensively used in the prevention and treatment of thromboembolic processes in humans, adverse effects of warfarin therapy have been recognized. Intestinal hemorrhage is one of the hazards of anticoagulant therapy, but the mechanisms of warfarin toxicity are virtually unknown. In this work, the effects of 30 days oral warfarin (0.35 mg/l and 3.5 mg/l) intake on rat's gut were examined. Both doses resulted in prolongation of prothrombin time. Systemic effects of higher warfarin dose (increases in plasma AST, proteinuria, hematuria, changes in peripheral blood hematological parameters) were seen. Warfarin intake resulted in histologically evident tissue damage, leukocyte infiltration and intestinal inflammation [increases in myeloperoxidase activity, malondialdehyde content, superoxide dismutase and catalase activity, proinflammatory cytokine (IFN-γ, IL-17) concentrations in intestinal homogenates]. In contrast, suppression of gut-draining mesenteric lymph node (MLN) cell activity [proliferation responsiveness, production of IFN-γ and IL-17 to T lymphocyte mitogen Concanavalin A stimulation] was noted. Inhibition of regulatory cytokine IL-10 production by MLN cells, suggests commitment of MLN to the suppression of all inflammatory activities and creation of the microenvironment which is non-permissive for induction of potentially harmful immune response. These novel findings indicate the need of staying alert for (adverse) effects of warfarin therapy.
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Affiliation(s)
- Ivana Mirkov
- Department of Ecology, Institute for Biological Research "Sinisa Stankovic", University of Belgrade, 142 Bulevar despota Stefana, 11000 Belgrade, Serbia
| | - Aleksandra Popov Aleksandrov
- Department of Ecology, Institute for Biological Research "Sinisa Stankovic", University of Belgrade, 142 Bulevar despota Stefana, 11000 Belgrade, Serbia
| | - Jelena Demenesku
- Department of Ecology, Institute for Biological Research "Sinisa Stankovic", University of Belgrade, 142 Bulevar despota Stefana, 11000 Belgrade, Serbia
| | - Marina Ninkov
- Department of Ecology, Institute for Biological Research "Sinisa Stankovic", University of Belgrade, 142 Bulevar despota Stefana, 11000 Belgrade, Serbia
| | - Dina Mileusnic
- Department of Ecology, Institute for Biological Research "Sinisa Stankovic", University of Belgrade, 142 Bulevar despota Stefana, 11000 Belgrade, Serbia
| | - Lidija Zolotarevski
- Institute for Pathology, Military Medical Academy, 17 Crnotravska, 11000 Belgrade, Serbia
| | - Vesna Subota
- Institute for Medical Biochemistry, Military Medical Academy, 17 Crnotravska, 11000 Belgrade, Serbia
| | - Dragan Kataranovski
- Department of Ecology, Institute for Biological Research "Sinisa Stankovic", University of Belgrade, 142 Bulevar despota Stefana, 11000 Belgrade, Serbia; Institute of Zoology, Faculty of Biology, University of Belgrade, 16 Studentski trg 11000 Belgrade, Serbia
| | - Milena Kataranovski
- Department of Ecology, Institute for Biological Research "Sinisa Stankovic", University of Belgrade, 142 Bulevar despota Stefana, 11000 Belgrade, Serbia; Institute of Physiology and Biochemistry, Faculty of Biology, University of Belgrade, 16 Studentski trg, 11000 Belgrade, Serbia.
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Subota V, Mirkov I, Demenesku J, Popov Aleksandrov A, Ninkov M, Mileusnic D, Kataranovski D, Kataranovski M. Transdermal toxicity of topically applied anticoagulant rodenticide warfarin in rats. ENVIRONMENTAL TOXICOLOGY AND PHARMACOLOGY 2016; 41:232-240. [PMID: 26742017 DOI: 10.1016/j.etap.2015.12.006] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/20/2015] [Revised: 12/11/2015] [Accepted: 12/14/2015] [Indexed: 06/05/2023]
Abstract
Occupational/accidental exposure data have showed hemorrhage as a result of transdermal exposure to warfarin, however, other effects are not known. In the present study, the impact of epicutaneous application of 10 μg or 100 μg of warfarin (three times, once a day) on peripheral blood polymorphonuclear (PMN) and mononuclear cells (PBMC) was examined in rats. Both doses resulted in prolongation of prothrombin time and changes in hematologic parameters. Increases in PMN intracellular myeloperoxidase (MPO) activity were seen at higher warfarin dose and both doses resulted in higher percentages of granular CD11b(+) cells. In contrast, a decrease in PMN TNF and IL-6 production (ELISA) and gene expression (RT-PCR) was observed. Epicutaneous application of warfarin resulted in decreased numbers of PBMC, higher numbers of mononuclear CD11b(+) cells, but without effect on PMBC cytokine production. The data obtained showed differential effects of transdermal exposure to warfarin depending on leukocyte type and activity.
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Affiliation(s)
- Vesna Subota
- Institute for Medical Biochemistry, Military Medical Academy, Crnotravska 17, 11000 Belgrade, Serbia
| | - Ivana Mirkov
- Department of Ecology, Institute for Biological Research "Sinisa Stankovic", University of Belgrade, Bulevar despota Stefana 142, 11000 Belgrade, Serbia
| | - Jelena Demenesku
- Department of Ecology, Institute for Biological Research "Sinisa Stankovic", University of Belgrade, Bulevar despota Stefana 142, 11000 Belgrade, Serbia
| | - Aleksandra Popov Aleksandrov
- Department of Ecology, Institute for Biological Research "Sinisa Stankovic", University of Belgrade, Bulevar despota Stefana 142, 11000 Belgrade, Serbia
| | - Marina Ninkov
- Department of Ecology, Institute for Biological Research "Sinisa Stankovic", University of Belgrade, Bulevar despota Stefana 142, 11000 Belgrade, Serbia
| | - Dina Mileusnic
- Department of Ecology, Institute for Biological Research "Sinisa Stankovic", University of Belgrade, Bulevar despota Stefana 142, 11000 Belgrade, Serbia
| | - Dragan Kataranovski
- Department of Ecology, Institute for Biological Research "Sinisa Stankovic", University of Belgrade, Bulevar despota Stefana 142, 11000 Belgrade, Serbia; Institute of Zoology, Faculty of Biology, University of Belgrade, Studentski trg 16, 11000 Belgrade, Serbia
| | - Milena Kataranovski
- Department of Ecology, Institute for Biological Research "Sinisa Stankovic", University of Belgrade, Bulevar despota Stefana 142, 11000 Belgrade, Serbia; Institute of Physiology and Biochemistry, Faculty of Biology, University of Belgrade, Studentski trg 16, 11000 Belgrade, Serbia.
