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668 Activation of Nrf2 promotes wound healing by expansion of hair follicle stem cell populations
Wound Healing and Tissue Remodelling | ABSTRACTS 664
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Thrombospondin 1 is a major activator of TGF-beta in Recessive Dystrophic Epidermolysis Bullosa VS Atanasova1, R Russell1, TG Webster1, j salas2 and A South1 1 Dermatology and Cutaneous Biology, Thomas Jefferson University, Philadelphia, PA and 2 Dermatology, University of Monterrey, Monterrey, Mexico Recessive dystrophic epidermolysis bullosa (RDEB) is a rare genetic skin disease caused by loss of function mutations in the gene encoding type VII collagen (COL7A1). RDEB is characterized by trauma induced wounds and excessive scarring, and one of the most frequent complications in RDEB is the development of fatal squamous cell carcinoma (SCC). Our published and unpublished data show that TGF-beta signaling is increased in RDEB, and others have shown that TGF-beta is a major disease modifier. We previously demonstrated that the matricellular protein thrombospondin-1 (TSP1) is increased in RDEB fibroblasts and that COL7A1 expression modulates TSP1. Here we investigated the role of TSP1 in TGF-beta activation in RDEB primary fibroblasts. Knock-down of TSP1 reduced phosphorylation of SMAD3 (a downstream target of TGF-beta signaling) in RDEB patient fibroblasts, but not in non-RDEB normal breast fibroblasts. Over-expression of COL7A1 in RDEB fibroblasts reduced intracellular TSP1 and phosphorylated SMAD3. Furthermore, peptide inhibition of TSP1 binding to the TGF-beta e latency associated protein complex decreased nuclear localization of phosphorylated SMAD3 in tissue engineered extracellular matrix (ECM) from RDEB fibroblasts compared with controls and to a similar extent as observed after SB-431542 inhibition of the TGF-beta type I receptor. Decreased nuclear phosphorylated SMAD3 correlated with a decrease in collagen fiber formation in tissue engineered ECM from RDEB fibroblasts treated with the peptide TSP1 inhibitor, as evaluated by picrosirius red staining followed by analyses of birefringent collagen fibrillar deposits using a polarizing microscope. These data suggest TSP1 is a major activator of TGF-beta signaling in RDEB and identify TSP1 as a potent therapeutic target.
6-formylindolo[3,2-b]carbazole (FICZ) accelerates skin wound healing via activation of ERK, but not aryl hydrocarbon receptor H Uchi1, S Morino-Koga2 and M Furue1 1 Dermatology, Kyushu University, Fukuoka, Japan and 2 Institute of Molecular Embryology and Genetics, Kumamoto, Japan Wound healing is an elaborate process composed of overlapping phases, such as proliferation and remodeling, and is delayed in several circumstances, including diabetes. Although several treatment strategies for chronic wounds, such as growth factors, have been applied, further alternatives are required. The skin, especially keratinocytes, is continually exposed to UV rays, which impairs wound healing. The 6-formylindolo[3,2-b]carbazole (FICZ) is a tryptophan photoproduct formed by UV exposure, indicating that FICZ might be one of the effectors of UV radiation. Therefore, the aim of our study is to determine the role of FICZ in wound healing. Here we showed that FICZ enhanced keratinocyte migration through MEK/ ERK activation, and promoted wound healing in various mouse models, including db/db mice, which exhibit wound healing impairments because of type 2 diabetes. Moreover, FICZ, the endogenous ligand of aryl hydrocarbon receptor (AhR), accelerated migration even in AhR knockdown condition, and also promoted wound healing in DBA/2 mice, bearing a lowaffinity AhR, suggesting that FICZ enhanced keratinocyte migration in an MEK/ERK-dependent, but AhR-independent, manner. The function of FICZ might indicate the possibility of its clinical use for intractable chronic wounds.
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Interferon-g deficiency attenuates skin fibrosis in a murine model of chronic graft-versus-host disease-like scleroderma A Saito, N Okiyama, N Kubota and M Fujimoto Dermatology, Tsukuba university, Tsukuba, Japan There are few effective treatments for sclerotic skin disorders including systemic sclerosis, chronic graft-versus-host disease (GVHD) and morphea. Because interferon (IFN)-g directly inhibits the proliferation of skin fibroblasts and the production of collagen in vitro, IFN-g is known as an antifibrotic factor. However, administration of recombinant IFN-g could not inhibit fibrosis in patients with systemic scleroderma. To elucidate the role of IFN-g on skin fibrosis, we established a new murine model of scleroderma. We found that chicken ovalbumin (OVA)-specific CD8 T cell (OT-I cell)-transferred transgenic mice that express membrane-bound OVA under the control of a keratin 14 promoter (K14-mOVA Tg mice), which develop acute GVHD-like mucocutaneous disease 14 days after the transfer, present chronic GVHD-like scleroderma 28 days after the transfer. While GFP+IFN-g-/-OT-I cells-transferred K14-mOVA Tg mice developed acute GVHD-like mucocutaneous disease as severe as GFP+OT-I cell-transferred mice 14 days after the transfer, GFP+IFN-g-/-OT-I cells-transferred K14-mOVA Tg mice developed significantly milder scleroderma than GFP+OT-I cell-transferred mice 28 days after the transfer, which were assessed by dermal thickness (the averages: 93.1 v.s. 118.6mm), the numbers of infiltrating aSMA+ fibroblasts (126 v.s. 291/HPF), and hydroxyproline contents (5.9 v.s. 10.9mg/mg). In addition, the levels of mRNA of Col1a2 (30.5 v.s. 90.0), aSMA (0.6 v.s. 1.3) 28 days after the transfer, TGFb (0.7 v.s. 1.3) 14 days after the transfer were significantly lower in the skins of GFP+IFN-g-/-OT-I cell-transferred K14-mOVA Tg mice than in those of GFP+OT-I cell-transferred mice by quantitative real-time PCR. Moreover, Infiltrating GFP+OT-I cells remained in the skins of K14-mOVA Tg mice while GFP+IFN-g-/-OT-I cells disappeared 21 days after the transfer, which correlated to the levels of mRNA of IFN-g-inducible chemokines (CXCL9, CXCL10 and CXCL11). Collectively, IFN-g may play a crucial role as a trigger in the pathogenesis of skin fibrosis.
A novel TGF-beta/Smad signaling inhibitor ameliorates bleomycin-induced skin fibrosis M Hasegawa1, VH Luong1, T Chino1, N Oyama1, Y Sasaki2, D Ogura2, S Niwa2, M Fujita3, Y Okamoto3, M Otsuka3 and H Ihn3 1 University of Fukui, Yoshida-gun, Japan, 2 Link Genomics, Inc., Chuo-ku, Japan and 3 Kumamoto University, Kumamoto, Japan Transforming growth factor-beta/Smad signaling is well known to play a critical role in the pathogenesis of systemic sclerosis (SSc). We previously reported an artificial molecule HPH15 that, as we now found, might be promising as anti-fibrotic agent. This study was aimed to clarify the effects of this drug for human skin fibroblasts and preclinical model of SSc. The effects of HPH-15 on migration, proliferation, release of extracellular matrix and TGF-beta signaling cascades of human dermal fibroblasts were analyzed. The antifibrotic properties of HPH-15 were also examined in bleomycin-induced skin fibrosis mouse model. HPH-15 suppressed the phospholyration of Smad3 induced by TGF-beta in human skin fibroblasts. HPH-15 inhibited the migration, proliferation, and expression of collagen and fibronectin in cultured human skin fibroblasts. In bleomycin-induced skin fibrosis model, the oral administration of HPH-15 protected the development of skin fibrosis and ameliorated the established skin fibrosis. In the treated group, the dermal infiltration of proinflammatory macrophages (CD11b+Ly6Chi) and M2 profibrotic macrophages (CD11b+CD206+ or CD11b+CD204+) was significantly decreased at the early and late stages, respectively. HPH15 treatment inhibited the mRNA levels of M2 macrophage markers such as arginase 1 and Ym-1 in the skin, but it inversely augmented those of Fli1 and KLF-5, the transcription factors that repress collagen synthesis. No apparent adverse effects were found through the study. Our results demonstrated several advantages of HPH-15, including oral bioavailability and a good safety profile, as well as its therapeutic availability. This TGF-beta/Smad inhibitor is a potential candidate for clinical trials in SSc.