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11
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Kongkhum S, Fungkrajai M, Prajan S, Bordeerat NK, Piumngam K, Siripurkpong P. High glucose enhances CD39 expression in vascular endothelial cells. ASIAN BIOMED 2014. [DOI: 10.5372/1905-7415.0802.291] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
Abstract
Background: Diabetes mellitus (DM) patients lose their ability to control normal blood glucose levels, resulting in high blood glucose levels (hyperglycemia). Hyperglycemia causes DM complications. This involves responses of vascular endothelial cells (VECs) to hyperglycemia, affecting inflammatory process and platelet activity. Ecto-enzyme CD39 is expressed on VECs, catalyzing the hydrolysis of ATP and ADP to AMP and, consequently, regulating inflammatory process and platelet activation.
Objective: We studied whether high glucose concentration has an effect on CD39 expression on VECs.
Methods: Cultured human umbilical vein endothelial cells (HUVEC) were used as a model of study. HUVEC were cultured in different glucose conditions (4, 9, 24, and 34 mM) for 24 hours. Cell viability was assessed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-based assay and expression of CD39 was examined by using SDS-PAGE and western blot techniques.
Results: HUVEC were cultured in normal (4 and 9 mM) or high (24 and 34 mM) glucose concentrations for short term (24 hours). The results showed that high glucose (24 and 34 mM) reduced cell viability to 89.5 ± 11.3 and 86.3 ± 13.5 (mean ± SD), compared with control (4 mM), respectively. High glucose also induced increases in CD39 expression in HUVEC.
Conclusion: High glucose decreases cell viability and increases CD39 expression in HUVEC, suggesting involvement of CD39 in cell responses to high glucose.
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Affiliation(s)
- Sudawadee Kongkhum
- Faculty of Allied Health Sciences, Thammasat University, Pathum Thani 12120, Thailand
| | - Mudtika Fungkrajai
- Faculty of Allied Health Sciences, Thammasat University, Pathumthani 12120, Thailand
| | - Sompoch Prajan
- Faculty of Allied Health Sciences, Thammasat University, Pathumthani 12120, Thailand
| | | | - Kanyanath Piumngam
- Faculty of Allied Health Sciences, Thammasat University, Pathumthani 12120, Thailand
| | - Pilaiwan Siripurkpong
- Faculty of Allied Health Sciences, Thammasat University, Pathumthani 12120, Thailand
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12
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Khlebtsov BN, Khanadeev VA, Panfilova EV, Pylaev TE, Bibikova OA, Staroverov SA, Bogatyrev VA, Dykman LA, Khlebtsov NG. New types of nanomaterials: powders of gold nanospheres, nanorods, nanostars, and gold-silver nanocages. ACTA ACUST UNITED AC 2013. [DOI: 10.1134/s1995078013020092] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/23/2022]
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13
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Popov A, Belij S, Subota V, Zolotarevski L, Mirkov I, Kataranovski D, Kataranovski M. Oral warfarin affects peripheral blood leukocyte IL-6 and TNFα production in rats. J Immunotoxicol 2012; 10:17-24. [PMID: 22793260 DOI: 10.3109/1547691x.2012.684159] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
Abstract
Warfarin is a Vitamin K (VK) antagonist that affects Vitamin K-dependent (VKD) processes, including blood coagulation, as well as processes unrelated to hemostasis such as bone growth, calcification, and growth of some cell types. In addition, warfarin exerts influence on some non-VKD-related activities, including anti-tumor and immunomodulating activity. With respect to the latter, both immune stimulating and suppressive effects have been noted in different experimental systems. To explore the in vivo immunomodulatory potential of warfarin on one type of activity (i.e., cytokine production) in two different immune cell populations (i.e., mononuclear or polymorphonuclear cells), effects of subchronic oral warfarin intake in rats on pro-inflammatory cytokine (i.e., TNFα, IL-6) production by peripheral blood mononuclear and polymorphonuclear cells (granulocytes) was examined. Differential effects of warfarin intake on TNFα and IL-6 were noted, depending on the type of peripheral blood leukocytes and on the cytokine examined. Specifically, a lack of effect on TNFα and a priming of IL-6 production by mononuclear cells along with a decrease in TNFα and a lack of effect on IL-6 in polymorphonuclear cells were seen in warfarin-exposed hosts. The cell- and cytokine-dependent effects from subchronic oral warfarin intake on peripheral blood leukocytes demonstrated in this study could, possibly, differentially affect reactions mediated by these cells. Ultimately, the observed effects in rats might have implications for those humans who are on long-term/prolonged warfarin therapy.
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Affiliation(s)
- Aleksandra Popov
- Department of Ecology, Institute for Biological Research 'Siniša Stanković', University of Belgrade, Belgrade, Serbia
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14
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Effects of subacute oral warfarin administration on peripheral blood granulocytes in rats. Food Chem Toxicol 2012; 50:1499-507. [PMID: 22342526 DOI: 10.1016/j.fct.2012.01.049] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/13/2011] [Revised: 01/30/2012] [Accepted: 01/31/2012] [Indexed: 11/22/2022]
Abstract
Warfarin affects mainly vitamin K dependent (VKD) processes, but the effects on some non-VKD-related activities such as tumor growth inhibition and mononuclear cell-mediated immune reactions were shown as well. In this study, the effect of subchronic (30 days) oral warfarin (0.35 mg/l and 3.5mg/l) intake on peripheral blood granulocytes in rats was investigated. Increase in prothrombin and partial thromboplastin time at high warfarin dose reflected its basic activity. Priming effect for respiratory burst was noted at both warfarin doses, while only high warfarin dose resulted in priming for adhesion, the rise in intracellular myeloperoxidase content/release and stimulation of nitric oxide production. Differential effects of high warfarin dose were noted on granulocyte cytokines IL-6 (lack of the effect), TNF-α (decreased release and mRNA expression) and IL-12 (increase in mRNA for IL-12 subunits p35 and p40). Changes in granulocytes seems not to rely on mitogen activated kinases p38 and ERK. Warfarin intake was associated with an increase in circulating IL-6, fibrinogen and haptoglobin and with changes in the activity of erythrocyte antioxidant enzymes superoxide dismutase and catalase. The effects of oral warfarin intake on peripheral blood granulocytes demonstrated in this study might be relevant for oral anticoagulant therapy strategies in humans.
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15
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Khlebtsov B, Panfilova E, Khanadeev V, Bibikova O, Terentyuk G, Ivanov A, Rumyantseva V, Shilov I, Ryabova A, Loshchenov V, Khlebtsov NG. Nanocomposites containing silica-coated gold-silver nanocages and Yb-2,4-dimethoxyhematoporphyrin: multifunctional capability of IR-luminescence detection, photosensitization, and photothermolysis. ACS NANO 2011; 5:7077-7089. [PMID: 21838309 DOI: 10.1021/nn2017974] [Citation(s) in RCA: 65] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/31/2023]
Abstract
We describe novel composite nanoparticles consisting of a gold-silver nanocage core and a mesoporous silica shell functionalized with the photodynamic sensitizer Yb-2,4-dimethoxyhematoporphyrin (Yb-HP). In addition to the long-wavelength plasmon resonance near 750-800 nm, the composite particles exhibited a 400-nm absorbance peak and two fluorescence peaks, near 580 and 630 nm, corresponding to bound Yb-HP. The fabricated nanocomposites generated singlet oxygen under 630-nm excitation and produced heat under laser irradiation at the plasmon resonance wavelength (750-800 nm). In particular, we observed enhanced killing of HeLa cells incubated with nanocomposites and irradiated by 630-nm light. Furthermore, an additional advantage of fabricated conjugates was an IR-luminescence band (900-1060 nm), originating from Yb(3+) ions of bound Yb-HP and located in the long-wavelength part of the tissue transparency window. This modality was used to control the accumulation and biodistribution of composite particles in mice bearing Ehrlich carcinoma tumors in a comparative study with intravenously injected free Yb-HP molecules. Thus, these multifunctional nanocomposites seem an attractive theranostic platform for simultaneous IR-luminescence diagnostic and photodynamic therapy owing to Yb-HP and for plasmonic photothermal therapy owing to Au-Ag nanocages.
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Affiliation(s)
- Boris Khlebtsov
- Institute of Biochemistry and Physiology of Plants and Microorganisms, Russian Academy of Sciences, 13 Prospekt Entuziastov, Saratov 410049, Russia
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16
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Lee JE, Lee RA, Kim KH, Lee JH. Induction of apoptosis with diallyl disulfide in AGS gastric cancer cell line. JOURNAL OF THE KOREAN SURGICAL SOCIETY 2011; 81:85-95. [PMID: 22066106 PMCID: PMC3204569 DOI: 10.4174/jkss.2011.81.2.85] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 02/16/2011] [Accepted: 05/04/2011] [Indexed: 11/30/2022]
Abstract
Purpose Diallyl disulfide (DADS) is a major organosulfur compound derived from garlic. It has been reported that DADS is able to inhibit the proliferation of several tumor cells. In this study, the effect of DADS was investigated in terms of the proliferation of AGS, gastric adenocarcinoma cell line at various concentrations. Methods The viability of cultured cells was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay. To detect the induction of apoptosis, Annexin V-FITC/propodium iodide (PI) staining assay was performed. Analysis of reactive oxygen species (ROS) and the distribution of cells in the cell cycle were measured by a flow cytometer. And using the Western blot analysis, the change of Fas, caspase-3, Bax, Bcl-2 activity was measured. Results The percentage of live AGS cells was decreased to 23% of that in the control group after 400 µM DADS treatment for 48 hours. The Annexin V positive/PI negative (apoptosis portion) area increased from low concentration of DADS to high concentration. When comparing among the DADS treatment groups, the amount of ROS production increased in a dose dependent manner. The percentage of sub-diploid DNA content increased from 8.71% at 50 µM to 25.74% at 400 µM DADS treatment group. The expressions of Fas, caspase-3, Bax were increased and that of Bcl-2 was decreased in a dose dependent manner. Conclusion DADS decreases the viability of AGS cell lines and induces apoptosis in a dose-dependent manner. But the relationship of the anti-proliferative effect of DADS and related molecular changes were not clearly proportional to the concentration of DADS.
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Affiliation(s)
- Jeong-Eun Lee
- Department of Surgery, Hansol Hospital, Seoul, Korea
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17
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Ye BQ, Geng ZH, Ma L, Geng JG. Slit2 regulates attractive eosinophil and repulsive neutrophil chemotaxis through differential srGAP1 expression during lung inflammation. JOURNAL OF IMMUNOLOGY (BALTIMORE, MD. : 1950) 2010; 185:6294-305. [PMID: 20944010 DOI: 10.4049/jimmunol.1001648] [Citation(s) in RCA: 56] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/19/2022]
Abstract
Directional migration of leukocytes is an essential step in leukocyte trafficking during inflammatory responses. However, the molecular mechanisms governing directional chemotaxis of leukocytes remain poorly understood. The Slit family of guidance cues has been implicated for inhibition of leuocyte migration. We report that Clara cells in the bronchial epithelium secreted Slit2, whereas eosinophils and neutrophils expressed its cell-surface receptor, Robo1. Compared to neutrophils, eosinophils exhibited a significantly lower level of Slit-Robo GTPase-activating protein 1 (srGAP1), leading to activation of Cdc42, recruitment of PI3K to Robo1, enhancment of eotaxin-induced eosinophil chemotaxis, and exaggeration of allergic airway inflammation. Notably, OVA sensitization elicited a Slit2 gradient at so-called bronchus-alveoli axis, with a higher level of Slit2 in the bronchial epithelium and a lower level in the alveolar tissue. Aerosol administration of rSlit2 accelerated eosinophil infiltration, whereas i.v. administered Slit2 reduced eosinophil deposition. In contrast, Slit2 inactivated Cdc42 and suppressed stromal cell-derived factor-1α-induced chemotaxis of neutrophils for inhibiting endotoxin-induced lung inflammation, which were reversed by blockade of srGAP1 binding to Robo1. These results indicate that the newly identified Slit2 gradient at the bronchus-alveoli axis induces attractive PI3K signaling in eosinophils and repulsive srGAP1 signaling in neutrophils through differential srGAP1 expression during lung inflammation.