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Activation of Nrf2 promotes wound healing by expansion of hair follicle stem cell populations SS Muzumdar1, H Hiebert1, E Haertel1, W Bloch2, S Werner1 and M Scha¨fer1 1 Institute of Molecular Health Sciences, ETH Zu¨rich, Zu¨rich, Switzerland and 2 Institute of Cardiology and Sports Medicine, German Sports University Cologne, Cologne, Germany Nrf2 is a transcription factor known to be crucial in orchestrating the cellular stress response by activating the expression of various detoxifying enzymes, antioxidant proteins and transporters. Therefore, pharmacological Nrf2 activation is a promising strategy to enhance the protection of the skin against environmental insults and for chemoprevention. In this study, we report that keratinocyte-specific activation of Nrf2 promotes wound healing through enhancing re-epithelialization. Surprisingly, this could not be explained by a direct influence of Nrf2 on wound keratinocytes since migration, proliferation and apoptosis in these cells was not affected. Instead, we observed that activation of Nrf2 led to an expansion of the junctional zone and upper isthmus hair follicle stem cell populations through enhancing their proliferation. It has been shown earlier that hair follicle stem cells peripheral to the wound serve as a reservoir of cells that get activated upon wounding and migrate into the wound to form the wound tongue. In fact, epidermal abrasion revealed a functional involvement of these stem cell populations in the Nrf2-mediated enhancement of re-epithelialization. Nrf2 activation most likely stimulated junctional zone and upper isthmus stem cell proliferation through upregulation of various members of the epidermal growth factor (EGF) family including the direct Nrf2 target epigen. Thus, our experiments suggest that Nrf2 activation enhances wound healing by stimulating hair follicle stem cell proliferation through the EGFR pathway. This provides a larger reservoir of keratinocytes, which migrate into the wound leading to a faster re-epithelialization. Activation of Nrf2 therefore provides a promising new strategy for enhancing wound healing.
Senescence-associated secretion of vesicular miR-23a-3p and its impact during the paracrine crosstalk of dermal and epidermal cells V Pils1,2, T Grillenberger2, J Schwestka2, I La¨mmermann1,2, R Weinmu¨llner1,2, I Perrotta4, M Mildner3, F Morizot5, F Gruber3,2, J Grillari1,2 and L Terlecki-Zaniewicz1,2 1 Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna, Austria, 2 Christian Doppler Laboratory for Biotechnology of Skin Aging, Vienna, Austria, 3 Departement of Dermatology, Medical University, Division for Biology and Pathobiology of the Skin, Vienna, Austria, 4 Department of Biology, Ecology and Earth Sciences, University of Calabria, Cosenza, Italy and 5 Department of Biology and Women Beauty, Chanel R&T, Pantin, France The chronic accumulation of senescent cells with age and their development of a senescenceassociated secretory phenotype (SASP) is thought to be one of the main contributors of age-associated skin deterioration. It is characterized by the secretion of pro-inflammatory cytokines and extracellular matrix remodeling enzymes among others that impair tissue homeostasis, while their transient presence enhances regeneration and wound healing. Recently, we identified miRNAs enclosed in extracellular vesicles (EV) as novel members of the SASP, which we termed ‘miR-SASP’. In the present study we aim to evaluate the vesicular crosstalk between dermal and epidermal cells and assessed the impact of the HDF derived miR-SASP on primary epidermal keratinocytes (NHEK). We confirmed the transfer of EVmiRNAs derived from HDF to NHEK in monolayers and in human skin equivalents and verified the presence of EVs in human skin sections by TEM. While the transient incubation with EVs derived from senescent HDF ameliorated the wound healing capacity of NHEK, we observed an impaired differentiation potential after long-term exposure. Finally, we identified the highly secreted miR-23a-3p as a crucial mediator of the miR-SASP by an enhanced healing capacity after its overexpression in NHEK. To summarize, we here uncovered the agerelated contribution of EV-enclosed miRNAs and their impact on NHEK functionality in vitro. The consequence of senescence-associated EV-miRNAs in human skin equivalents and in vivo remains to be investigated.