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Affiliation(s)
- Bu-Qing Ye
- Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China
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18
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Park WS. Effect of Lactobacillus pentosus-Fermented Artemisiae Argi Folium on Nitric Oxide Production of Macrophage impaired with Various Toxicants. J Pharmacopuncture 2009. [DOI: 10.3831/kpi.2009.12.4.089] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022] Open
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19
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Optical probes for detection and quantification of neutrophils’ oxidative burst. A review. Anal Chim Acta 2009; 649:8-23. [DOI: 10.1016/j.aca.2009.06.063] [Citation(s) in RCA: 125] [Impact Index Per Article: 7.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/14/2009] [Revised: 06/25/2009] [Accepted: 06/29/2009] [Indexed: 11/20/2022]
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20
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Zhao JF, Sun AH, Ruan P, Zhao XH, Lu MQ, Yan J. Vibrio vulnificus cytolysin induces apoptosis in HUVEC, SGC-7901 and SMMC-7721 cells via caspase-9/3-dependent pathway. Microb Pathog 2009; 46:194-200. [PMID: 19167479 DOI: 10.1016/j.micpath.2008.12.005] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/28/2008] [Revised: 12/16/2008] [Accepted: 12/30/2008] [Indexed: 11/28/2022]
Abstract
Vibrio vulnificus cytolysin (VVC) is known to be a pore-forming toxin which shows cytotoxicity for mammalian cells in culture and induces apoptosis in endothelial cells. In order to determine whether VVC induces apoptosis in vascular endothelial cells and tumor cells, the cytotoxicity induced by recombinant VVC (rVVC) and its potential mechanism in HUVEC, SGC-7901 and SMMC-7721 cells were investigated. Our study demonstrated that rVVC induced the release of intracellular K(+) from all the target cells, yet lactate dehydrogenase was not released by rVVC. It indicates that osmotic lysis might not contribute to the cytolysin-induced cytotoxicity. The study also demonstrated that rVVC induced apoptosis in HUVEC, SGC-7901 and SMMC-7721 cells in time- and dosage-dependent manners, which was associated with the activation of caspase-9 and -3, but not caspase-8. During the apoptotic process of the target cells, rVVC labeled with FITC was monitored to attach initially to the surface of the cells and entered the cytoplasma subsequently. These findings suggest that VVC may be not only a pore-forming toxin, but also a transmembrane toxin with powerful ability to induce apoptosis in human vascular endothelial cells and tumor cells.
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Affiliation(s)
- Jin-fang Zhao
- Basic Medical Microbiology Division, State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Hangzhou 310003, Zhejiang, PR China
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21
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Kataranovski M, Mirkov I, Vrankovic J, Kataranovski D, Subota V. Percutaneous Toxicity of Anticoagulant Warfarin in Rats. Cutan Ocul Toxicol 2008; 27:29-40. [DOI: 10.1080/15569520701860999] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/22/2022]
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22
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Huang Y, Hu XQ, Shen DP, Chen YF, Xu PF. Synthesis of 1H-imidazo[1,2-b]-1,2,4-triazol-6-amines via multicomponent reaction. Mol Divers 2007; 11:73-80. [PMID: 17566867 DOI: 10.1007/s11030-007-9059-3] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/22/2006] [Accepted: 03/14/2007] [Indexed: 10/23/2022]
Abstract
A simple and effective method for the synthesis of bioactive imidazo[1,2,4]triazole analogues has been developed utilizing the MCR technique, which involved the reaction of aminotriazoles, isocyanides and aldehydes. The products were characterized by (1)H, (13)C NMR spectroscopy, MS and elemental analysis. Interestingly, compounds 19d, 21a, 21b and 21h displayed good cytotoxic activities at 10(-4) M concentration in the inhibition of tumor cell growth as evaluated by the MTT method.
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Affiliation(s)
- Yan Huang
- State Key Laboratory of Applied Organic Chemistry, College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, P.R. China
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Seledtsov VI, Rabinovich SS, Kashchenko EA, Fel'de MA, Banul NV, Poveshchenko OV, Astrakov SV, Savchenko SA, Kafanova MY, Seledtsova GV, Kozlov VA. Immunological and clinical aspects of cell therapy in the treatment of aftereffects of craniocerebral injury. Bull Exp Biol Med 2006; 141:121-3. [PMID: 16929982 DOI: 10.1007/s10517-006-0110-7] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
Cell suspension consisting of cells from immature nervous and hemopoietic tissues was transplanted subarachnoidally to patients with craniocerebral injury aftereffects. In some patients cell therapy led to immune sensitization to donor antigens, detected by the leukocyte migration inhibition test. No signs of tissue-destructive autoimmune reactions were detected in patients receiving cell therapy. Follow-up of 56 patients showed that cell therapy was associated with significant improvement of the neurological status. No serious complications of this treatment modality were observed. Presumably, cell therapy is a safe method which can be used in the treatment of craniocerebral injury aftereffects.
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Affiliation(s)
- V I Seledtsov
- Institute of Clinical Immunology, Siberian Division of Russian Academy of Medical Sciences
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24
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Kwon KB, Kim EK, Han MJ, Shin BC, Park YK, Kim KS, Lee YR, Park JW, Park BH, Ryu DG. Induction of Apoptosis by Radix Paeoniae Alba Extract through Cytochrome c Release and the Activations of Caspase-9 and Caspase-3 in HL-60 Cells. Biol Pharm Bull 2006; 29:1082-6. [PMID: 16754997 DOI: 10.1248/bpb.29.1082] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Radix Paeoniae Alba has been used as a constituent of herbal medicine prescriptions for the treatment of inflammation, cancer, and other diseases. The aim of this study was to investigate the mechanism of Radix Paeoniae Alba extract (RPAE)-induced apoptosis in HL-60 leukemic cells. We observed that RPAE induced apoptotic changes in a dose-dependent manner, which was confirmed by DNA fragmentation and poly-(ADP-ribose) polymerase (PARP) cleavage. We also found release of cytochrome c from mitochondria to the cytosol in RPAE-treated HL-60 cells. The caspases, caspase-9 and -3, but not caspase-8, were found to be activated in response to RPAE treatment, and the caspase-3 inhibitor, Ac-DEVD-CHO, and the caspase-9 inhibitor, z-LEHD-FMK, but not the caspase-8 inhibitor, z-IETD-FMK, attenuated RPAE-induced DNA fragmentation and PARP cleavage. These results suggest that RPAE-induced apoptosis is stimulated by the release of cytochrome c to the cytosol, by procaspase-9 processing, and via a caspase-3 dependent mechanism.
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Affiliation(s)
- Kang Beom Kwon
- Department of Physiology, School of Oriental Medicine, Wonkwang University, Iksan, Jeonbuk, Korea
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Kataranovski M, Prokić V, Kataranovski D, Zolotarevski L, Majstorović I. Dermatotoxicity of epicutaneously applied anticoagulant warfarin. Toxicology 2005; 212:206-18. [PMID: 15990214 DOI: 10.1016/j.tox.2005.04.018] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/30/2005] [Revised: 04/26/2005] [Accepted: 04/27/2005] [Indexed: 02/03/2023]
Abstract
Dermatotoxic effects of epicutaneous application of a first-generation anticoagulant, warfarin (WF) were examined in rats. Selected parameters of skin activity were determined 24h following warfarin application, including metabolic viability of skin explants, some aspects of oxidative activity in skin tissue homogenates and inflammatory/immune relevant activity of epidermal cells from warfarin-treated skin. No changes in skin metabolic viability (MTT reduction) were noted ex vivo following WF application, suggesting the absence of immediate toxicity for skin. In contrast, increased formation of malondialdehyde (MDA), with a decrease in protein and non-protein thiols in homogenates of warfarin-treated skin was demonstrated, pointing to prooxidant activity in warfarin-treated skin. Increased costimulatory activity of epidermal cells isolated from warfarin-exposed skin in Con-A-stimulated T-cell activation/proliferation assay was noted, reflecting proinflammatory and immune-modulating capacity of warfarin for epidermis. No evident differences in skin histology between control and warfarin-treated skin were found at that time point, while striking changes in tissue integrity, cellularity and appearance 72 h following WF application were noted. The observed histological picture probably reflects a regenerative/inflammatory program related to oxidant/inflammation-type warfarin-evoked injury to the skin. Presented data demonstrate the potential of epicutaneously applied warfarin to modulate local skin activity in rats.