www.jidonline.org S307
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Thrombospondin 1 is a major activator of TGF-beta in Recessive Dystrophic Epidermolysis Bullosa VS Atanasova1, R Russell1, TG Webster1, j salas2 and A South1 1 Dermatology and Cutaneous Biology, Thomas Jefferson University, Philadelphia, PA and 2 Dermatology, University of Monterrey, Monterrey, Mexico Recessive dystrophic epidermolysis bullosa (RDEB) is a rare genetic skin disease caused by loss of function mutations in the gene encoding type VII collagen (COL7A1). RDEB is characterized by trauma induced wounds and excessive scarring, and one of the most frequent complications in RDEB is the development of fatal squamous cell carcinoma (SCC). Our published and unpublished data show that TGF-beta signaling is increased in RDEB, and others have shown that TGF-beta is a major disease modifier. We previously demonstrated that the matricellular protein thrombospondin-1 (TSP1) is increased in RDEB fibroblasts and that COL7A1 expression modulates TSP1. Here we investigated the role of TSP1 in TGF-beta activation in RDEB primary fibroblasts. Knock-down of TSP1 reduced phosphorylation of SMAD3 (a downstream target of TGF-beta signaling) in RDEB patient fibroblasts, but not in non-RDEB normal breast fibroblasts. Over-expression of COL7A1 in RDEB fibroblasts reduced intracellular TSP1 and phosphorylated SMAD3. Furthermore, peptide inhibition of TSP1 binding to the TGF-beta e latency associated protein complex decreased nuclear localization of phosphorylated SMAD3 in tissue engineered extracellular matrix (ECM) from RDEB fibroblasts compared with controls and to a similar extent as observed after SB-431542 inhibition of the TGF-beta type I receptor. Decreased nuclear phosphorylated SMAD3 correlated with a decrease in collagen fiber formation in tissue engineered ECM from RDEB fibroblasts treated with the peptide TSP1 inhibitor, as evaluated by picrosirius red staining followed by analyses of birefringent collagen fibrillar deposits using a polarizing microscope. These data suggest TSP1 is a major activator of TGF-beta signaling in RDEB and identify TSP1 as a potent therapeutic target.
6-formylindolo[3,2-b]carbazole (FICZ) accelerates skin wound healing via activation of ERK, but not aryl hydrocarbon receptor H Uchi1, S Morino-Koga2 and M Furue1 1 Dermatology, Kyushu University, Fukuoka, Japan and 2 Institute of Molecular Embryology and Genetics, Kumamoto, Japan Wound healing is an elaborate process composed of overlapping phases, such as proliferation and remodeling, and is delayed in several circumstances, including diabetes. Although several treatment strategies for chronic wounds, such as growth factors, have been applied, further alternatives are required. The skin, especially keratinocytes, is continually exposed to UV rays, which impairs wound healing. The 6-formylindolo[3,2-b]carbazole (FICZ) is a tryptophan photoproduct formed by UV exposure, indicating that FICZ might be one of the effectors of UV radiation. Therefore, the aim of our study is to determine the role of FICZ in wound healing. Here we showed that FICZ enhanced keratinocyte migration through MEK/ ERK activation, and promoted wound healing in various mouse models, including db/db mice, which exhibit wound healing impairments because of type 2 diabetes. Moreover, FICZ, the endogenous ligand of aryl hydrocarbon receptor (AhR), accelerated migration even in AhR knockdown condition, and also promoted wound healing in DBA/2 mice, bearing a lowaffinity AhR, suggesting that FICZ enhanced keratinocyte migration in an MEK/ERK-dependent, but AhR-independent, manner. The function of FICZ might indicate the possibility of its clinical use for intractable chronic wounds.