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Affiliation(s)
- Milena Kataranovski
- Institute for Medical Research, Military Medical Academy, Crnotravska 17, 11000 Belgrade, Serbia and Montenegro.
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26
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Kim EK, Kwon KB, Shin BC, Seo EA, Lee YR, Kim JS, Park JW, Park BH, Ryu DG. Scopoletin induces apoptosis in human promyeloleukemic cells, accompanied by activations of nuclear factor κB and caspase-3. Life Sci 2005; 77:824-36. [PMID: 15936354 DOI: 10.1016/j.lfs.2005.02.003] [Citation(s) in RCA: 59] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/09/2004] [Accepted: 02/26/2005] [Indexed: 11/20/2022]
Abstract
Scopoletin (6-methoxy-7-hydroxycoumarin) is a phenolic coumarin and a member of the phytoalexins. In this study we investigated whether scopoletin caused apoptosis in HL-60 promyelocytic cells and, if so, by what mechanisms. We found that scopoletin induced apoptosis as confirmed by a characteristic ladder pattern of discontinuous DNA fragments in a dose-dependent manner. The signal cascade activated by scopoletin included the heterodimeric redox-sensitive transcription factor NF-kappaB, which exhibited an upregulation of nuclear factor-kappa B (NF-kappaB) translocation to the nucleus by increase of IkappaBalpha degradation. In addition, scopoletin activated caspase-3 as was evidenced by both the proteolytic cleavage of the proenzyme and increased protease activity. Activation of caspase-3 resulted in the cleavage of 116 kDa poly(ADP-ribose) polymerase (PARP) to 85 kDa cleavage product in time-and dose-dependent fashions. Prior treatment of the cells with pyrrolidine dithiocarbamate, a potent inhibitor of NF-kappaB activation, or Ac-DEVD-CHO, a specific caspase-3 inhibitor, prevented scopoletin-induced caspase-3 activation, PARP cleavage, and finally DNA fragmentation. Taken together, these results suggest that scopoletin induces NF-kappaB activation, which, in turn, causes activation of caspase-3, degradation of PARP, and eventually leads to apoptotic cell death in HL-60 cells.
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Affiliation(s)
- Eun-Kyung Kim
- Department of Physiology, School of Oriental Medicine, Wonkwang University, Iksan, Chonbuk 570-749, South Korea
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Seledtsov VI, Kafanova MY, Rabinovich SS, Poveshchenko OV, Kashchenko EA, Fel'de MA, Samarin DM, Seledtsova GV, Kozlov VA. Cell Therapy of Cerebral Palsy. Bull Exp Biol Med 2005; 139:499-503. [PMID: 16027889 DOI: 10.1007/s10517-005-0330-2] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/25/2022]
Abstract
The paper presents the results of a controlled study of cell therapy in 30 patients with severe forms of cerebral palsy. A cell suspension from immature nervous and hemopoietic tissues was injected into the subarachnoidal space of a recipient through a spinal puncture. Immune sensitization to donor antigens (detected by suppression of lymphocyte migration) was noted in few patients. In none patients laboratory and clinical signs of tissue-destructive autoimmune reactions were observed. One year after treatment activity of the major psychomotor functions in treated patients considerably surpassed the normal. No delayed complications of cell therapy were noted. These findings suggest that cell therapy is an effective, safe, and immunologically justified method of therapy for patients with cerebral palsy.
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Affiliation(s)
- V I Seledtsov
- Institute of Clinical Immunology, Siberian Division of the Russian Academy of Medical Sciences, Russia.
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Kwon KB, Kim EK, Lim JG, Jeong ES, Shin BC, Jeon YS, Kim KS, Seo EA, Ryu DG. Molecular mechanisms of apoptosis induced by Scorpio water extract in human hepatoma HepG2 cells. World J Gastroenterol 2005; 11:943-7. [PMID: 15742393 PMCID: PMC4250782 DOI: 10.3748/wjg.v11.i7.943] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To clarify the mechanism underlying the anti-mutagenic and anti-cancer activities of Scorpio water extract (SWE).
METHODS: Human hepatoma HepG2 cells were incubated with various concentrations of SWE. After 24-h incubation, cytotoxicity and apoptosis evaluations were determined by MTT and DNA fragmentation assay, respectively. After treatment with SWE, mitochondrial membrane potential (MMP) was determined by measuring the retention of the dye 3,3’-dihexyloxacarbocyanine (DiOC6(3)) and the protein expression including cytochrome C and poly-(ADP-ribose) polymerase (PARP) were measured by Western blotting. Caspase-3 and -9 enzyme activities were measured using specific fluorescence dyes such as Ac-DEVD-AFC and Ac-LEHD-AFC.
RESULTS: We found that treatment with SWE induced apoptosis as confirmed by discontinuous DNA fragmentation in cultured human hepatoma HepG2 cells. Our investigation also showed that SWE-induced apoptosis of HepG2 cells were associated with intracellular events including disruption of MMP, increased translocation of cytochrome C from mitochondria to cytosol, activation of caspase-3, and PARP. Pre-treatment of N-acetyl-Asp-Glu-Val-Asp-CHO (Ac-DEVD-CHO), a caspase-3 specific inhibitor, or cyclosporin A (CsA), an inhibitor of MMP disruption, completely abolished SWE-induced DNA fragmentation.
CONCLUSION: These results suggest that SWE possibly causes mitochondrial damage, leading to cytochrome C release into cytosol and activation of caspases resulting in PARP cleavage and execution of apoptotic cell death in HepG2 cells. These results further suggest that Scorpio may be a valuable agent of therapeutic intervention of human hepatomas.
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Affiliation(s)
- Kang-Beom Kwon
- Department of Physiology, School of Oriental Medicine, Wonkwang University, Iksan, 570-749 Republic of Korea
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Milena K, Marija V, Dragan K. Peripheral Blood Granulocyte Activity Following Epicutaneous Application of Sodium Dodecyl Sulphate (SDS) in Rats. ACTA ACUST UNITED AC 2004. [DOI: 10.1081/cus-200037205] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/03/2022]
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30
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Kataranovski M, Vlaski M, Kataranovski D, Tosić N, Mandić-Radić S, Todorović V. Immunotoxicity of epicutaneously applied anticoagulant rodenticide warfarin: evaluation by contact hypersensitivity to DNCB in rats. Toxicology 2003; 188:83-100. [PMID: 12748043 DOI: 10.1016/s0300-483x(03)00047-7] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/23/2022]
Abstract
The immunotoxicity of epicutaneously administered anticoagulant rodenticide warfarin (WF) was examined in this work by using experimental contact hypersensitivity (CHS) reaction to hapten dinitrochlorobenzene (DNCB). WF (0.05 and 0.5 mg/kg) administration 24 h before the induction of CHS does not change expression of CHS evaluated by ear swelling assay. Regional draining lymph node response during sensitization phase was characterized by decreased cellularity but increased spontaneous and IL-2 stimulated proliferation of draining lymph node cells (DLC). No changes in IL-2 production and in numbers of CD25(+) cells were noted and even decreased proliferative index (ratio of IL-2 stimulated to unstimulated DLC proliferation) was detected. Increase in granulocyte activity (MTT reduction and adhesion to plastic) was noted following application of WF solely with further increase following subsequent application of DNCB, when granulocyte activation (NBT reduction) was noted also. Access of WF into general circulation might be responsible for observed changes, what was supported by ex vivo changes in DLC and granulocyte functions assessed before initiation of sensitization and by in vitro effect of exogenous WF as well. Differential effects of WF on lymphocytes and granulocytes noted in this study highlight the need for simultaneous testing of both cell type activity what might constitute a more integrated approach in immunotoxicity studies.
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Affiliation(s)
- Milena Kataranovski
- Institute for Medical Research, Military Medical Academy, Crnotravska 17, 11000, Belgrade, Yugoslavia.
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31
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Sulowska Z, Majewska E, Krawczyk K, Klink M, Tchórzewski H. Influence of opioid peptides on human neutrophil apoptosis and activation in vitro. Mediators Inflamm 2002; 11:245-50. [PMID: 12396476 PMCID: PMC1781664 DOI: 10.1080/096293502900000104] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/27/2022] Open
Abstract
BACKGROUND It has been shown that cells of the immune system release opioid peptides and possess receptors for them. The concentrations of opioid peptides in the peripheral circulation rapidly increase during inflammation and acute stress response. AIMS The effect of opioid peptides Met-enkephalin (M-ENK) and beta-endorphin (beta-END) on the oxidative metabolism of normal human neutrophils and their death by apoptosis in vitro was investigated. METHODS Isolated from peripheral blood, neutrophils were incubated in the presence or absence of 10(-6) to 10(-10) M of M-ENK and beta-END for 12 and 18 h. Apoptosis of neutrophils was determined in vitro by flow cytometric analysis of cellular DNA content and Annexin V-FITC protein binding to the cell surface. The MTT-reduction assay was employed to estimate the oxidative metabolism of neutrophils. RESULTS Treatment with M-ENK caused a significant increase in apoptotic cells after 18 h of culture: *0 M (control) versus 10(-10) M, p < or = 0.02; **10(-10) M versus 10(-10) M, p < or = 0.02. Treatment with beta-END caused a significant increase in apoptotic cells after 12 h of culture: 0 M versus 10(-8) M, p < or = 0.03; **0 M versus 10(-10) M, p < or = 0.04. We found the significant increase in MTT reduction by neutrophils in the presence of M-ENK and beta-END both before and after the culture. However, the ability of neutrophils to reduce the MTT salt to formazan decreased significantly after the culture. CONCLUSIONS We observed that the in vitro effect of opioid peptides on the neutrophil survival and their functional state was time and dose dependent. The presence of antioxidants in the culture medium modifies neutrophil survival.
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Affiliation(s)
- Zofia Sulowska
- Microbiology and Virology Center, Polish Academy of Sciences, Lodz, Poland
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32
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Andreopoulos D, Kasi LP, Asimacopoulos PJ, Jhingran SG, Cole W, Yang D, Kim EE. Selective in vitro labeling of white blood cells using 99mTc-labeled liposomes. Nucl Med Biol 2002; 29:185-90. [PMID: 11823123 DOI: 10.1016/s0969-8051(01)00299-2] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022]
Abstract
We describe a method by which endocytosis-based radiolabeling of WBC is achieved using 99mTc-liposomes of optimal size and charge, and of a composition that assures both in vitro (whole blood) and intracellular stability of the radiopharmaceutical. In our study, excellent in vitro stability of 99mTc-liposomes with 95% labeling efficiency was observed with >90% stability up to 6 h and a minimum of 85% after 24 h of incubation either in normal saline or serum. Total WBC labeling efficiency using 99mTc-liposomes determined by radio-thin layer chromatographic analysis was 30.6 +/- 2.21%, 20.89 +/- 1.31% for monocytes and 9.7 +/- 1.74% for polymorphonuclears. Negligible activity was bound to red blood cells. The procedure did not affect the cell viability and the separation of the free 99mTc-liposomes from the cells was done by centrifugation.
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33
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Kwon KB, Yoo SJ, Ryu DG, Yang JY, Rho HW, Kim JS, Park JW, Kim HR, Park BH. Induction of apoptosis by diallyl disulfide through activation of caspase-3 in human leukemia HL-60 cells. Biochem Pharmacol 2002; 63:41-7. [PMID: 11754872 DOI: 10.1016/s0006-2952(01)00860-7] [Citation(s) in RCA: 106] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
Diallyl disulfide (DADS), a component of garlic (Allium sativum), has been known to exert potent chemopreventative activity against colon, lung, and skin cancers. However, its molecular mechanism of action is still obscure. The present study demonstrated that DADS induces apoptosis of human leukemia HL-60 cells in a concentration- and time-dependent manner with an IC50 for cell viability of less than 25 microM. DADS activated caspase-3 as evidenced by both the proteolytic cleavage of the proenzyme and increased protease activity. Activation of caspase-3 was maximal at 3 hr and led to the cleavage of 116 kDa poly(ADP-ribose) polymerase (PARP), resulting in the accumulation of an 85 kDa cleavage product. Both activation of caspase-3 and cleavage of PARP were blocked by pretreatment with either antioxidants or a caspase-3 inhibitor, but not a caspase-1 inhibitor. DADS increased the production of intracellular hydrogen peroxide, which was blocked by preincubation with catalase. These results indicate that DADS-induced apoptosis is triggered by the generation of hydrogen peroxide, activation of caspase-3, degradation of PARP, and fragmentation of DNA. The induction of apoptosis by DADS may be the pivotal mechanism by which its chemopreventative action against cancer is based.