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667
Interferon-g deficiency attenuates skin fibrosis in a murine model of chronic graft-versus-host disease-like scleroderma A Saito, N Okiyama, N Kubota and M Fujimoto Dermatology, Tsukuba university, Tsukuba, Japan There are few effective treatments for sclerotic skin disorders including systemic sclerosis, chronic graft-versus-host disease (GVHD) and morphea. Because interferon (IFN)-g directly inhibits the proliferation of skin fibroblasts and the production of collagen in vitro, IFN-g is known as an antifibrotic factor. However, administration of recombinant IFN-g could not inhibit fibrosis in patients with systemic scleroderma. To elucidate the role of IFN-g on skin fibrosis, we established a new murine model of scleroderma. We found that chicken ovalbumin (OVA)-specific CD8 T cell (OT-I cell)-transferred transgenic mice that express membrane-bound OVA under the control of a keratin 14 promoter (K14-mOVA Tg mice), which develop acute GVHD-like mucocutaneous disease 14 days after the transfer, present chronic GVHD-like scleroderma 28 days after the transfer. While GFP+IFN-g-/-OT-I cells-transferred K14-mOVA Tg mice developed acute GVHD-like mucocutaneous disease as severe as GFP+OT-I cell-transferred mice 14 days after the transfer, GFP+IFN-g-/-OT-I cells-transferred K14-mOVA Tg mice developed significantly milder scleroderma than GFP+OT-I cell-transferred mice 28 days after the transfer, which were assessed by dermal thickness (the averages: 93.1 v.s. 118.6mm), the numbers of infiltrating aSMA+ fibroblasts (126 v.s. 291/HPF), and hydroxyproline contents (5.9 v.s. 10.9mg/mg). In addition, the levels of mRNA of Col1a2 (30.5 v.s. 90.0), aSMA (0.6 v.s. 1.3) 28 days after the transfer, TGFb (0.7 v.s. 1.3) 14 days after the transfer were significantly lower in the skins of GFP+IFN-g-/-OT-I cell-transferred K14-mOVA Tg mice than in those of GFP+OT-I cell-transferred mice by quantitative real-time PCR. Moreover, Infiltrating GFP+OT-I cells remained in the skins of K14-mOVA Tg mice while GFP+IFN-g-/-OT-I cells disappeared 21 days after the transfer, which correlated to the levels of mRNA of IFN-g-inducible chemokines (CXCL9, CXCL10 and CXCL11). Collectively, IFN-g may play a crucial role as a trigger in the pathogenesis of skin fibrosis.
A novel TGF-beta/Smad signaling inhibitor ameliorates bleomycin-induced skin fibrosis M Hasegawa1, VH Luong1, T Chino1, N Oyama1, Y Sasaki2, D Ogura2, S Niwa2, M Fujita3, Y Okamoto3, M Otsuka3 and H Ihn3 1 University of Fukui, Yoshida-gun, Japan, 2 Link Genomics, Inc., Chuo-ku, Japan and 3 Kumamoto University, Kumamoto, Japan Transforming growth factor-beta/Smad signaling is well known to play a critical role in the pathogenesis of systemic sclerosis (SSc). We previously reported an artificial molecule HPH15 that, as we now found, might be promising as anti-fibrotic agent. This study was aimed to clarify the effects of this drug for human skin fibroblasts and preclinical model of SSc. The effects of HPH-15 on migration, proliferation, release of extracellular matrix and TGF-beta signaling cascades of human dermal fibroblasts were analyzed. The antifibrotic properties of HPH-15 were also examined in bleomycin-induced skin fibrosis mouse model. HPH-15 suppressed the phospholyration of Smad3 induced by TGF-beta in human skin fibroblasts. HPH-15 inhibited the migration, proliferation, and expression of collagen and fibronectin in cultured human skin fibroblasts. In bleomycin-induced skin fibrosis model, the oral administration of HPH-15 protected the development of skin fibrosis and ameliorated the established skin fibrosis. In the treated group, the dermal infiltration of proinflammatory macrophages (CD11b+Ly6Chi) and M2 profibrotic macrophages (CD11b+CD206+ or CD11b+CD204+) was significantly decreased at the early and late stages, respectively. HPH15 treatment inhibited the mRNA levels of M2 macrophage markers such as arginase 1 and Ym-1 in the skin, but it inversely augmented those of Fli1 and KLF-5, the transcription factors that repress collagen synthesis. No apparent adverse effects were found through the study. Our results demonstrated several advantages of HPH-15, including oral bioavailability and a good safety profile, as well as its therapeutic availability. This TGF-beta/Smad inhibitor is a potential candidate for clinical trials in SSc.