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Affiliation(s)
- Kang-Beom Kwon
- Department of Physiology, School of Oriental Medicine, Wonkwang University, 570-749, Iksan, Republic of Korea
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34
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Kwon KB, Yang JY, Ryu DG, Rho HW, Kim JS, Park JW, Kim HR, Park BH. Vibrio vulnificus cytolysin induces superoxide anion-initiated apoptotic signaling pathway in human ECV304 cells. J Biol Chem 2001; 276:47518-23. [PMID: 11591724 DOI: 10.1074/jbc.m108645200] [Citation(s) in RCA: 56] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
Previous studies showed that exposure to Vibrio vulnificus cytolysin (VVC) caused characteristic morphologic changes and dysfunction of vascular structures in lung. VVC showed cytotoxicity for mammalian cells in culture and acted as a vascular permeability factor. In this study, the underlying mechanisms of VVC-induced cytotoxicity was investigated on ECV304 cell, a human vascular endothelial cell line. When cells were exposed to 0.4 hemolytic units (HU) of VVC, consecutive apoptotic events were observed; the elevation of superoxide anion (O (-.)(2)), the release of cytochrome c, the activation of caspase-3, the cleavage of poly(ADP-ribose) polymerase, and the DNA fragmentation. The pretreatment with 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO), O(-.) 2) scavenger, completely abolished O(-.)(2) levels and downstream apoptotic events. Moreover, pretreatment with cyclosporin A (CsA), a mitochondrial permeability transition inhibitor, was capable of attenuating O(-.)(2)-mediated cytochrome c release and caspase-3 activation, and consequent apoptosis. Apoptosis, as demonstrated by oligonucleosomal DNA fragmentation and fluorescence microscopy, was induced 24 h after VVC treatment, which was also prevented by caspase-3 inhibitor, Ac-DEVD-CHO. Caspase-1 inhibitor, Ac-YVAD-CHO, did not protect ECV 304 cells from apoptosis. These results suggest a scenario where VVC-induced apoptosis is triggered by the generation of O(-.)(2), release of cytochrome c from mitochondria, activation of caspase-3, degradation of poly(ADP-ribose) polymerase, and DNA fragmentation. The induction of apoptosis in endothelial cells by VVC may provide a pivotal mechanism for understanding the pathophysiology of septicemia.
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Affiliation(s)
- K B Kwon
- Department of Physiology, School of Oriental Medicine, Won-Kwang University, Iksan 570-749, Republic of Korea
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35
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Kataranovski M, Drasković-Pavlović B, Jovcić G, Milojević G, Todorović V, Colić M, Popović P. Peripheral blood granulocyte activity following contact sensitization of rats with dinitrochlorobenzene. Toxicology 2001; 162:121-36. [PMID: 11337111 DOI: 10.1016/s0300-483x(01)00365-1] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
Contact hypersensitivity (CHS) reaction is a classic example of a cell-mediated reaction. As the afferent phase of the reaction includes inflammation, CHS is a suitable model for investigating non-specific immunity. Some aspects of granulocyte activity in the afferent phase of experimentally induced CHS to dinitrochlorobenzene (DNCB) in two genetically different rat strains, AO and DA were examined in this study. A shift in the ratio of granulocytes to lymphocytes in favour of granulocytes and an increase in granulocyte survival were noted in DA rats. Granulocytes from both strains demonstrated increased levels of NBT reduction and an increase in their adhesion to plastic. Decreased granulocyte adhesion in the presence of monoclonal antibodies to beta2 integrins (anti-CD11b/c and anti-CD18) points to the contribution of these molecules to granulocyte adhesiveness during the sensitization phase of CHS. Stimulation of adhesion in the presence of anti-CD11a antibody, points to a differential modulation of adhesion molecule activity during the afferent phase of CHS. Changes in functional activity of granulocytes demonstrated in this study might contribute to the development of CHS in rats.
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Affiliation(s)
- M Kataranovski
- Institute for Medical Research, Military Medical Academy, Crnotravska 17, Belgrade, Yugoslavia.
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36
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Andreopoulos D, Kasi LP. 99mTc-labelled diphytanoylphosphatidylcholine liposomes: in vitro and in vivo studies. J Microencapsul 1997; 14:427-36. [PMID: 9229342 DOI: 10.3109/02652049709033827] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/04/2023]
Abstract
The uniquely structured diphytanoylphosphatidylcholine (DphPC) forms liposomes more stable than conventional straight chain phospholipids. In this study DphPC and pegylated DphPC (DphPC-PEG) liposomes were radiolabelled and evaluated in vitro and in vivo. 99mTc-DphPC liposomes were found to be nontoxic to human white blood cells in vitro. In addition 99mTc labelled DphPC-PEG liposomes were evaluated as a nonspecific infection imaging agent in a mouse model. Infection sites were imaged within 30 min postinjection, and the radiopharmaceutical exhibited a remarkable in vivo stability. As their biodistribution and pharmacokinetic patterns can be size-modulated, DphPC-based lipsomes are excellent candidates for diagnostic and therapeutic applications.
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Affiliation(s)
- D Andreopoulos
- Department of Nuclear Medicine, University of Texas, M.D. Anderson Cancer Center, Houston 77030, USA
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37
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Mazurik VK, Mikhailov VF, Ushenkova LN, Raeva NF. DNA reparation and the $$O\mathop .\limits_2^ - $$ -producing system of nonspecific defense of the organism under conditions of induced radioresistance-producing system of nonspecific defense of the organism under conditions of induced radioresistance. Bull Exp Biol Med 1997. [DOI: 10.1007/bf02766184] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/22/2022]
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Habtemariam S. Catechols and quercetin reduce MTT through iron ions: A possible artefact in cell viability assays. Phytother Res 1995. [DOI: 10.1002/ptr.2650090816] [Citation(s) in RCA: 21] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
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van de Loosdrecht AA, Beelen RH, Ossenkoppele GJ, Broekhoven MG, Langenhuijsen MM. A tetrazolium-based colorimetric MTT assay to quantitate human monocyte mediated cytotoxicity against leukemic cells from cell lines and patients with acute myeloid leukemia. J Immunol Methods 1994; 174:311-20. [PMID: 8083535 DOI: 10.1016/0022-1759(94)90034-5] [Citation(s) in RCA: 493] [Impact Index Per Article: 15.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
The MTT-colorimetric monocyte mediated cytotoxicity assay, based upon the ability of living cells to reduce 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide (MTT) into formazan, was evaluated using leukemic cells from five representative human leukemic cell lines and from 28 patients with acute myeloid leukemia (AML). An excellent linearity between absorbance and leukemic cell number was observed up to 5 x 10(4) cells/well and 50 x 10(4) cells/well for all cell lines and patients samples tested, respectively, in a 96-wells microtiter culture system. A huge variability in the susceptibility of leukemic cells to purified and IFN-gamma-activated human monocytes could be observed at effector-to-target cell (E:T) ratios of 1. The mean signal-to-noise ratio of the MTT assay for monocyte-leukemic cell mixtures from patients was 2.69 +/- 0.39 at E:T 1. In conclusion, the MTT based monocyte mediated cytotoxicity assay should be useful for studying the susceptibility of a variety of leukemic cells from cell lines and from patients with AML to monocytes in a rapid, sensitive and semi-automated manner.