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Activation of Nrf2 promotes wound healing by expansion of hair follicle stem cell populations SS Muzumdar1, H Hiebert1, E Haertel1, W Bloch2, S Werner1 and M Scha¨fer1 1 Institute of Molecular Health Sciences, ETH Zu¨rich, Zu¨rich, Switzerland and 2 Institute of Cardiology and Sports Medicine, German Sports University Cologne, Cologne, Germany Nrf2 is a transcription factor known to be crucial in orchestrating the cellular stress response by activating the expression of various detoxifying enzymes, antioxidant proteins and transporters. Therefore, pharmacological Nrf2 activation is a promising strategy to enhance the protection of the skin against environmental insults and for chemoprevention. In this study, we report that keratinocyte-specific activation of Nrf2 promotes wound healing through enhancing re-epithelialization. Surprisingly, this could not be explained by a direct influence of Nrf2 on wound keratinocytes since migration, proliferation and apoptosis in these cells was not affected. Instead, we observed that activation of Nrf2 led to an expansion of the junctional zone and upper isthmus hair follicle stem cell populations through enhancing their proliferation. It has been shown earlier that hair follicle stem cells peripheral to the wound serve as a reservoir of cells that get activated upon wounding and migrate into the wound to form the wound tongue. In fact, epidermal abrasion revealed a functional involvement of these stem cell populations in the Nrf2-mediated enhancement of re-epithelialization. Nrf2 activation most likely stimulated junctional zone and upper isthmus stem cell proliferation through upregulation of various members of the epidermal growth factor (EGF) family including the direct Nrf2 target epigen. Thus, our experiments suggest that Nrf2 activation enhances wound healing by stimulating hair follicle stem cell proliferation through the EGFR pathway. This provides a larger reservoir of keratinocytes, which migrate into the wound leading to a faster re-epithelialization. Activation of Nrf2 therefore provides a promising new strategy for enhancing wound healing.
Senescence-associated secretion of vesicular miR-23a-3p and its impact during the paracrine crosstalk of dermal and epidermal cells V Pils1,2, T Grillenberger2, J Schwestka2, I La¨mmermann1,2, R Weinmu¨llner1,2, I Perrotta4, M Mildner3, F Morizot5, F Gruber3,2, J Grillari1,2 and L Terlecki-Zaniewicz1,2 1 Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna, Austria, 2 Christian Doppler Laboratory for Biotechnology of Skin Aging, Vienna, Austria, 3 Departement of Dermatology, Medical University, Division for Biology and Pathobiology of the Skin, Vienna, Austria, 4 Department of Biology, Ecology and Earth Sciences, University of Calabria, Cosenza, Italy and 5 Department of Biology and Women Beauty, Chanel R&T, Pantin, France The chronic accumulation of senescent cells with age and their development of a senescenceassociated secretory phenotype (SASP) is thought to be one of the main contributors of age-associated skin deterioration. It is characterized by the secretion of pro-inflammatory cytokines and extracellular matrix remodeling enzymes among others that impair tissue homeostasis, while their transient presence enhances regeneration and wound healing. Recently, we identified miRNAs enclosed in extracellular vesicles (EV) as novel members of the SASP, which we termed ‘miR-SASP’. In the present study we aim to evaluate the vesicular crosstalk between dermal and epidermal cells and assessed the impact of the HDF derived miR-SASP on primary epidermal keratinocytes (NHEK). We confirmed the transfer of EVmiRNAs derived from HDF to NHEK in monolayers and in human skin equivalents and verified the presence of EVs in human skin sections by TEM. While the transient incubation with EVs derived from senescent HDF ameliorated the wound healing capacity of NHEK, we observed an impaired differentiation potential after long-term exposure. Finally, we identified the highly secreted miR-23a-3p as a crucial mediator of the miR-SASP by an enhanced healing capacity after its overexpression in NHEK. To summarize, we here uncovered the agerelated contribution of EV-enclosed miRNAs and their impact on NHEK functionality in vitro. The consequence of senescence-associated EV-miRNAs in human skin equivalents and in vivo remains to be investigated.
www.jidonline.org S307