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Affiliation(s)
- A A van de Loosdrecht
- Department of Hematology (BR238), University Hospital, Vrije Universiteit, Amsterdam, Netherlands
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40
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Ciapetti G, Cenni E, Pratelli L, Pizzoferrato A. In vitro evaluation of cell/biomaterial interaction by MTT assay. Biomaterials 1993; 14:359-64. [PMID: 8507779 DOI: 10.1016/0142-9612(93)90055-7] [Citation(s) in RCA: 296] [Impact Index Per Article: 9.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
The tetrazolium-based colorimetric assay (MTT test) measures only in vitro living cells and the results are directly related to the number of viable cultured cells. It has been adopted in immunological investigations, cancer research and, recently, biocompatibility evaluation. We used the MTT method with minor modifications to fit it to an in vitro study of biomaterial-cell interactions. The MTT assay was confirmed to be feasible, rapid and reproducible. Moreover, it showed a good correlation with other in vitro proliferation assays, such as the 3H-thymidine uptake assay. By using the MTT method and the ASTM procedure for extracting biomaterials, we quantified the in vitro cell compatibility of different metals and polymers.
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Affiliation(s)
- G Ciapetti
- Laboratory for Biocompatibility Research on Implant Materials, Istituti Ortopedici Rizzoli, Bologna, Italy
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41
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Wan CP, Myung E, Lau BH. An automated micro-fluorometric assay for monitoring oxidative burst activity of phagocytes. J Immunol Methods 1993; 159:131-8. [PMID: 8445246 DOI: 10.1016/0022-1759(93)90150-6] [Citation(s) in RCA: 164] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/30/2023]
Abstract
A micro-fluorometric assay using 2',7'-dichlorofluorescein diacetate (DCFH-DA) to monitor oxidative burst (OB) in phagocytes has been developed. This assay is based on the oxidation of nonfluorescent DCFH-DA to highly fluorescent 2',7'-dichlorofluorescein (DCF) both intracellularly and extracellularly. A murine macrophage cell line, J774, and a human monocytic cell line, Mono Mac 6, were used as models. The cells were harvested from tissue culture flasks, washed, counted and adjusted to desired concentrations. They were then dispensed into a 96-well flat-bottom tissue culture plate. After adding DCFH-DA and an agent eliciting OB, the plates were incubated in 5% CO2 at 37 degrees C for various periods. The intensity of fluorescence was measured directly in the wells of the tissue culture plate with the cells in situ using a computerized microplate fluorometer at 485 nm excitation and 530 nm emission. This assay provided a rapid measurement of oxidative burst of phagocytes. The automated micro-fluorometric assay may be suitable for screening the immunomodulating activities of various biological and pharmacological substances.
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Affiliation(s)
- C P Wan
- Department of Microbiology, School of Medicine, Loma Linda University, CA 92350
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42
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Oez S, Birkmann J, Kalden JR. Clonal growth of functionally normal and deficient neutrophils from the bone marrow of a patient with variant chronic granulomatous disease. Lack of reconstitution of oxidative burst defect by G-CSF, GM-CSF, and IFN-gamma in vitro. Ann Hematol 1993; 66:21-5. [PMID: 7679293 DOI: 10.1007/bf01737685] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/26/2023]
Abstract
To evaluate the effect of colony-stimulating factors and interferon gamma on the oxidative burst capacity of neutrophils in chronic granulomatous disease (CGD) we studied the neutrophils of a patient with variant CGD both from peripheral blood and from bone marrow culture on day 7 and 14. The results revealed that preincubation of peripheral neutrophils for 24 h in medium containing recombinant human granulocyte colony-stimulating factor (rhG-CSF), recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF), and recombinant human interferon gamma (rhIFN-gamma) alone or in combination did not improve the maximal oxidative burst activity measured by MTT assay. The colonies of this patient formed in agar assay were either composed of predominantly nitroblue tetrazolium (NBT)-positive cells or completely unable to reduce NBT. Despite variable colony numbers in the presence of different cytokines, the rate of NBT-positive colonies was less than 17% of the total number of colonies. However, more than 72% of the colonies were NBT positive in controls. In liquid culture, bone marrow cells yielded a comparable rate of NBT-positive and -negative populations at day 7. These data indicate that rhG-CSF, rhGM-CSF, and rhIFN-gamma alone or rhG-CSF and rhGM-CSF in combination with rhIFN-gamma are not able to reconstitute the oxidative burst defect in CGD in vitro. Indeed, regarding colony-forming capacity, the bone marrow cells from the patient responded to CSFs as well as those from control donors did. This fact may warrant the administration of hematopoietic growth factors, at least in variant CGD, in order to enhance the absolute number of functionally normal neutrophils.
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Affiliation(s)
- S Oez
- 5. Medizinische Klinik, Klinikum der Stadt Nürnberg, Federal Republic of Germany
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43
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Oez S, Welte K, Platzer E, Kalden JR. A simple assay for quantifying the inducible adherence of neutrophils. Immunobiology 1990; 180:308-15. [PMID: 1697843 DOI: 10.1016/s0171-2985(11)80294-9] [Citation(s) in RCA: 30] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022]
Abstract
By making use of the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) reducing ability of stimulated human neutrophils, we developed an alternative method to quantify the adherence of neutrophils in vitro. After discarding non-adherent neutrophils, the adherent cells were exposed to MTT solution containing 10 ng/ml phorbol 12-myristate 13-acetone (PMA). Subsequently, MTT reduced by this simultaneous stimulation was measured optically and used to calculate percent of adhesion. In these experiments, heat-inactivated autologous serum and tumor necrosis factor alpha (rhTNF-alpha) were observed to promote the adherence to polystyrene surfaces. In contrast, minimal or no effects on neutrophil adherence were achieved in case of treatment with native autologous serum, recombinant human granulocyte colony stimulating factor (rhG-CSF), recombinant human granulocyte-macrophage colony stimulating factor (rhGM-CSF), or recombinant human interferon gamma (rhIFN-gamma).
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Affiliation(s)
- S Oez
- Medizinische Klinik III, Universität Erlangen-Nürnberg, Federal Republic of Germany
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